Advances in Botanical Research, Volume 51: Plant Innate Immunity

Advances in BOTANICAL RESEARCH VOLUME 51 Advances in BOTANICAL RESEARCH Series Editors JEAN-CLAUDE KADER Laborato...

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Advances in

BOTANICAL RESEARCH

VOLUME 51

Advances in

BOTANICAL RESEARCH Series Editors JEAN-CLAUDE KADER

Laboratoire Physiologie Cellulaire et Molećulaire des Plantes, CNRS, Universite´ de Paris, Paris, France

MICHEL DELSENY

Laboratoire Geńome et De´veloppement des Plantes, CNRS IRD UP, Universite´ de Perpignan, Perpignan, France

PLANT INNATE IMMUNITY Editor L. C. VAN LOON Plant-Microbe Interactions, Institute of Environmental Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier

Academic Press is an imprint of Elsevier 32 Jamestown Road, LondonNW17BY, UK 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands First edition 2009 Copyright © 2009, Elsevier Ltd. All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher. Permissions may be sought directly from Elsevier's Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made ISBN: 978-0-12-374834-8 ISSN: 0065-2296 For information on all Academic Press publications visit our Web site at www.elsevierdirect.com Printed and bound in USA 09 10 10 9 8 7 6 5 4 3 2 1

CONTENTS

CONTRIBUTORS TO VOLUME 51 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xi

PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xv

CONTENTS OF VOLUMES 35–50 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xix

PAMP-Triggered Basal Immunity in Plants ¨ RNBERGER AND BIRGIT KEMMERLING THORSTEN NU I. II. III. IV. V.

The Concept of Plant Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Signals Mediating the Activation of Plant Defense Responses . . . . . . . . . . . . . . Receptors Mediating Pattern Recognition in Plant Immunity . . . . . . . . . . . . . . Signal Transduction in PTI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Suppression of PTI—A Major Virulence Strategy of Phytopathogenic Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 4 15 21 25 27 28 28

Plant Pathogens as Suppressors of Host Defense JEAN-PIERRE ME´TRAUX, ROBERT WILSON JACKSON, ESTHER SCHNETTLER AND ROB W. GOLDBACH I. II. III. IV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Suppressors Produced by Fungal and Oomycete Pathogens . . . . . . . . . . . . . . . . Suppressors Produced by Bacterial Pathogens. . . . . . . . . . . . . . . . . . . . . . . . . . . . RNA Silencing, the Plant’s Innate Immune System Against Viruses . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

40 42 48 65 74 74

From Nonhost Resistance to Lesion-Mimic Mutants: Useful for Studies of Defense Signaling ANDREA LENK AND HANS THORDAL-CHRISTENSEN I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Defense Induction Mediated by PAMPs and EVectors . . . . . . . . . . . . . . . . . . . .

92 93

vi

CONTENTS III. IV. V. VI. VII.

Signaling Downstream of Pathogen Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . Commonalities in the Defense Response of Host and Nonhost Resistance . . . . What is the Explanation for Nonhost Resistance?. . . . . . . . . . . . . . . . . . . . . . . . . Lesion-Mimic Mutants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mutant Screens Without Pathogens for Finding Genes in Defense Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

96 99 104 107 108 112 112 112

Action at a Distance: Long-Distance Signals in Induced Resistance MARC J. CHAMPIGNY AND ROBIN K. CAMERON I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Time to Flower—Signaling Events in the Vegetative to Flowering Transition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Mechanisms of Signaling During the Wound Response . . . . . . . . . . . . . . . . . . . . IV. Long-Distance Signaling in SAR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Systemic Induced Susceptibility (SIS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Signaling During ISR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Techniques to Further Elucidate Long-Distance Signaling. . . . . . . . . . . . . . . . . . VIII. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

124 125 132 138 150 151 153 155 156

Systemic Acquired Resistance R. HAMMERSCHMIDT I. II. III. IV. V. VI.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Biological Spectrum of SAR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Induction of SAR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Systemic Biochemical Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . How SAR Protects Plants Against Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Comments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

174 177 177 185 188 209 209 209

Rhizobacteria-Induced Systemic Resistance ¨ FTE DAVID DE VLEESSCHAUWER AND MONICA HO I. II. III. IV.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Signalling in Rhizobacteria-Induced Systemic Resistance . . . . . . . . . . . . . . . . . . . Final Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

224 233 258 265 266

CONTENTS

vii

Plant Growth-Promoting Actions of Rhizobacteria STIJN SPAEPEN, JOS VANDERLEYDEN AND YAACOV OKON I. II. III. IV.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modes of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Agricultural Aspects and Relevance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

284 285 304 308 309 310

Interactions Between Nonpathogenic Fungi and Plants M. I. TRILLAS AND G. SEGARRA I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Interactions Between Plants and Endophytic Fungi . . . . . . . . . . . . . . . . . . . . . . . III. Interactions Between Plants and Free-Living Opportunistic Symbiotic Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Overview of Plant Defense Mechanisms Induced by Nonpathogenic Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

322 323 332 347 350

Priming of Induced Plant Defense Responses UWE CONRATH I. II. III. IV. V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Types of IR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Priming is a Mechanism of IR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Relevance of Priming in Plant Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

362 362 367 379 384 384 384

Transcriptional Regulation of Plant Defense Responses MARCEL C. VAN VERK, CHRISTIANE GATZ AND HUUB J. M. LINTHORST I. II. III. IV.

Plant Immune Signaling Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Defense Signaling Regulatory Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transcription Factors Regulating Plant Defense Gene Expression. . . . . . . . . . . Regulation of Plant Defenses at the Chromosomal Level . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

398 400 407 420 426 426

viii

CONTENTS

Unexpected Turns and Twists in Structure/Function of PR-Proteins that Connect Energy Metabolism and Immunity MEENA L. NARASIMHAN, RAY A. BRESSAN, MATILDE PAINO D’URZO, MATTHEW A. JENKS AND TESFAYE MENGISTE I. Historical Perspective Leading to the Recognition of Innate Immunity in Plants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Roles of PR-proteins Revealed by Studies of PR gene Expression . . . . . . . . . . . III. PR-5 Protein Structure Reveals the Primitive Relationship Between Pathogen Defense and Energy Balance . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Directions in Which Current Classification or Definition of PR-proteins May Change in the Coming Years as Advanced Functional Studies Progress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

440 444 450 472 474

Role of Iron in Plant–Microbe Interactions P. LEMANCEAU, D. EXPERT, F. GAYMARD, P. A. H. M. BAKKER AND J.-F. BRIAT I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Strategies of Iron Acquisition and Homeostasis by Plants and Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Reciprocal Interactions Between Plants and Microorganisms During Their Saprophytic Life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Reciprocal Interactions Between Plants and Microorganisms During Pathogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

492 494 505 518 530 532

Adaptive Defense Responses to Pathogens and Insects LINDA L. WALLING I. II. III. IV. V. VI. VII. VIII.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Co‐evolution of Defense Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Portals of Entry and Activation of Defenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Perceiving Pathogen and Pest Visitations: The Role of Microbial and Herbivore Elicitors and Molecular Patterns . . . . . . . . . . . . . . . . . Integrating Signals and Activating Defenses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adaptations to Unfriendly Hosts: Effectors and Evasion Tactics . . . . . . . . . . . . Effector-Triggered Immunity: Resistance to Pathogens and Pests . . . . . . . . . . . . Summary and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

552 554 556 564 576 581 589 593 595 595

CONTENTS

ix

Plant Volatiles in Defence MERIJN R. KANT, PETRA M. BLEEKER, MICHIEL VAN WIJK, ROBERT C. SCHUURINK AND MICHEL A. HARING I. II. III. IV. V. VI.

Introduction to Volatile Organic Compounds (VOCs) From Plants . . . . . . . . . Herbivore-Produced Elicitors and Suppressors of Plant VOC Emission . . . . . . Biosynthesis of Plant VOCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Volatile Metabolism in Plant Trichomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Volatile Defence Hormones MeJA, MeSA and Ethylene. . . . . . . . . . . . . . . . . . . VOC Signals Are Influenced by Abiotic Factors and Plant Developmental Stage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Natural Variation in VOC Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. VOC-Mediated Specificity of Indirect Defences . . . . . . . . . . . . . . . . . . . . . . . . . . IX. VOCs as Alarm Signals for Neighbouring Plants . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

614 616 619 624 627 632 635 639 643 651

Ecological Consequences of Plant Defence Signalling MARTIN HEIL AND DALE R. WALTERS I. II. III. IV. V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Signalling at Three Different Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Costs of Induced Resistance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Resistance Induced by Mutualistic Micro-organisms . . . . . . . . . . . . . . . . . . . . . . Defence Signalling at the Level of Plant Individual, Community and Evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Conclusions and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

668 669 680 687

Author Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

717

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

739

690 698 699 699

CONTRIBUTORS TO VOLUME 51

P. A. H. M. BAKKER Plant–Microbe Interactions, Institute of Environmental Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands PETRA M. BLEEKER Department of Plant Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands RAY A. BRESSAN Plant Stress Genomics and Technology Research Center, King Abdullah University for Science and Technology, Jeddah 21534, Saudi Arabia, World Class University Program, Gyeonsang National University, Republic of Korea, and Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA J.-F. BRIAT CNRS, Universite´ Montpellier II, SupAgro, INRA, UMR5004, Biochimie et Physiologie Molećulaire des Plantes, Place Pierre Viala, F-34060 Montpellier cedex I, France ROBIN K. CAMERON Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1 MARC J. CHAMPIGNY Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, B.C., Canada V5A 1S6 UWE CONRATH Plant Biochemistry and Molecular Biology Group, Department of Plant Physiology, RWTH Aachen University, Aachen 52056, Germany DAVID DE VLEESSCHAUWER Laboratory of Phytopathology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Gent, Belgium D. EXPERT INRA, AgroParisTech, Universite´ Paris 6, UMR217, Interactions Plantes Pathoge`nes, 16 rue Claude Bernard, F-75005 Paris, France CHRISTIANE GATZ Department of General and Developmental Plant Physiology, Albrecht-von-Haller-Institut, Untere Karspu¨le 2, 37073 Go¨ttingen, Germany F. GAYMARD CNRS, Universite´ Montpellier II, SupAgro, INRA, UMR5004, Biochimie et Physiologie Molećulaire des Plantes, Place Pierre Viala, F-34060 Montpellier cedex I, France ROB W. GOLDBACH Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands R. HAMMERSCHMIDT Department of Plant Pathology, 107 Center for Integrated Plant Systems Building, Michigan State University, East Lansing, MI 48824-1311, USA

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CONTRIBUTORS

MICHEL A. HARING Department of Plant Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands MARTIN HEIL Departamento de Ingenierıá Gene´tica, CINVESTAV— Irapuato. Km. 9.6 Libramiento Norte, 36821 Irapuato, Guanajuato, Me´xico ¨ FTE Laboratory of Phytopathology, Department of Crop MONICA HO Protection, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Gent, Belgium ROBERT WILSON JACKSON School of Biological Sciences, University of Reading, Reading RG6 6AJ, Berks, United Kingdom MATTHEW A. JENKS Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA MERIJN R. KANT Department of Population Biology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands BIRGIT KEMMERLING Eberhard-Karls-Universita¨t Tu¨bingen, Zentrum fu¨r Molekularbiologie der Pflanzen (ZMBP), Auf der Morgenstelle 5, D-72076 Tu¨bingen, Germany P. LEMANCEAU INRA, Universite´ de Bourgogne, UMR1229, Microbiologie du Sol et de l’Environnement, CMSE, 17 rue Sully, BV 86510, F-21034 Dijon cedex, France ANDREA LENK Department of Agriculture and Ecology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark HUUB J. M. LINTHORST Institute Biology Leiden, Sylviusweg 72, 2333 BE Leiden, The Netherlands TESFAYE MENGISTE Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA JEAN-PIERRE ME´TRAUX Department of Biology, University of Fribourg, CH-1700 Fribourg, Switzerland MEENA L. NARASIMHAN Plant Stress Genomics and Technology Research Center, King Abdullah University for Science and Technology, Jeddah 21534, Saudi Arabia ¨ RNBERGER Eberhard-Karls-Universita¨t Tu¨bingen, THORSTEN NU Zentrum fu¨r Molekularbiologie der Pflanzen (ZMBP), Auf der Morgenstelle 5, D-72076 Tu¨bingen, Germany YAACOV OKON Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel MATILDE PAINO D’URZO Plant Stress Genomics and Technology Research Center, King Abdullah University for Science and Technology, Jeddah 21534, Saudi Arabia

CONTRIBUTORS

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ESTHER SCHNETTLER Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands ROBERT C. SCHUURINK Department of Plant Physiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands G. SEGARRA Departament Biologia Vegetal, Facultat Biologia, Universitat de Barcelona, Avinguda Diagonal 645, 08028 Barcelona, SPAIN STIJN SPAEPEN Department of Microbial and Molecular Systems, Centre of Microbial and Plant Genetics, K.U.Leuven, Kasteelpark Arenberg 20—Box 2460, 3001 Heverlee, Belgium HANS THORDAL-CHRISTENSEN Department of Agriculture and Ecology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark M. I. TRILLAS Departament Biologia Vegetal, Facultat Biologia, Universitat de Barcelona, Avinguda Diagonal 645, 08028 Barcelona, SPAIN JOS VANDERLEYDEN Department of Microbial and Molecular Systems, Centre of Microbial and Plant Genetics, K.U.Leuven, Kasteelpark Arenberg 20—Box 2460, 3001 Heverlee, Belgium MARCEL C. VAN VERK Institute Biology Leiden, Sylviusweg 72, 2333 BE Leiden, The Netherlands LINDA L. WALLING Department of Botany and Plant Sciences, Center for Plant Cell Biology, University of California, Riverside, CA 92521, USA DALE R. WALTERS Crop & Soil Systems Research Group, Scottish Agricultural College, King’s Buildings, Edinburgh EH9 3JG, United Kingdom MICHIEL VAN WIJK Department of Population Biology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands

PREFACE: PLANT INNATE IMMUNITY

Plants flourish in almost all natural environments, even though they are surrounded by potentially harmful organisms. Because of their sessile nature, they have to cope eVectively with these threats. During evolution they have developed eVective mechanisms to counteract microbial invasion and animal attack. Apart from morphological adaptations, they rely on inducible defenses that are activated in response to infection or attack and limit proliferation of, and tissue colonization by the attacker. Most potential pathogens are halted by the expression of an integrated set of defense responses, comprising the reinforcement of plant cell walls, the synthesis of antimicrobial secondary metabolites (phytoalexins, toxins), and the accumulation of so-called pathogenesis-related proteins (peptides and low-molecular-weight proteins with toxic or lytic properties). Plant innate immunity is a collective term to describe this complex of interconnected mechanisms that plants use to withstand potential pathogens and herbivores. The last decade has seen a rapid advance in our understanding of the induction, signal-transduction, and expression of resistance responses to oomycetes, fungi, bacteria, viruses, nematodes, and insects. The present volume is aimed at providing an overview of these processes and mechanisms. It has become clear that plants have evolved sophisticated mechanisms to deal with environmental challenges. To ward oV attack by diVerent types of organisms, they possess a surveillance system that recognizes common microbial components, such as bacterial lipopolysaccharides and flagellin, or fungal constituents comprising ergosterol and chitin. These so-called MAMPS or PAMPS (microbe/pathogen-associated molecular patterns) bind to receptors on or in plant cells, upon which a signaling cascade is activated that results in a basal resistance against potential pathogens. EVective pathogens are able to evade or suppress basal resistance by secreting eVector molecules (see Fig. 1), and may even interfere with plant defense signaling pathways to their advantage. In turn, plants may activate additional defensive mechanisms, triggered either as a result of the presence of specific resistance (R) genes or through boosting of general defense responses. Even harmless organisms that are perceived through conserved MAMPs will activate the innate immune system. This may explain why plants can develop an enhanced defensive capacity upon a primary infection or attack, which may be maintained for long periods. This induced resistance is often expressed systemically and can protect the plant against further infection by the same or unrelated attackers. So-called

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PREFACE

High ETS

PTI

Pathogen effectors

Priming

Amplitude of defense response

Threshold for HR

Level of induced resistance

Level of basal resistance

Low PAMPs

Fig. 1. The basics of plant innate immunity. Healthy plants constitutively express low levels of defenses. Upon contact with a potential pathogen, conserved pathogenassociated molecular patterns (PAMPs) are recognized, leading to inducible defense responses conferring PAMP-triggered immunity (PTI). PTI may be suppressed in turn by the pathogen through the secretion of eVector molecules, resulting in eVectortriggered susceptibility (ETS). Once inducible defense responses have been activated, the tissue is primed to react more eVectively against further infection (induced resistance). Priming leads to an enhanced defensive capacity, in which the amplitude of defense responses typical of basal resistance is shifted to the higher level of induced resistance. Only rarely does induced resistance lead to a hypersensitive reaction (HR) by which the pathogen is completely halted. Adapted by permission from Macmillan Publishers Ltd: Nature 444, 323–329, copyright 2006; courtesy of Christos Zamioudis.

systemic acquired resistance (SAR) results from limited primary infection by a pathogen, whereas induced systemic resistance (ISR) can be triggered by nonpathogenic organisms that colonize root or leaf surfaces. These forms of induced resistance are phenotypically similar in that they both confer an enhanced defensive capacity on plants that is manifested by an earlier and stronger defense response upon pathogen challenge. However, the two forms are mechanistically diVerent in being based on diVerent molecular mechanisms. ISR-eliciting bacteria and fungi can also promote plant growth by mechanisms that may, or may not, be linked to the induced resistance, and in which the element iron can play an important role.

PREFACE: PLANT INNATE IMMUNITY

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Even though all types of resistance to pathogens in plants are genetically determined, the term ‘‘plant innate immunity’’ is used preferentially to denote basal resistance mechanisms. Plants lack the adaptive system of generating specific antibodies against the intruder, with which we as humans are so familiar. It has been pointed out that R gene-mediated resistance is adaptive in the sense that it evolves with time to stabilize host–pathogen relationships in genetically evolving populations. Because the underlying eVector–R protein relationship is highly specific, R gene-mediated resistance appears superficially similar to adaptive immunity in animals. However, in plants, R gene-mediated resistance is expressed through the same defense responses as those that are active in basal resistance, but on a much grander scale, often culminating in a hypersensitive reaction (HR), in which the tissue surrounding the initial point of infection rapidly necroses. The HR is particularly eVective against biotrophic pathogens, which parasitize on living host tissue, and far less so against necrotrophic pathogens that obtain their nutrients from killed tissues. Hence, plants employ diVerent strategies to deal with biotrophic and necrotrophic pathogens, as they also do against insects with diVerent feeding modes. In this book, aspects specific to R gene-mediated resistance are mentioned only briefly, as this type of resistance is essentially superimposed on basal resistance. It has been intensively studied for many years and been the subject of many excellent reviews. In contrast, concepts and mechanisms of basal resistance have emerged only relatively recently. It has become clear that the activation of appropriate defense responses is critically dependent on the integration of diverse signals from the environment: elicitors from pathogenic and nonpathogenic microorganisms, compounds released from plants as a result of damage by insects, and even volatiles from neighboring plants. The various resistance responses rely on the activation, by a combination of signaling compounds, of transcriptional regulators that determine the type of resistance expressed. An important component in the expression of resistance is the synthesis of pathogenesis-related proteins. These are commonly used as markers of resistance, as they possess potential antimicrobial and antiherbivore properties. Pathogens and insects are resisted through partially diVerent signaling pathways that can act either synergistically or antagonistically depending on the plant species and the attacker involved. Activation of these defenses is metabolically costly, and has ecological consequences by influencing plant fitness. These consequences need to be taken into account when aiming at exploiting plant innate immunity to harness the plant’s own natural defense mechanisms for durable and environmental-friendly crop protection. L. C. van Loon

CONTENTS OF VOLUMES 35–50 Series Editor (Volumes 35–44) J.A. CALLOW School of Biosciences, University of Birmingham, Birmingham, United Kingdom

Contents of Volume 35 Recent Advances in the Cell Biology of Chlorophyll Catabolism H. THOMAS, H. OUGHAM and S. HORTENSTEINER The Microspore: A Haploid Multipurpose Cell A. TOURAEV, M. PFOSSER and E. HEBERLE-BORS The Seed Oleosins: Structure Properties and Biological Role J. NAPIER, F. BEAUDOIN, A. TATHAM and P. SHEWRY Compartmentation of Proteins in the Protein Storage Vacuole: A Compound Organelle in Plant Cells L. JIANG and J. ROGERS Intraspecific Variation in Seaweeds: The Application of New Tools and Approaches C. MAGGS and R. WATTIER Glucosinolates and Their Degradation Products R. F. MITHEN

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CONTENTS OF VOLUMES 35–50

Contents of Volume 36 PLANT VIRUS VECTOR INTERACTIONS Edited by R. Plumb Aphids: Non-Persistent Transmission T. P. PIRONE and K. L. PERRY Persistent Transmission of Luteoviruses by Aphids B. REAVY and M. A. MAYO Fungi M. J. ADAMS Whitefly Transmission of Plant Viruses J. K. BROWN and H. CZOSNEK Beetles R. C. GERGERICH Thrips as Vectors of Tospoviruses D. E. ULLMAN, R. MEIDEROS, L. R. CAMPBELL, A. E. WHITFIELD, J. L. SHERWOOD and T. L. GERMAN Virus Transmission by Leafhoppers, Planthoppers and Treehoppers (Auchenorrhyncha, Homoptera) E. AMMAR and L. R. NAULT Nematodes S. A. MacFARLANE, R. NEILSON and D. J. F. BROWN Other Vectors R. T. PLUMB


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Contents of Volume 37 ANTHOCYANINS IN LEAVES Edited by K. S. Gould and D. W. Lee Anthocyanins in Leaves and Other Vegetative Organs: An Introduction D. W. LEE and K. S. GOULD Le Rouge et le Noir: Are Anthocyanins Plant Melanins? G. S. TIMMINS, N. M. HOLBROOK and T. S. FEILD Anthocyanins in Leaves: History, Phylogeny and Development D. W. LEE The Final Steps in Anthocyanin Formation: A Story of Modification and Sequestration C. S. WINEFIELD Molecular Genetics and Control of Anthocyanin Expression B. WINKEL-SHIRLEY Differential Expression and Functional Significance of Anthocyanins in Relation to Phasic Development in Hedera helix L. W. P. HACKETT Do Anthocyanins Function as Osmoregulators in Leaf Tissues? L. CHALKER-SCOTT The Role of Anthocyanins for Photosynthesis of Alaskan Arctic Evergreens During Snowmelt S. F. OBERBAUER and G. STARR Anthocyanins in Autumn Leaf Senescence D. W. LEE A Unified Explanation for Anthocyanins in Leaves? K. S. GOULD, S. O. NEILL and T. C. VOGELMANN

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Contents of Volume 38 An Epidemiological Framework for Disease Management C. A. GILLIGAN Golgi-independent Trafficking of Macromolecules to the Plant Vacuole D. C. BASSHAM Phosphoenolpyruvate Carboxykinase: Structure, Function and Regulation R. P. WALKER and Z.-H. CHEN Developmental Genetics of the Angiosperm Leaf C. A. KIDNER, M. C. P. TIMMERMANS, M. E. BYRNE and R. A. MARTIENSSEN A Model for the Evolution and Genesis of the Pseudotetraploid Arabidopsis thaliana Genome Y. HENRY, A. CHAMPION, I. GY, A. PICAUD, A. LECHARNY and M. KREIS

Contents of Volume 39 Cumulative Subject Index Volumes 1–38

Contents of Volume 40 Starch Synthesis in Cereal Grains K. TOMLINSON and K. DENYER The Hyperaccumulation of Metals by Plants M. R. MACNAIR Plant Chromatin — Learning from Similarities and Differences J. BRZESKI, J. DYCZKOWSKI, S. KACZANOWSKI, P. ZIELENKIEWICZ and A. JERZMANOWSKI


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The Interface Between the Cell Cycle and Programmed Cell Death in Higher Plants: From Division unto Death D. FRANCIS The Importance of Extracellular Carbohydrate Production by Marine Epipelic Diatoms G. J. C. UNDERWOOD and D. M. PATERSON Fungal Pathogens of Insects: Cuticle Degrading Enzymes and Toxins A. K. CHARNLEY

Contents of Volume 41 Multiple Responses of Rhizobia to Flavonoids During Legume Root Infection JAMES E. COOPER Investigating and Manipulating Lignin Biosynthesis in the Postgenomic Era CLAIRE HALPIN Application of Thermal Imaging and Infrared Sensing in Plant Physiology and Ecophysiology HAMLYN G. JONES Sequences and Phylogenies of Plant Pararetroviruses, Viruses, and Transposable Elements CELIA HANSEN and J. S. HESLOP-HARRISON

Role of Plasmodesmata Regulation in Plant Development ARNAUD COMPLAINVILLE and MARTIN CRESPI

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Contents of Volume 42 Chemical Manipulation of Antioxidant Defences in Plants ROBERT EDWARDS, MELISSA BRAZIER-HICKS, DAVID P. DIXON and IAN CUMMINS The Impact of Molecular Data in Fungal Systematics P. D. BRIDGE, B. M. SPOONER and P. J. ROBERTS Cytoskeletal Regulation of the Plane of Cell Division: An Essential Component of Plant Development and Reproduction HILARY J. ROGERS Nitrogen and Carbon Metabolism in Plastids: Evolution, Integration, and Coordination with Reactions in the Cytosol ALYSON K. TOBIN and CAROLINE G. BOWSHER

Contents of Volume 43 Defensive and Sensory Chemical Ecology of Brown Algae CHARLES D. AMSLER and VICTORIA A. FAIRHEAD Regulation of Carbon and Amino Acid Metabolism: Roles of Sucrose Nonfermenting-1-Related Protein Kinase-1 and General Control Nonderepressible-2-Related Protein Kinase NIGEL G. HALFORD Opportunities for the Control of Brassicaceous Weeds of Cropping Systems Using Mycoherbicides AARON MAXWELL and JOHN K. SCOTT Stress Resistance and Disease Resistance in Seaweeds: The Role of Reactive Oxygen Metabolism MATTHEW J. DRING Nutrient Sensing and Signalling in Plants: Potassium and Phosphorus ANNA AMTMANN, JOHN P. HAMMOND, PATRICK ARMENGAUD and PHILIP J. WHITE


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Contents of Volume 44 Angiosperm Floral Evolution: Morphological Developmental Framework PETER K. ENDRESS Recent Developments Regarding the Evolutionary Origin of Flowers MICHAEL W. FROHLICH Duplication, Diversification, and Comparative Genetics of Angiosperm MADS-Box Genes VIVIAN F. IRISH Beyond the ABC-Model: Regulation of Floral Homeotic Genes LAURA M. ZAHN, BAOMIN FENG and HONG MA Missing Links: DNA-Binding and Target Gene Specificity of Floral Homeotic Proteins RAINER MELZER, KERSTIN KAUFMANN ¨ NTER THEIßEN and GU Genetics of Floral Development in Petunia ANNEKE RIJPKEMA, TOM GERATS and MICHIEL VANDENBUSSCHE Flower Development: The Antirrhinum Perspective BRENDAN DAVIES, MARIA CARTOLANO and ZSUZSANNA SCHWARZ-SOMMER Floral Developmental Genetics of Gerbera (Asteraceae) TEEMU H. TEERI, MIKA KOTILAINEN, ANNE UIMARI, SATU RUOKOLAINEN, YAN PENG NG, URSULA MALM, ¨ NEN, SUVI BROHOLM, ROOSA LAITINEN, ¨ LLA EIJA PO PAULA ELOMAA and VICTOR A. ALBERT Gene Duplication and Floral Developmental Genetics of Basal Eudicots ELENA M. KRAMER and ELIZABETH A. ZIMMER

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Genetics of Grass Flower Development CLINTON J. WHIPPLE and ROBERT J. SCHMIDT Developmental Gene Evolution and the Origin of Grass Inflorescence Diversity SIMON T. MALCOMBER, JILL C. PRESTON, RENATA REINHEIMER, JESSIE KOSSUTH and ELIZABETH A. KELLOGG Expression of Floral Regulators in Basal Angiosperms and the Origin and Evolution of ABC-Function PAMELA S. SOLTIS, DOUGLAS E. SOLTIS, SANGTAE KIM, ANDRE CHANDERBALI and MATYAS BUZGO The Molecular Evolutionary Ecology of Plant Development: Flowering Time in Arabidopsis thaliana KATHLEEN ENGELMANN and MICHAEL PURUGGANAN A Genomics Approach to the Study of Ancient Polyploidy and Floral Developmental Genetics JAMES H. LEEBENS-MACK, KERR WALL, JILL DUARTE, ZHENGUI ZHENG, DAVID OPPENHEIMER and CLAUDE DEPAMPHILIS Series Editors (Volume 45– ) JEAN-CLAUDE KADER Laboratoire Physiologie Cellulaire et Molećulaire des Plantes, CNRS, Universite´ de Paris, Paris, France MICHEL DELSENY Laboratoire Geńome et De´veloppement des Plantes, CNRS IRD UP, Universite´ de Perpignan, Perpignan, France

Contents of Volume 45 RAPESEED BREEDING History, Origin and Evolution S. K. GUPTA and ADITYA PRATAP


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Breeding Methods B. RAI, S. K. GUPTA and ADITYA PRATAP The Chronicles of Oil and Meal Quality Improvement in Oilseed Rape ABHA AGNIHOTRI, DEEPAK PREM and KADAMBARI GUPTA Development and Practical Use of DNA Markers KATARZYNA MIKOLAJCZYK Self-Incompatibility RYO FUJIMOTO and TAKESHI NISHIO Fingerprinting of Oilseed Rape Cultivars ´ ˇ URN and JANA ZˇALUDOVA VLADISLAV C Haploid and Doubled Haploid Technology L. XU, U. NAJEEB, G. X. TANG, H. H. GU, G. Q. ZHANG, Y. HE and W. J. ZHOU Breeding for Apetalous Rape: Inheritance and Yield Physiology LIXI JIANG Breeding Herbicide-Tolerant Oilseed Rape Cultivars PETER B. E. MCVETTY and CARLA D. ZELMER Breeding for Blackleg Resistance: The Biology and Epidemiology W. G. DILANTHA FERNANDO, YU CHEN and KAVEH GHANBARNIA Development of Alloplasmic Rape MICHAL STARZYCKI, ELIGIA STARZYCKI and JAN PSZCZOLA Honeybees and Rapeseed: A Pollinator–Plant Interaction D. P. ABROL

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Genetic Variation and Metabolism of Glucosinolates NATALIA BELLOSTAS, ANNE DORTHE SØRENSEN, JENS CHRISTIAN SØRENSEN and HILMER SØRENSEN Mutagenesis: Generation and Evaluation of Induced Mutations SANJAY J. JAMBHULKAR Rapeseed Biotechnology VINITHA CARDOZA and C. NEAL STEWART, JR. Oilseed Rape: Co-existence and Gene Flow from Wild Species RIKKE BAGGER JØRGENSEN Evaluation, Maintenance, and Conservation of Germplasm RANBIR SINGH and S. K. SHARMA Oil Technology ¨ US BERTRAND MATTHA

Contents of Volume 46 INCORPORATING ADVANCES IN PLANT PATHOLOGY Nitric Oxide and Plant Growth Promoting Rhizobacteria: Common Features Influencing Root Growth and Development ´ NICA CREUS, MARIÁ CELESTE MOLINA-FAVERO, CECILIA MO LUCIANA LANTERI, NATALIA CORREA-ARAGUNDE, MARIÁ CRISTINA LOMBARDO, CARLOS ALBERTO BARASSI and LORENZO LAMATTINA How the Environment Regulates Root Architecture in Dicots ´ RIE LEFEBVRE, PHILIPPE MARIANA JOVANOVIC, VALE LAPORTE, SILVINA GONZALEZ-RIZZO, CHRISTINE LELANDAIS-BRIE`RE, FLORIAN FRUGIER, CAROLINE HARTMANN and MARTIN CRESPI


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Aquaporins in Plants: From Molecular Structure to Integrated Functions OLIVIER POSTAIRE, LIONEL VERDOUCQ and CHRISTOPHE MAUREL Iron Dynamics in Plants JEAN-FRANC ¸ OIS BRIAT Plants and Arbuscular Mycorrhizal Fungi: Cues and Communication in the Early Steps of Symbiotic Interactions VIVIENNE GIANINAZZI-PEARSON, NATHALIE SE´JALON-DELMAS, ANDREA GENRE, SYLVAIN JEANDROZ and PAOLA BONFANTE Dynamic Defense of Marine Macroalgae Against Pathogens: From Early Activated to Gene-Regulated Responses AUDREY COSSE, CATHERINE LEBLANC and PHILIPPE POTIN

Contents of Volume 47 INCORPORATING ADVANCES IN PLANT PATHOLOGY The Plant Nucleolus ´ EZ-VA ´ SQUEZ AND FRANCISCO JAVIER MEDINA JULIO SA Expansins in Plant Development DONGSU CHOI, JEONG HOE KIM AND YI LEE Molecular Biology of Orchid Flowers: With Emphasis on Phalaenopsis WEN-CHIEH TSAI, YU-YUN HSIAO, ZHAO-JUN PAN, CHIACHI HSU, YA-PING YANG, WEN-HUEI CHEN AND HONG-HWA CHEN

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Molecular Physiology of Development and Quality of Citrus ´ S, JOSE´ M. FRANCISCO R. TADEO, MANUEL CERCO COLMENERO-FLORES, DOMINGO J. IGLESIAS, MIGUEL A. NARANJO, GABINO RIÓS, ESTHER CARRERA, OMAR RUIZ-RIVERO, IGNACIO LLISO, RAPHAE¨ L MORILLON, PATRICK OLLITRAULT AND MANUEL TALON Bamboo Taxonomy and Diversity in the Era of Molecular Markers MALAY DAS, SAMIK BHATTACHARYA, PARAMJIT SINGH, TARCISO S. FILGUEIRAS AND AMITA PAL

Contents of Volume 48 Molecular Mechanisms Underlying Vascular Development JAE-HOON JUNG, SANG-GYU KIM, PIL JOON SEO AND CHUNG-MO PARK Clock Control Over Plant Gene Expression ANTOINE BAUDRY AND STEVE KAY Plant Lectins ELS J. M. VAN DAMME, NAUSICAA LANNOO AND WILLY J. PEUMANS Late Embryogenesis Abundant Proteins MING-DER SHIH, FOLKERT A. HOEKSTRA AND YUE-IE C. HSING

Contents of Volume 49 Phototropism and Gravitropism in Plants MARIA LIA MOLAS AND JOHN Z. KISS


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Cold Signalling and Cold Acclimation in Plants ERIC RUELLAND, MARIE-NOELLE VAULTIER, ALAIN ZACHOWSKI AND VAUGHAN HURRY Genome Evolution in Plant Pathogenic and Symbiotic Fungi GABRIELA AGUILETA, MICHAEL E. HOOD, GUISLAINE REFRE´GIER AND TATIANA GIRAUD

Contents of Volume 50 Aroma Volatiles: Biosynthesis and Mechanisms of Modulation During Fruit Ripening BRUNO G. DEFILIPPI, DANIEL MANRI´QUEZ, ´ LEZ-AGU ¨ ERO KIETSUDA LUENGWILAI AND MAURICIO GONZA Jatropha curcas: A Review NICOLAS CARELS You are What You Eat: Interactions Between Root Parasitic Plants and Their Hosts LOUIS J. IRVING AND DUNCAN D. CAMERON Low Oxygen Signaling and Tolerance in Plants FRANCESCO LICAUSI AND PIERDOMENICO PERATA Roles of Circadian Clock and Histone Methylation in the Control of Floral Repressors RYM FEKIH, RIM NEFISSI, KANA MIYATA, HIROSHI EZURA AND TSUYOSHI MIZOGUCHI

PAMP-Triggered Basal Immunity in Plants

¨ RNBERGER AND BIRGIT KEMMERLING THORSTEN NU

Eberhard-Karls-Universita¨t Tu¨bingen, Zentrum fu¨r Molekularbiologie der Pflanzen (ZMBP), Auf der Morgenstelle 5, D-72076 Tu¨bingen, Germany

I. The Concept of Plant Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Signals Mediating the Activation of Plant Defense Responses . . . . . . . . . . . . . A. Pathogen-Associated Molecular Patterns................................... B. Damage-Associated Molecular Patterns .................................... C. Pathogen-Derived Toxins as Triggers of Plant Immunity................ III. Receptors Mediating Pattern Recognition in Plant Immunity . . . . . . . . . . . . . IV. Signal Transduction in PTI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Suppression of PTI—A Major Virulence Strategy of Phytopathogenic Bacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 4 4 11 12 15 21 25 27 28 28

ABSTRACT Significant progress has recently been made in our understanding of the molecular mechanisms that underpin a plant’s ability to cope with microbial infection. A new concept has derived thereof that provides evidence for a functional link between different types of microbial resistance in plants and their evolutionary relationship. Research on microbial elicitor-induced plant noncultivar-specific defenses and Corresponding authors: Email: [email protected], [email protected] Advances in Botanical Research, Vol. 51 Copyright 2009, Elsevier Ltd. All rights reserved.

0065-2296/09 $35.00 DOI: 10.1016/S0065-2296(09)51001-4

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¨ RNBERGER AND B. KEMMERLING T. NU

microbial avirulence factor-induced host plant cultivar-specific defenses had coexisted for a long time, without providing an integrated model for plant disease resistance. Research milestones that have significantly reshaped our view on plant immunity comprise the realization of conceptual and mechanistic similarities in animal and plant immunity (including the adoption of the term ‘‘immunity’’ into the plant literature), the identification of plant pattern-recognition receptors (PRRs) recognizing pathogen- or microbe-derived molecular patterns (PAMP/MAMP), and the finding that PAMP-triggered immunity (PTI) is a biologically important element of plant disease resistance. Moreover, microbial infection strategies that have evolved for the suppression of PTI underline the importance of this element of the plant immune system.

I. THE CONCEPT OF PLANT IMMUNITY The recent literature on plant defense, disease resistance or susceptibility has adopted a terminology that is very different from that of some time ago. This phenomenon is characterized by the use of such terms as innate immunity, pathogen-associated molecular pattern, pattern-recognition receptors (PRRs), effectors etc. This terminology, which has been deliberately chosen because of obvious cross-kingdom parallels in the molecular concept of immunity, has in a large part replaced the phytopathological vocabulary that has dominated the literature for many years. However, is this development a justified one? As there is still some confusion in the field on this issue, we see a need to address the adequacy of an immunity-associated terminology for the description of plant disease resistance. In general, the term immunity describes the ability of an organism to withstand microbial infection, disease or other disadvantageous biological invasion. As this definition is valid for all multicellular eukaryotic systems, it is correct to refer to the ability of plants to cope with microbial infections as an immune response. Notwithstanding early discussions on analogies between plant disease resistance genes and the major histocompatibility complex of animal systems (Dangl, 1992), the true force that initiated this paradigm shift has been the recent reappreciation of innate immunity in insects and jawed vertebrates as a major element for the containment of microbial infection and for sufficient functioning of the adaptive immune system in animals. Since then, countless landmark papers have highlighted striking similarities in the molecular organization of nonself recognition and antimicrobial defense systems in animals and plants and thus, have substantiated and lent justification to this development (Ausubel, 2005; Chisholm et al., 2006; Jones and Dangl, 2006; Nu¨rnberger et al., 2004). Such similarities are found in the nature of the microbial patterns that are recognized by the innate immune systems in both lineages, extend to the molecular architecture of

PAMP-TRIGGERED BASAL IMMUNITY IN PLANTS

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pattern recognition complexes, and culminate in the production of antimicrobial products that eventually halt microbial infections. In the light of such information, the adoption of an immunity-based terminology by molecular plant pathologists is a logical consequence—at least in hindsight. Related terminologies in plant and animal immunity should, however, be taken with caution as significant differences between the systems remain. For example, the term ‘‘innate’’ in conjunction with plant immunity appears to be dispensable, as all immunity-associated traits of plants are inheritable. In animal systems, the term is useful as it discriminates between germline-encoded innate immunity and adaptive immunity, the latter being characterized by the large antigen receptor reservoir resulting from genomic recombination and by clonal expansion of particular lymphocyte populations (Medzhitov, 2007). Adaptive immunity, however, does not exist in plants, and the term ‘‘innate’’ in conjunction with plant immunity is misleading because it implies the existence of another type of plant immunity that is of non‐innate nature. Plants provide multiple habitats that can be invaded by microorganisms, including the roots, leaves, flowers or the vascular system. Some of these niches, such as the rhizosphere, are colonized constitutively by a vast microflora. However, the consequence of microbial colonization on host fitness strictly depends on the microbial strategy to adapt to the host environment. Different symbioses represent cases in which the impact of infection is positive and mutually beneficial to both partners. The relationship between two living organisms in which one benefits, and the other is not significantly affected has been described in plant–microbe associations, but the term commensalism (commonly used in animal ecology to refer to this phenomenon) is unusual in the plant literature. In other cases, microbial colonization can be disadvantageous to the host, and microbes that interact in an antagonistic manner with their hosts, are referred to as pathogens. The ability of infectious pathogens to strive in the specific environment of the host is governed by virulence factors that enable host ingress, the establishment of stable infection structures, suppression and evasion of host defenses, microbial nutrition and proliferation. In turn, microbial infections are considered to be the driving force that shapes the plant immune system during evolution. In brief, the plant immune system consists of two evolutionarily linked branches. The evolutionarily more ancient, primary plant immune response is referred to as PAMP-triggered immunity (PTI) and is based upon the recognition of the invariant structures of microbial surfaces termed PAMPs (Ausubel, 2005; Chisholm et al., 2006; Jones and Dangl, 2006; Nu¨rnberger et al., 2004; Zipfel and Felix, 2005). PAMP-induced immune responses are important for immunity to microbial infection of whole plant species (species

4


or nonhost immunity) and for basal immunity in susceptible host plant cultivars (Bittel and Robatzek, 2007; Nu¨rnberger and Lipka, 2005). Suppression of PTI by microbial effectors (effector-triggered susceptibility, ETS) is assumed to be the key for successful pathogens to grow and multiply in a potentially hostile plant environment (Alfano and Collmer, 2004; Chisholm et al., 2006; Jones and Dangl, 2006). As a consequence of a co‐evolutionary arms race, co‐evolution between susceptible hosts and virulent pathogens, individual plant cultivars have acquired resistance (R) proteins that guard microbial effector-mediated perturbations of host cell functions and thus trigger plant immune responses. This type of plant defense is referred to as effector-triggered immunity (ETI) and is synonymous with pathogen race/ host plant cultivar-specific plant disease resistance (Ausubel, 2005; Chisholm et al., 2006; Jones and Dangl, 2006). The following chapters focus on the current knowledge about PTI. Readers interested in the molecular basis of ETS/ETI are referred to the relevant chapters of this monograph, as well as to a wealth of original and review literature that is available on this topic.

II. SIGNALS MEDIATING THE ACTIVATION OF PLANT DEFENSE RESPONSES A. PATHOGEN-ASSOCIATED MOLECULAR PATTERNS

Activation of inducible host defenses strictly depends on the recognition of potential microbial invaders, regardless of their aggressive potential. Microbial sensing is based on the detection of molecular structures (‘‘patterns’’) that are unique to microorganisms and that enable the host to discriminate between microbial nonself and host-derived self (Akira et al., 2006; Ferrandon et al., 2007; Medzhitov, 2007). In 1997, Medzhitov and Janeway provided a terminology to describe the elements and processes implicated in innate immunity in various animal systems (Medzhitov and Janeway, 1997). The authors referred to PAMPs as triggers of immune responses in organisms as diverse as human, mice, crustaceans, and insects. Immune defenses in jawed vertebrates comprise proinflammatory cytokine production mediating inflammatory responses (referred to as activation of the ‘‘inflammasome’’), as well as the production and secretion of antimicrobial, proteinaceous defensins (Akira et al., 2006; Medzhitov, 2007). Likewise, insects such as Drosophila melanogaster produce a large blend of antimicrobial peptides that constitute the major executive element in insect innate immunity (Ferrandon et al., 2007; Girardin et al., 2002). Lipopolysaccharides (LPS) derived from Gram-negative bacteria, peptidoglycans from both Gram-positive and


5

Gram-negative bacteria, eubacterial flagellin, unmethylated bacterial DNA fragments, as well as fungal cell wall-derived glucans, chitins, mannans and proteins are well-characterized patterns that trigger innate immune responses in numerous vertebrate and nonvertebrate organisms (Aderem and Ulevitch, 2000; Ferrandon et al., 2007; Girardin et al., 2002; Medzhitov, 2007). Undoubtedly, the PAMP-terminology had an enormous impact on molecular plant pathology, which was mainly because many of the microbe-associated patterns with immunity-stimulating features were long known as (general) elicitors of cultivar‐nonspecific defenses in many plants (Boller, 1995; Vorwerk et al., 2004; Zipfel and Felix, 2005) (Table I). For example, peptidoglycans and muropeptides derived from Gram-positive and Gramnegative bacteria, different structural elements of LPS from Gram-negative bacteria, or an N-terminal 22-amino acid fragment of eubacterial flagellin (flg22) are potent inducers of defense-associated responses in various plant species (Erbs et al., 2008; Felix et al., 1999; Gust et al., 2007; Newman et al., 2002). A comprehensive summary of such PAMPs with proven immunitystimulating activities in plants is listed in Table I. The intriguing aspect of that insight was that it implies a common evolutionary concept of microbial pattern recognition that generally underlies activation of antimicrobial counter-defense in multicellular eukaryotes. In addition, such insight also suggested a relevant role of elicitor recognition in plant immunity that had been predicted for a long time. However, the mere existence of such recognition events did not provide evidence for a causal link between elicitorinduced plant defenses and plant disease resistance or immunity. This gap was only filled when the contribution of PRR-mediated basal resistance to overall plant immunity was unequivocally documented (Zipfel et al., 2004). It is needless to say that the term PAMP is a misnomer, because such structures are not only found on pathogenic microbes, but are also characteristic of nonpathogenic microorganisms (Ausubel, 2005). There have been attempts in the recent (plant, but not animal) literature to introduce more correct terms, such as MAMP (Ausubel, 2005) or MIMP (microbe-induced molecular patterns) (Mackey and McFall, 2006). While these terms have their merits and justification, we prefer the further use of the ‘‘historical’’ term PAMP simply for the reason of maintaining the understanding among the communities of animal and plant immunologists. PAMPs constitute abundant, conserved structures (patterns) that are typical of whole classes of pathogens (Medzhitov and Janeway, 1997). In addition, such patterns appear to be absent in eukaryotic host organisms, but are indispensable for the microbial lifestyle. Because of such characteristics PAMPs are considered to be favorite determinants for microbe detection by host-encoded nonself recognition systems. General elicitors of plant defenses

TABLE I Known Inducers of PAMP-Triggered Immunity

PAMP Lipopolysaccharide Peptidoglycan Flagellin

Origin Gram-negative bacteria (Xanthomonas, Pseudomonas) Gram-positive and Gram-negative bacteria Gram-negative bacteria

Elongation factor (EF-Tu)

Gram-negative bacteria

Harpin (HrpZ)

Gram-negative bacteria (Pseudomonas, Erwinia)

Cold-shock protein

Gram-negative bacteria Gram-positive bacteria

Necrosis-inducing proteins (NLP)

Bacteria (Bacillus spp.), fungi (Fusarium spp.), oomycetes (Phytophthora spp., Pythium spp.)

Minimal structural motif required for defense activation

Sensitive plants

Lipid A, lipooligosaccharides

Pepper, tobacco

Muropeptides

Arabidopsis, tomato

flg22 (amino-terminal fragment of flagellin) elf18 (N-acetylated amino-terminal fragment of EF-Tu) Undefined

Arabidopsis, tomato

RNP-1 motif (aminoterminal fragment of the cold-shock protein) Undefined

References Meyer et al. (2001); Newman et al. (2002); Zeidler et al. (2004) Erbs et al. (2008); Felix and Boller (2003); Gust et al. (2007) Felix et al. (1999)

Arabidopsis and other Brassicaceae

Kunze et al. (2004)

Arabidopsis, cucumber, tobacco, tomato Solanaceae

He et al. (1993); Lee et al. (2001a); Wei et al. (1992) Felix and Boller (2003)

Dicotyledonous plants

Bailey (1995); Fellbrich et al. (2002); Mattinen et al. (2004); Pemberton and Salmond (2004); Qutob et al. (2002); Veit et al. (2001)

Transglutaminase

Oomycetes (Phytophthora spp.)

Pep-13 motif (surfaceexposed epitope of the transglutaminase)

Cellulose‐binding elicitor lectin (CBEL) Lipid-transfer proteins (elicitins) Xylanase

Oomycetes (Phytophthora spp.) Oomycetes (Phytophthora spp., Pythium spp.) Fungi (Trichoderma spp.)

Conserved Cellulose‐ binding domain Undefined

Invertase

Yeast

-glucans

Sulfated fucans Chitin

Fungi (Pyricularia oryzae) Oomycetes (Phytophthora spp.) Brown algae Brown algae All fungi

Ergosterol Cerebrosides A, C Oligouronides Cellodextrins Cutin monomers

All fungi Fungi (Magnaporthe spp.) Plant cell wall pectins Plant cell wall cellulose Plant cuticle

Siderophores

Pseudomonas fluorescens

TKLGE pentapeptide (surface-exposed epitope of the xylanase) N-mannosylated peptide (fragment of the invertase) Tetraglucosyl glucitol Branched hepta-glucoside Linear oligo--glucosides Fucan oligosaccharide Chitin oligosaccharides (degree of polymerisation > 3) Sphingoid base Oligomers Oligomers Dodecan-1-ol Undefined

Grapevine, Nicotiana benthamiana, parsley, potato, tobacco Tobacco, Arabidopsis Tobacco, turnip

Brunner et al. (2002); Nu¨rnberger et al. (1994)

Gaulin et al. (2006) Osman et al. (2001)

Tobacco, tomato

Enkerli et al. (1999); Ron and Avni (2004)

Tomato

Basse et al. (1993)

Rice, tobacco, Fabaceae

Fliegmann et al. (2004); Klarzynski et al. (2000); Yamaguchi et al. (2000)

Tobacco Arabidopsis, barley, rice, tomato, wheat Tomato Rice Arabidopsis, tobacco Grapevine Apple, cucumber, tomato Tobacco

Klarzynski et al. (2003) Baureithel et al. (1994); Ito et al. (1997) Granado et al. (1995) Koga et al. (1998) Darvill et al. (1994) Aziz et al. (2007) Fauth et al. (1998) Van Loon et al. (2008)

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meet these characteristics (Brunner et al., 2002; Felix and Boller, 2003). Pep-13 (Nu¨rnberger et al., 1994), a surface-exposed peptide motif present in cell wall transglutaminases (TGase) of various Phytophthora species (Brunner et al., 2002) may serve as a recognition determinant for the activation of defense in plants, including parsley, potato, grapevine and Nicotiana benthamiana (Halim et al., 2005; H.-H. Kassemeyer and T. Romeis, personal communication). Pep-13 sequences are conserved among Phytophthora TGases, but are not found in any proteins of higher eukaryotic origin. The Pep-13 motif is essential for elicitor activity and for TGase activity of the protein, and individual TGase isoforms containing the Pep-13 motif are expressed at all stages of the life cycle of Phytophthora infestans, including plant infection, suggesting that these enzymes play pivotal roles in Phytophthora biology (Fabritius and Judelson, 2003). A similar set of investigations was conducted on cold-shock-inducible RNA-binding proteins that are found in various Gram-positive bacteria (RNP-1) and that induce defense responses in tobacco. As shown for Pep-13, this elicitor also met the characteristics of a PAMP (Felix and Boller, 2003). A conserved central peptide (csp22) within RNP-1 was found in all bacterial RNP-1 orthologs tested. This region proved indispensable not only for the RNA-binding activity of the protein, but was also shown to be necessary and sufficient for its defense-inducing potential. It is reasonable to assume that other microbe-specific structures, such as fungal chitin, oomycete glucans, bacterial flagellin and the bacterial elongation factor, EF-Tu (Kunze et al., 2004), are indispensable for the microbial host as well, and are thus supposed to be conceptually equivalent to PAMPs that trigger innate immunity in animal systems (Medzhitov and Janeway, 1997). Indeed, very recently it was shown that the N-terminal fragment of bacterial flagellin, flg22, was not only sufficient to trigger well-known PAMP responses in Arabidopsis thaliana (Felix et al., 1999), but also for the proper function of flagellar stability and bacterial motility (Naito et al., 2008). A mutation in a conserved aspartic acid residue in flagellin of Pseudomonas syringae pv. tabaci was shown to result not only in weaker PAMP activity, but also in reduced bacterial mobility and fitness (Naito et al., 2008). Intimate contact between pathogen and host surfaces during attempted infection inevitably results in near-simultaneous exposure of various microbial patterns to the repertoire of cognate host PRRs. It is envisaged that activation of inducible plant defenses is likely the result of recognition of complex patterns that build the microbial surface (Nu¨rnberger and Lipka, 2005; Zipfel and Felix, 2005) rather than that of individual recognition events. For example, the cell walls of many phytopathogenic fungi harbor chitins, N-mannosylated glycopeptides and ergosterol, all of which have been


9

reported to trigger plant defense responses (Basse et al., 1993; Baureithel et al., 1994; Granado et al., 1995). Various phytopathogenic Gram-negative bacteria harbor plant defense-stimulating LPS and flagellin and produce HrpZ (harpins), bacterial effector proteins that may function as pathogenicity factors during bacterial infection of plants and that are translocated into the plant apoplast via the bacterial type III secretion system (TTSS) upon contact with plants (Felix et al., 1999; He et al., 1993; Lee et al., 2001a; Newman et al., 2002; Wei et al., 1992). Moreover, phytopathogenic oomycetes of the genera Phytophthora and Pythium possess defense-eliciting heptaglucan structures, elicitins and other cell wall proteins (Fellbrich et al., 2002; Kamoun, 2001; Mitho¨fer et al., 2000; Qutob et al., 2002; Veit et al., 2001). Although not all plant species may recognize and respond to all of these signals, plant cells have recognition systems for multiple patterns derived from individual microbial species. This is exemplified by tobacco and Arabidopsis cells, which recognize Ps. syringae-derived harpins and flagellin (Desikan et al., 1999; Felix et al., 1999), and tomato cells that have the abilities to perceive fungal chitin fragments, glycopeptides and ergosterol (Boller, 1995). Obviously, complex pattern recognition in plants is another phenomenon that resembles the activation of innate defense responses in animals. For example, innate immune responses in humans are activated by Gram-negative bacteria-derived LPS, flagellin and unmethylated CpG dinucleotides, which are characteristic of bacterial DNA (Aderem and Ulevitch, 2000; Akira et al., 2006; Medzhitov, 2007). It is currently open whether recognition of multiple signals derived from one pathogen may mediate more sensitive perception or, alternatively, if redundant recognition systems may act as independent back-up systems in the same or different tissues. It was shown that peptidoglycans from Gram-positive bacteria act synergistically on inflammatory cytokine production in human mononuclear macrophages when added simultaneously with Gram-negative bacteria-derived LPS (Wolfert et al., 2002). Similarly, simultaneous application of Ps. syringae-derived LPS and HrpZ1 resulted in synergistic activation of antimicrobial phytoalexin production in parsley cells (our unpublished data). In contrast, bacterial flagellin and EF-Tu activate a common set of signaling events and defense responses, but with additive rather than synergistic effects (Zipfel et al., 2006). Further experiments using different PAMP combinations from the same microbial sources, as well as combinations of individual PAMPs at very low concentrations are needed to prove if synergistic or additive effects are triggered by perception of PAMP combinations. In either case, however, may eukaryotic hosts take advantage of concomitant recognition of microbial patterns.

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PAMPs have recently been characterized as triggers of various plant immunity-associated responses that, however, would not include apoptotic-like, programmed cell death (hypersensitive response, HR). This places PAMPtriggered responses in contrast to ETI responses in resistant host plant cultivars that frequently involve HR (Jones and Dangl, 2006). However, like ETI, that does not always rely on host HR, some microbe-derived PAMPs were shown to trigger HR cell death in a plant cultivar-nonspecific manner. For example, a pentapeptide-motif found within fungal cell wall-associated xylanases is sufficient for the HR elicitor activity of the intact protein (Rotblat et al., 2002). Moreover, flagellin preparations from various, but not all Pseudomonas species exhibited cell-death-inducing activities in tobacco and rice (Che et al., 2000; Hann and Rathjen, 2007), but not in Arabidopsis, whereas Pep-13 triggered cell death in potato, but not in parsley (Halim et al., 2005). Very recently, the flagellin peptide flg22 was also reported to trigger cell death responses in Arabidopsis (Naito et al., 2008). This is contrary to previous reports on such activities of flg22, which raises the suspicion that additional, yet elusive environmental conditions may impinge on the occurrence of PAMP-triggered cell death in plants. Clearly, statements regarding cell death activities of PAMPs need to be made with reference to the experimental system, and cannot be made in a grossly generalizing manner. PAMPs are considered to be building blocks of microbial surfaces that are constitutively present. However, phytopathogenic Gram-negative bacterial species of the genera Pseudomonas, Erwinia, and Ralstonia produce HrpZ, HrpN or PopA proteins (‘‘harpins’’), that are massively secreted only upon attempted invasion of plants (Alfano and Collmer, 2004). Although secreted in a TTSS-dependent manner, unlike typical TTSS effectors ‘‘harpins’’ appear not to be translocated into plant host cells, but to be targeted to the apoplastic plant/bacteria interface. Harpins have been shown to contribute to microbial virulence (Bauer et al., 1995) and to form ion-conducting pores in synthetic and plant lipid-bilayer systems (Lee et al., 2001b; Racape´ et al., 2005). This suggested that these proteins are ‘‘helper proteins’’ that facilitate nutrient delivery into the apoplastic space or effector delivery into host cells as part of the TTSS effector translocon. HrpZ has recently been assigned a function in effector delivery in Ps. syringae, thus rendering the latter assumption more likely (Kvitko et al., 2007). Remarkably, all ‘‘harpins’’ have been shown to trigger noncultivar-specific plant immunity, including HR cell death, in various plant species (Alfano and Collmer, 2004; Lee et al., 2001a). A structure–function analysis of the HrpZ protein from Ps. syringae pv. phaseolicola revealed that the full-length protein was required for its virulence-associated pore-forming activity, while a C-terminal portion of the protein was sufficient for the activation of plant immune responses (Engelhardt et al., 2008). Since the C-terminal portion was also sufficient to


11

bind to a previously identified HrpZ binding site in tobacco and parsley membranes (Lee et al., 2001a), it is assumed that HrpZ plays dual roles in plant–microbe interactions as a virulence-promoting factor and as a trigger of PTI (Engelhardt et al., 2008). B. DAMAGE-ASSOCIATED MOLECULAR PATTERNS

Breakdown products of the plant cell wall, also called ‘‘endogenous elicitors,’’ have long been known to elicit plant immune responses (Vorwerk et al., 2004). Plant cell wall-derived oligogalacturonide fragments (pectic fragments from primary cell walls), cellulose fragments (cellodextrins) or cutin monomers stimulate plant immune responses that are indistinguishable from those triggered by microbe-derived PAMPs (Aziz et al., 2007; Darvill et al., 1994; Fauth et al., 1998). Such plant-derived elicitors are likely released by glucohydrolytic activities from attacking microbes, and are thought to be conceptually equivalent to animal tissue-derived ‘‘danger’’ or ‘‘alarm’’ signals. Animal host-derived patterns are produced either upon microbial infection, or as a result of mechanical injury or necrotic cell death (called damage-associated molecular patterns, DAMPs), and act as mediators of cell damage or distress, perception of which eventually culminates in the activation of innate immune responses (Gallucci and Matzinger, 2001; Matzinger, 2002; Seong and Matzinger, 2004). There exists a large body of animal hostderived immunostimulators, comprising glucose-starvation proteins, fibronectins, hyaluronan, heat-shock proteins (Hsp), cardiolipin and -defensins, many of which are sensed through PRRs that also recognize ‘‘classical’’ PAMPs. For example, TLR4 (human Toll-like receptor 4) recognizes bacterial LPS, host-derived Hsp70, and breakdown products of host hyaluronan (Matzinger, 2007). Common to all these signals is that they are not released in/to the blood or lymph system in intact and healthy tissues and, therefore, do not normally get into contact with PRRs on specialized immune cells that patrol the body for the presence of microbial patterns as well as for such determinants of damaged host-self. Because these endogenous immunostimulators are difficult to fit into self/nonself discrimination models (referred to as the ‘‘stranger’’ model), it has been proposed that animal innate immune cells may rather recognize ‘‘danger’’ signals that comprise both, nonself representing microbial PAMPs and damaged self-representing DAMPs (‘‘danger’’ model) (Matzinger, 2002). Infection by microbes usually also inflicts tissue damage and, thus, microbial PAMPs together with tissuederived DAMPs might constitute a more powerful trigger of defense responses than either alone. It is quite conceivable that activation of plant immunity by pectin, cellulose or cutin fragments constitutes a phenomenon that is conceptually similar to DAMP-induced animal immunity.

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¨ RNBERGER AND B. KEMMERLING T. NU C. PATHOGEN-DERIVED TOXINS AS TRIGGERS OF PLANT IMMUNITY

Microbial toxin-induced plant immunity is a virtual paradox that has been known for a long time. Phytopathogenic microorganisms produce numerous cytolytic toxins that function as major virulence factors (Glazebrook, 2005; Van’t Slot and Knogge, 2002). Phytopathogenic necrotrophic fungi, for example, synthesize host-selective and host-nonselective compounds that facilitate killing of host plant tissue (Gijzen and Nu¨rnberger, 2006; Qutob et al., 2006; Van’t Slot and Knogge, 2002; Wolpert et al., 2002). An intriguing characteristic of many of these mycotoxins is that they not only cause damage, but also trigger plant immunity-associated cellular responses. Certain Fusarium spp. produce the sphinganine toxin, fumonisin B1 (FB1), that elicits cytolysis of plant and animal cells most probably through competitive inhibition of ceramide synthase, an enzyme involved in sphingolipid biosynthesis (Tolleson et al., 1999; Wang et al., 1996). However, in addition to cell death, FB1 triggers accumulation of reactive oxygen species (ROS), deposition of callose, defense-related gene expression and production of the phytoalexin camalexin in Arabidopsis (Asai et al., 2000; Stone et al., 2000). Likewise, the cell death-inducing toxins fusicoccin from Fusicoccum amygdali, or AAL-toxin from Alternaria alternata trigger expression of pathogenesis-related (PR) genes in tomato or Arabidopsis, respectively (Gechev et al., 2004; Schaller and Oecking, 1999), whilst the host selective cell death-inducing toxin victorin from Cochliobolus victoriae elicits the production of avenanthramide phytoalexins in oat (Tada et al., 2005). It is most important to state though, that it is uncertain in virtually all cases whether toxin-induced plant immune responses are an unavoidable consequence of toxin action or, alternatively, if toxins have a second role as PAMP-like factors that trigger plant defenses in a host-PRRdependent fashion. Necrosis and ethylene-inducing protein 1 (Nep1)-like proteins (NLPs) are proteins that have been scrutinized for their molecular mode of action (Bae et al., 2006; Fellbrich et al., 2002; Mattinen et al., 2004; Pemberton et al., 2005; Qutob et al., 2002; Veit et al., 2001). NLPs trigger a multifaceted plant immune response in various dicotyledonous plants, but not in monocotyledons. NLPs are found in multiple bacterial, fungal and oomycete species, most of which favor a (hemi)biotrophic, necrotrophic or saprophytic lifestyle (Qutob et al., 2006). NLP sequences are not present in higher eukaryotes, including plants, but were shown to be important virulence factors in Erwinia spp. (Mattinen et al., 2004; Pemberton et al., 2005), incapacitation of which resulted in severely reduced bacterial infection rates. Thus, NLPs appear to fulfill the criteria of a ‘‘classical’’ PAMP. However, unlike PAMPs, NLPs are not ‘‘on display’’ on the microbial surface, but are produced strongly at later


13

stages of infection. In the hemibiotroph oomycete, Phytophthora sojae, NLP production was strongest during the transition from the biotrophic to the necrotrophic stage of infection (Qutob et al., 2002). It is notable that a Phytophthora-derived NLP restored virulence of NLP-deficient Erwinia carotovora mutants, suggesting that NLPs from both sources share the same molecular mode of action (our unpublished data). Other characteristics of NLPs further distinguish them from ‘‘classical’’ PAMPs. In most PAMPS characterized so far, small ‘‘antigenic’’ epitopes within the intact molecule were identified as sufficient for the immunomodulatory activities of these compounds (Table I). However, no such specific motives could be identified within various NLPs (Fellbrich et al., 2002; Gijzen and Nu¨rnberger, 2006). Moreover, NLPs cause cytolytic cell death in dicotyledonous plants that is genetically different from the programmed cell death that is characteristic of the HR (Qutob et al., 2006), whereas many PAMPs either do not induce HR at all or trigger a HR that involves activation of salicylic acid (SA)-dependent responses similar to those occurring during R gene-mediated resistance (Jones and Dangl, 2006; Kamoun, 2001). NLP-induced plant PR gene expression was also SA-dependent (Fellbrich et al., 2002). Furthermore, NLPs disrupt plasma membrane vesicles prepared from dicot plants, but not those from monocot plants, indicating that an intact cell is not required for NLP-induced cell death (our unpublished data). Altogether, these findings suggest that NLP-induced cell death is a symptom of impending disease, not a plant defense response. Indeed, the cytolytic activity of NLPs and the broad spectrum of NLP-sensitive plants rather suggest that these proteins are microbial toxins with defense-activating potential. The elucidation of the 3D-structure of a Pythium aphanidermatum NLP (C. Oecking and H. U. Seitz, personal communication) and the computational modeling of additional NLP folds revealed a high degree of structural conservation between prokaryotic and eukaryotic NLPs. NLP structures closely resemble those of known proteinaceous cytolytic toxins, called actinoporins. Actinoporins bind to lipid docking sites in animal host target membranes, insert into membranes, form ion-conducting pores and subsequently mediate target cell lysis (Parker and Feil, 2005). Structure–activity relationship analyses performed on amino acid residues that are highly conserved among all NLPs, suggested a close correlation between the ability of mutant proteins to cause cytolysis, to restore virulence in NLP-deficient Erwinia, and to induce plant immune responses (our unpublished data). This is very important as it indicates that a common fold of a cytolytic toxin mediates both, microbial attack and plant immunity. NLPs are distinguished from other known phytotoxins by their wide distribution across taxa and their broad spectrum of activity against

14


dicotyledonous plants. In particular, the production of orthologous cytolytic toxins in prokaryotic and eukaryotic phytopathogenic microbes is unprecedented and suggests that NLPs constitute an evolutionarily ancient toxin fold that has been retained preferentially in organisms with a hemibiotrophic or necrotrophic lifestyle. These findings also suggest that toxin-induced interference with cell integrity may culminate in plant immune responses. Importantly, microbial toxin-induced innate immunity is also known from animal systems. Cytolytic bacterial (pneumolysin), fungal (nigericin) or marine (maitotoxin) toxins are triggers of the mammalian inflammasome (Mariathasan et al., 2006; Srivastava et al., 2005). Toxin-mediated activation of mammalian immune responses is based upon the recognition of endogenous, host-derived compounds that are released as a result of toxin-induced host cell damage and subsequent formation of DAMPs (Matzinger, 2002, 2007). Consequently, mammalian inflammasome activation is now considered to be activated not only upon perception of microbial patterns, but also by the action of toxins (Dostert et al., 2008; Mariathasan et al., 2006; Martinon et al., 2004; Srivastava et al., 2005). We suggest that plant cells are also capable of sensing toxin-induced cellular changes. NLP-driven membrane disruption may result in the release of host-derived molecules that serve as endogenous DAMPs. Alternatively, NLP-induced disturbance of the cellular ion homeostasis or membrane potential may signal activation of plant defenses. Remarkably, NLP-induced Ca2þ influx as well as Kþ efflux that mimicked synthetic ionophore-induced ion fluxes in plant cells were reported (Fellbrich et al., 2002), which themselves were shown to trigger plant defense-associated responses in a non-receptor-mediated manner (Jabs et al., 1997). Our structure-based analyses suggest that NLPs are cytolytic toxins that trigger plant immunity-associated defenses through interference with plant tissue integrity. Hence, disturbed host integrity as a common signal for the activation of immune defenses adds to the list of conceptual similarities in the organization of innate immunity in the animal and plant lineages. Peptaibols, the products of nonribosomal peptide synthetases, are linear peptide antibiotics produced by various fungal genera, including Trichoderma. These compounds are assumed to contribute to the protection of fungi against bacterial infections (Engelberth et al., 2000; Viterbo et al., 2007). Several hundred different peptaibols have already been identified, numerous of which were shown to possess plant immunity-stimulating potential (Viterbo et al., 2007). The antibiotic functions of peptaibols have been assigned to their membrane insertion and pore-forming abilities (Engelberth et al., 2000; Viterbo et al., 2007). It is not known whether ion-pore formation is the trigger for immune activation in peptaibol-sensitive plants. Thus, these


15

molecules may initiate the release of host-derived DAMPs, may stimulate plant immune responses directly via their ionophore-like activity and disturbance of host cell homeostasis, or, may alternatively be recognized by yet elusive PRRs.

III. RECEPTORS MEDIATING PATTERN RECOGNITION IN PLANT IMMUNITY Vertebrate immune cells sense microbe- or host damage-derived patterns by a family of receptors that resemble the Drosophila Toll protein and are thus referred to as Toll-like receptors (TLRs) (Aderem and Ulevitch, 2000; Akira et al., 2006; Cook et al., 2004; Ferrandon et al., 2007; Girardin et al., 2002; Medzhitov, 2007). TLRs are composed of extracytoplasmic leucine-richrepeat (LRR) domains, a transmembrane domain and a cytoplasmic TIR domain (Drosophila Toll and human interleukin-1 receptor) (Cook et al., 2004; Underhill and Ozinsky, 2002). Human cell types implicated in innate immunity (mucosal epithelia, phagocytes) express a total of 13 different TLRs that are often implicated in the recognition of various structurally different patterns (Akira et al., 2006; Ferrandon et al., 2007; Medzhitov, 2007). For example, whereas TLR4 recognizes bacterial LPS, host-derived Hsp70 and hyaluronan, TLR2 recognizes approximately 15 microbial and host-encoded agonists (Akira et al., 2006). This is probably brought about by stimulus-specific assembly of multicomponent perception complexes in which individual TLRs represent subunits that supposedly mediate transmembrane signaling. For the sake of correctness, it should be mentioned that several other receptor types have also been implicated in inflammasome activation in animal innate immunity (Ishii et al., 2008) and therefore TLRs represent only one single class of PRRs in animals. Proteinaceous binding sites for microbial patterns have been detected in plasma membrane preparations from various plants, but biochemical purification of these proteins proved notoriously difficult (Montesano et al., 2003; Nu¨rnberger et al., 2004). The first successful purification of a PAMP binding site was reported from soybean membranes that mediated recognition of 1,6--linked, 1,3--branched heptaglucosides (HG) from the cell walls of the phytopathogenic oomycete, Ph. sojae (Mitho¨fer et al., 1999; Umemoto et al., 1997). This 75-kDa HG-binding protein (HGP) conferred glucan binding to transgenic tobacco plants, but lacked a transmembrane or membrane attachment domain (Umemoto et al., 1997). This binding protein was shown to harbor an intrinsic endoglucanase activity that was capable of releasing small oligomeric 1,3--D-oligoglucosides from complex glucans

16


(Fliegmann et al., 2004). Thus, during attempted infection plant glucanases may release oligoglucoside fragments from the oomycete cell wall that constitute suitable ligands for a yet unknown transmembrane glucan receptor. The lack of functional domains for transmembrane signaling in HGP and the proposed existence of multimeric glucan recognition systems in different Fabaceae (Mitho¨fer et al., 1999) suggest that plant PAMP perception system architecture may be as multimeric as in animal cells (Akira et al., 2006; Ferrandon et al., 2007; Medzhitov, 2007). The Arabidopsis FLS2 (FLAGELLIN SENSING 2) gene encodes a plasma membrane LRR-receptor kinase (LRR-RK) that recognizes bacterial flagellin via its extracytoplasmic LRR domain (Go´mez-Go´mez and Boller, 2000). FLS2 is the only flagellin receptor in Arabidopsis because the loss of FLS2 resulted in complete flagellin insensitivity (Go´mez-Go´mez and Boller, 2000), the presence of flagellin binding sites correlated with flagellin sensitivity in all ecotypes tested (Bauer et al., 2001), and the expression of Arabidopsis FLS2 in tomato conferred Arabidopsis flagellin signaling specificity to this plant (flagellin recognition specificities in tomato and Arabidopsis are subtly different) (Robatzek et al., 2007). Flagellin perception is widespread among Solanaceae and Brassicaceae (Go´mez-Go´mez and Boller, 2002), but is lacking, for example, in Umbelliferaceae (our unpublished data). Importantly, flagellin-induced immune responses are necessary for the restriction of the growth of the adapted (virulent) Ps. syringae pv. tomato strain DC3000 (Pst), as fls2 mutants were more susceptible to this pathogen (Zipfel et al., 2004). Thus, bacterial pattern recognition through the PRR FLS2 contributes to basal plant immunity against adapted pathogens (restriction of growth) and likely to species immunity against nonadapted pathogens (halt of growth). This is a most noteworthy finding because this is unequivocal evidence that PTI contributes indeed measurably to plant resistance. It should, however, be stated that not in all cases inactivation of individual PRR may result in statistically significant reduction of overall basal immunity (residual immunity in susceptible hosts) to adapted (virulent) pathogens. This is because the overall aggressiveness of virulent strains may simply override subtle effects brought about by PTI, an immunity that has been rendered insufficient by adapted pathogens. However, the use of partially ‘‘disarmed’’ pathogenic strains (lacking individual effectors, such as Pst AvrPto/AvrPtoB; Shan et al., 2008), nonpathogenic strains (avirulent or TTSS-deficient strains), or nonadapted strains (for which a given plant species is not a host) in infection assays on plants lacking individual PRRs may be a suitable way to demonstrate experimentally the role of these receptors in plant basal immunity. Both plants and animals possess flagellin perception systems that mediate the activation of appropriate innate immune responses (Fig. 1). The human

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Flagellin epitopes

Bacteria

Animals

Plants TLR5 FLS2

BAK1 Plasma membrane MyD88 P

P

IRAK

Innate immune responses

Fig. 1. PAMP perception in plants and animals. Different epitopes of bacterial flagellin are perceived by LRR-type PRRs in plants (FLS2 supported by its co-receptor BAK1) and animals (TLR5). While the plant receptor utilizes cytoplasmic kinase domains for transduction of the signal across the plasma membrane, the animal TLR carries a TIR-(TOLL-Interleukin receptor) domain that interacts via adapter proteins (MyD88, Myeloid differentiation factor 88) with a cytoplasmic kinase (IRAK, Interleukin1-receptor associated kinase). The evolutionary conservation of motives, such as LRR and protein kinase domains, in eukaryotic PPRs probably reflects their biochemical suitability to mediate protein–protein interactions, as well as to initiate intracellular signaling cascades. The fact that both flagellin receptors perceive different epitopes of bacterial flagellin together with the differences in domain structures of FLS2 and TLR5 indicates that flagellin perception in both lineages arose as the result of convergent evolution.

flagellin receptor TLR5 (Hayashi et al., 2001) is the conceptual counterpart of plant FLS2. Beside the striking fact that flagellin perception exists in both lineages, a structural comparison between FLS2 and TLR5 revealed conservation of the modular structure among both receptors. Although amino acid sequences of both receptors differ considerably, both proteins carry extracytoplasmic LRR domains that are linked to a cytoplasmic portion (Fig. 1). Whereas the cytoplasmic portion of FLS2 represents a functional serine/ threonine protein kinase itself, the TIR domain of TLR5 forms a complex with the Interleukin-1-receptor-associated kinase, IRAK, and the adaptor

18


protein, MyD88 (Hayashi et al., 2001), and thereby links flagellin perception to cytoplasmic protein kinase activity. Thus, the domain or module structure of flagellin perception appears to be strongly conserved across kingdom borders. However, differences in the cytoplasmic domains of both receptors also suggest that flagellin perception systems arose independently, and are the result of convergent evolution rather than of divergent evolution. This view (Ausubel, 2005) is further substantiated by the absence of LRR receptor-mediated immunity in unicellular eukaryotes (the supposed phylogenetic divergence point of animals and plants), and by the fact that the antigenic epitopes within flagellin required for FLS2 activation (the conserved flg22 motif within the N-terminal region of bacterial flagellin) and TLR5 (two helical structures within the central portion of flagellin) are grossly different (Go´mez-Go´mez and Boller, 2002) (Fig. 1). Plants possess approximately 235 LRR-RKs (Shiu et al., 2004), many of which are expected to serve as PRRs in PAMP perception (Nu¨rnberger and Kemmerling, 2006). This assumption is based on the fact that transcript levels encoding multiple LRR-RK-encoding genes increased upon pathogen infection or PAMP treatment. For example, bacterial infection of Arabidopsis plants resulted in enhanced transcript levels of 49 LRR-RK-encoding genes (Kemmerling et al., 2007), while flagellin treatment led to increased transcript accumulation for 28 LRR-RK-encoding genes (Navarro et al., 2004). Expression of flagellin-responsive LRR-RK genes was also triggered by other PAMPs, including bacterial LPS and fungal chitin (Thilmony et al., 2006; Zhang et al., 2002). The latter was astounding as it suggested that different PAMPs triggered a generic plant response that could potentially facilitate or improve recognition of different microbial species. An N-terminal, acetylated 18-amino acid fragment (elf18) of Escherichia coli elongation factor Tu (EF-Tu) was identified as another PAMP that triggered plant immunity-associated responses in Arabidopsis (Kunze et al., 2004). Based upon identical response patterns observed in Arabidopsis seedlings treated with flg22 or elf18, it was assumed that the EF-Tu receptor might be structurally related to FLS2 (Zipfel et al., 2006). Screening a collection of T-DNA insertion lines impaired in the expression of flg22responsive LRR-RK genes, yielded an elf18-insensitive mutant line (Zipfel et al., 2006). Insensitivity to elf18 was restored by ectopic expression of the EFR (EF RECEPTOR) gene in this mutant. Moreover, expression of the EFR gene in N. benthamiana conferred elf18 sensitivity that was absent from wild-type plants. EFR possesses an extracytoplasmic LRR domain that is linked to a cytoplasmic serine/threonine kinase domain and is, therefore, closely similar to FLS2 (Go´mez-Go´mez and Boller, 2000; Zipfel et al., 2006). As EFR and FLS2 group into the same clade of LRR-RK genes (LRR XII


19

clade) (Shiu and Bleecker, 2001; Shiu et al., 2004), it is assumed that more members of this particular clade encode ‘‘orphan’’ PRRs that sense yet unknown microbial patterns. EFR-mediated sensing of bacteria did not contribute measurably to basal immunity of Arabidopsis against Pst infections, but limited Agrobacterium tumefaciens-mediated transformation of this plant (Zipfel et al., 2006). Mutant efr lines consistently showed 50-fold higher expression levels upon transformation with a pBIN1935S::-GLUCURONIDASE (GUS) reporter gene construct. This finding is also important from a biotechnological point of view, as it indicates that suppression of PRR-mediated microbial pattern recognition might be a valuable strategy to develop efficient transformation protocols for crop plants that are difficult to transform by Agrobacterium-mediated technologies. Regardless of the importance of LRR-RKs as PRRs in plant immunity, the existence of other types of plant PRRs is expected (Bittel and Robatzek, 2007; Nu¨rnberger and Kemmerling, 2006). For example, fungal chitin perception in rice occurs by a plasma membrane LysM (lysine motif) receptor protein (LysM-P) carrying an extracytoplasmic LysM domain that is linked to a very small cytoplasmic domain (Kaku et al., 2006). Biochemical evidence suggests that the LysM domain directly mediates binding of oligomeric chitooligosaccharide fragments (Ito et al., 1997; Kaku et al., 2006). It is currently unknown whether the very short cytoplasmic tail of this protein contributes to the initiation of an intracellular signaling cascade. Alternatively, LysM-P may also form complexes with transmembrane proteins carrying cytoplasmic signaling domains. An Arabidopsis LysM-RK (CERK1) has recently been found to be implicated in chitin perception (Miya et al., 2007). Mutants defective in the expression of the CERK1 gene lacked any chitininducible immune responses, such as production of ROS (oxidative burst) or defense-related gene expression. It is unclear whether the LysM domain of CERK1 binds chitin physically. Therefore, it remains to be seen whether or not chitin perception and signaling are brought about by the same or distinct proteins. In any case, chitin recognition in plants appears to engage at least two types of LysM-domain proteins (LysM-P, LysM-RK). The LysM (lysine motif) domain consists of approximately 40 amino acid residues and has originally been found in a variety of bacterial enzymes involved in cell wall biosynthesis and degradation (Bateman and Bycroft, 2000). The finding that plant LysM domain-containing proteins bind fungal chitin (CERK1, plant chitinases) is interesting from an evolutionary point of view, because the bacterial LysM domain is known as a general peptidoglycan-binding module that is present in a number of peptidoglycan-modifying enzymes, such as Es. coli murein transglycosylase D (Bateman and Bycroft, 2000). Peptidoglycans, also known as murein, are polymers consisting of

20


sugars and amino acids that form a mesh-like layer in the cell walls of Gram-negative and Gram-positive bacteria. The sugar component consists of alternating residues of -(1,4)-linked N-acetylglucosamine and N-acetylmuramic acid residues (a heteroglycan). Attached to the N-acetylmuramic acid is a peptide chain of three to five amino acids that can be cross-linked to the peptide chain of another strand forming a 3D mesh-like layer. Apparently, during evolution plant LysM domain-containing proteins have acquired the ability to bind fungus-derived homoglycans consisting of -(1,4)-linked N-acetylglucosamine (chitin) that are structurally related to the carbohydrate backbones of bacterial peptidoglycan. Moreover, the LysM motif is present in the Lotus japonicus lipochitooligosaccharide (Nod-factor) receptor kinases LjNFR1 and LjNFR2 that mediate the establishment of symbiosis between leguminous plants and rhizobacteria (Radutoiu et al., 2003). This suggests that plants utilize LysM domain host receptors as a general module for chitin-based self/nonself discrimination in both, symbiotic and antagonistic plant–microbe interactions. A more general assumption would be that carbohydrate ligands are preferentially recognized by LysM domain-containing PRRs, whereas proteinaceous ligands may be recognized preferably by LRR domain-containing proteins. As peptidoglycans are triggers of immune responses in Arabidopsis and tobacco (Erbs et al., 2008; Felix and Boller, 2003; Gust et al., 2007), it will be important to test whether plant LysM domain-containing proteins may (in addition to their ability to sense fungal chitin fragments) also recognize and respond to peptidoglycan. At least six peptidoglycan perception systems in humans [TLR2, NOD1 (Nucleotide binding oligomerization domain), NOD2] and Drosophila immune cells [PGRP (Peptidoglycan recognition protein)-SA, PGRP-LC, PGRP-SC1B] that recognize different fragments of bacterial peptidoglycans (Akira et al., 2006; Ferrandon et al., 2007) are known. However, none of these proteins is a LysM-P or LysM-RK, suggesting that LysMmediated carbohydrate recognition in eukaryotic innate immunity has primarily, and probably independently, evolved in plants. A plasma membrane-anchored extracellular LRR-protein (LRR-P) lacking a cytoplasmic signaling domain has been implicated in the recognition of a fungal xylanase and subsequent activation of noncultivar-specific immunity in tomato (Ron and Avni, 2004). As plant genomes harbor multiple LRR-P-encoding sequences (the Arabidopsis genome contains 57 LRRP-encoding sequences) (Wang et al., 2008), it is conceivable that these proteins constitute another class of PRRs that are mechanistically similar to animal LRR-P-type PAMP receptors (Bittel and Robatzek, 2007; Nu¨rnberger and Kemmerling, 2006). Whether ligand perception and intracellular signal transduction through plant LRR-Ps (similar to LysM-P) requires additional components remains, however, to be shown.


21

IV. SIGNAL TRANSDUCTION IN PTI PAMP-mediated activation of PRRs transduces ligand-encoded information across the plasma membrane and initiates a host signaling cascade that culminates in the execution of pathogen-nonspecific immune responses. Previously obtained pharmacological evidence suggested that protein kinase activity is required to trigger very rapid PAMP responses in plants, such as influxes of Hþ and Ca2þ across the plasma membrane (Felix and Boller, 2003; Felix et al., 1991, 1999; Nu¨rnberger et al., 1994). The fls2-17 allele of the flagellin receptor FLS2 carries a point mutation in the protein kinase domain that confers insensitivity to flagellin and loss of autophosphorylation activity of FLS2 in vitro (Go´mez-Go´mez et al., 2001). This mutation also abolished the binding of flg22 to the LRR domain of FLS2, suggesting that the overall stability or conformation of the receptor was altered by this single mutation. A point mutation in a putative phosphorylation site of FLS2 also led to flagellin insensitivity, which further documents the importance of protein kinase activity for the activation of flagellin-inducible plant responses (Robatzek et al., 2006). Moreover, overexpression of KAPP, a kinase-associated protein phosphatase that is supposed to regulate the function of several transmembrane RKs, resulted in flagellin insensitivity and reduced flagellin binding to FLS2 (Go´mez-Go´mez et al., 2001). Thus, the current knowledge suggests that phosphorylation of FLS2 (by its own intrinsic PK activity or by another, yet unknown, PK) is a crucial element of flagellin sensing/signaling. More recently, BAK1 (BRI1-associated receptor kinase 1), an LRR-RK that was previously shown to control plant growth by hormone-dependent heterodimerization with the plant brassinosteroid (BR) hormone receptor, BRI1 (brassinosteroid-insenstive 1; an LRR-RK itself) (Li et al., 2002; Wang et al., 2001), has been implicated in FLS2 and EFR function (Chinchilla et al., 2007). BAK1 mutants were (partially) insensitive to both flg22 and elf18. Flg22-dependent rapid heterodimerization of FLS2 and BAK1 was demonstrated by co-immunoprecipitation experiments (Chinchilla et al., 2007), suggesting that BAK1 function follows the same mode of action in activation of both, FLS2 and BRI1. In addition to its role as a positive regulator of PTI and plant growth, BAK1 appears to fulfill other functions. bak1 mutants were recently shown to have altered disease-resistance phenotypes to biotrophic and necrotrophic pathogens, that are likely to be the consequence of infection-induced deregulated cell death control (Kemmerling et al., 2007). Thus, in addition to its role as a positive regulator of PTI, BAK1 may act as a negative regulator of plant cell death. Both plant immunityassociated functions of BAK1 are independent of the BR activity, because

22


several mutants impaired either in BR sensitivity or biosynthesis were not impaired in flagellin sensitivity or infection-induced runaway cell death (Chinchilla et al., 2007; Kemmerling et al., 2007). Thus, BAK1 has BRindependent, immunity-associated functions in addition to its well-established, BR-dependent role in plant development (Morillo and Tax, 2006). BAK1 represents a second example of a plant LRR-RK with dual functions in plant development and immunity, as the LRR-RK ERECTA was previously implicated in both flower development and plant pathogen resistance (Godiard et al., 2003; Llorente et al., 2005). Dual roles for receptor proteins in development and immunity are also known from animal systems. For example, the Drosophila receptor TOLL controls embryonic patterning in larvae and immunity against fungal infections in adult insects (Lemaitre et al., 1996). Attenuation and termination of PRR function in animal immunity is achieved mainly by the activities of negative regulators. In Arabidopsis, KAPP-mediated inactivation of FLS2 has been proposed to be such a mechanism (Go´mez-Go´mez et al., 2001). More recently, proteasomedependent, ligand-induced endocytosis of FLS2 has been demonstrated and proposed to be an additional way to shut down PRR activity in plant immunity (Robatzek et al., 2006). In addition, internalization of FLS2 into endosome-like compartments may contribute to flg22 signaling. PTI signaling pathways further employ altered cytoplasmic Ca2þ levels, ROS, nitric oxide (NO) and several mitogen-activated protein kinase (MAPK) cascades (Jonak et al., 2002; Nu¨rnberger et al., 2004; Zhang and Klessig, 2001). Many of these components are important for PAMP-induced activation of innate immune responses in animal cells (Barton and Medzhitov, 2003) also, lending further support to the view of conceptual and mechanistic conservation in the molecular architecture of eukaryotic innate immunity across kingdom borders. NO production has been observed in both PAMP-treated plants and during ETI in resistant host plants (Clarke et al., 2000; Delledonne et al., 1998; Durner et al., 1998). Although there is no evidence for a plant ortholog of human NO SYNTHASE (hNOS ), pharmacological hNOS inhibitors blocked both infection and elicitor-stimulated NO production, cell death and defense gene activation in plants (Delledonne et al., 1998; Durner et al., 1998). Zeidler et al. (2004) reported that AtNOS1, a plant-specific NOS previously associated with hormone signaling in plants (Guo et al., 2003), mediated LPS-induced NO production and PR gene expression in Arabidopsis. Importantly, inactivation of the AtNOS1 gene did not only abrogate LPS-induced NO production in these plants, but also dramatically enhanced susceptibility of the mutant to


23

Pst infection. However, the role of AtNOS1 as a bona fide plant NO synthase was recently questioned, as the 3D-fold of the protein suggested similarities to mitochondrial GTPase involved in mitochondrial biogenesis (Zemojtel et al., 2006). Thus, it appears likely that reduced levels of NO in Atnos1 mutants and the observed immunity-associated phenotypes are due rather to mitochondrial dysfunction affecting NO production (mitochondria are a major source of NO) than to a lack of NO synthase activity (Zemojtel et al., 2006). NO-mediated protein nitrosylation has been found in PAMPtreated plant cells (Lindermayr et al., 2005, 2006). Recently, NO-mediated nitrosylation of NPR1 (Nonexpressor of PR genes 1), a key molecule in the establishment of SA-dependent systemic acquired resistance, and of AtSABP3, a SA-binding protein with intrinsic carbonic anhydrase activity and a proven role in basal immunity to bacterial infection, were shown to be necessary for the biological contribution of both proteins to different types of disease resistance (Tada et al., 2008; Wang et al., 2009). This strongly suggests that infection-induced nitrosylation bursts mediate redox changes in the plant cell that affect biological activities of proteins through posttranslational nitrosylation events (Wang et al., 2009). MAPKs are central points of cross-talk in plant signaling cascades, including those that protect against microbial invasion (Go´mez-Go´mez and Boller, 2002; Jonak et al., 2002; Nakagami et al., 2005; Zhang and Klessig, 2001). Various fungus- or bacteria-derived PAMPs and phytopathogenic microbes activate MAPK enzyme activities in a transient fashion. For example, Arabidopsis AtMPK3 and AtMPK6 are responsive to PAMP treatment or infection (Jonak et al., 2002; Zhang and Klessig, 2001). Silencing of MPK6 resulted in remarkably compromised disease resistance in Arabidopsis (Menke et al., 2004). In PAMP-treated parsley cells, PcMPK3 and PcMPK6 translocate into the nucleus (Lee et al., 2004; Ligterink et al., 1997) and contribute to WRKY transcription factor-dependent PR gene expression (Eulgem et al., 1999; Kroj et al., 2003). Using an Arabidopsis protoplast transient expression system, Asai et al. (2002) identified a flg22induced MAP kinase cascade consisting of the MAPK kinase kinase MEKK1, the MAPK kinases MKK 4/5 and MAPK 3/6, and WRKY transcription factors acting downstream of FLS2, and proposed a role of this cascade in bacterial and fungal resistance (Asai et al., 2002). Importantly, PAMP-triggered MAPK pathways regulate PTI-associated responses both in a positive and a negative manner. The flg22 peptide was recently shown to activate the MEKK1/MKK1/MPK4 pathway that suppresses various pathogen defense responses, including callose deposition and PR gene expression (Ichimura et al., 2006; Suarez-Rodriguez et al., 2007). Thus, fine-tuning of

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PTI-associated responses is brought about by two MAPK pathways that exert positive and negative regulatory activities. Surprisingly, flg22-induced activation of the MPK4 pathway did not require MEKK1 kinase activity, suggesting that the protein may exert biological functions as a scaffold or structural protein (Suarez-Rodriguez et al., 2007). The only known substrates that are directly phosphorylated by AtMPK6 are two isoforms of 1-aminocyclopropane-1-carboxylic acid synthase (ACS), the rate-limiting enzyme of ethylene (ET) biosynthesis (Liu and Zhang, 2004). Phosphorylation of ACS2 and ACS6 by MPK6 led to the accumulation of the ACS proteins, elevated levels of cellular ACS activity, increased ET production and ET-induced plant phenotypes. Causal links between MAPK activation, expression of PR genes, and the initiation of programmed cell death were suggested by a set of loss- and gain-of-function experiments performed in tobacco or Arabidopsis, respectively (Jonak et al., 2002; Nu¨rnberger et al., 2004; Ren et al., 2002; Zhang and Klessig, 2001). Surprisingly little is known about the role of plant hormones in the activation of PTI-associated immune responses. Classical hormones implicated in the establishment of ETI, such as SA, jasmonic acid (JA) or ET, appear not to be required for flg22-induced defense responses and basal immunity to Pst (Zipfel et al., 2004).Thus, although flg22 triggers ET biosynthesis in Arabidopsis (Felix et al., 1999), this may not be necessary for the activation of PTI in PAMP-treated cells, but might contribute to the activation of defenses in plant cells remote from the site of PAMP application. Flagellin treatment further induces a plant microRNA (miRNA) that negatively regulates transcript levels of the auxin receptors TIR1, AFB (Auxin binding factor) 2, and AFB3 (Navarro et al., 2006). Moreover, repression of auxin signaling restricted growth of Pst, suggesting that the growth-promoting hormone, auxin, is a disease susceptibility factor and that miRNAmediated suppression of auxin signaling is an element of PTI. Suppression of auxin-mediated growth as a consequence of activated immunity might reflect the functionality of an innate, built-in trade-off between plant immunity and growth programs. Expression of genes encoding several components implicated in basal immunity of host plants or species resistance is enhanced by various PAMPs (Bittel and Robatzek, 2007). Gene products, such as PENETRATION 1 (PEN1), PEN2, PEN3, ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), and PHYTOALEXIN DEFICIENT 4 (PAD4), are thus likely to contribute to PAMP-triggered immune responses in infected plants. For more details on these proteins the reader is referred to the relevant chapters of this issue.


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V. SUPPRESSION OF PTI—A MAJOR VIRULENCE STRATEGY OF PHYTOPATHOGENIC BACTERIA Infection of host plants by virulent pathogens requires tolerance to or active evasion of the host immune system. A major strategy of virulent pathogens to facilitate infections in susceptible host plants is effector-mediated suppression of PTI. This phenomenon was first documented by showing that TTSSdeficient mutants of Pst (incapable of effector delivery into host cells) triggered a number of plant defenses (callose deposition, PR gene expression) that were suppressed by virulent Pst (Hauck et al., 2003). Activation of these responses by TTSS-deficient bacteria was proposed to be mediated through PAMP perception/PRR activities, and, later on, such responses were indeed reported to be triggered by various PAMPs (He et al., 2007). Among the first bacterial effectors shown to suppress PTI-associated responses were AvrPto and AvrPtoB (He et al., 2006). In a screen that aimed at identifying Pst effectors that suppress flg22 responses in Arabidopsis protoplasts, both effectors, but not several others, were identified to block PAMP-induced signal transduction cascades upstream of MAPK activities (He et al., 2006). In these experiments, it remained unclear whether these effectors inhibited a regulator of PTI pathways or interfered directly with components of PAMPtriggered signaling pathways. The 3D-structure elucidation-based identification of AvrPto as a Ser/Thr protein kinase inhibitor (Xing et al., 2007) favored the idea that soluble protein kinases and/or cytoplasmic protein kinase domains of transmembrane LRR-RKs could serve as direct targets for bacterial effector activities in planta. Indeed, AvrPto has very recently been shown to bind to BAK1 in vivo, thereby inhibiting flg22-induced heterodimerization of FLS2 and BAK1 (Shan et al., 2008) (Fig. 2A). As BAK1 is implicated in the function of additional PRRs, such as EFR (Chinchilla et al., 2007), interference with BAK1 appears to be a powerful strategy pursued by virulent pathogens to suppress PTI and to infect susceptible host plants (Shan et al., 2008). Very recently, the ubiquitin ligase activity of AvrPtoB was shown to mediate ubiquitinylation and subsequent destabilization (presumably by degradation via the host 26S proteasome) of FLS2 (Go¨hre et al., 2008) (Fig. 2B). Thus, multiple microbial effectors target PRR function, thereby underlining the major importance of plant basal defenses for plant immunity, as well as suppression of PTI as a major step towards the establishment of infection (Fig. 2). Different modes of interference with PTI were proposed for Ps. syringae effectors AvrRpt2 and AvrRpm1 that inhibit PAMP-induced signaling and compromise host basal immunity through manipulation of a regulator of PTI, RPM1-INTERACTING PROTEIN 4 (RIN4) (Kim et al., 2005).


26

A

FLS2

flg22

BAK1

AvrPto B P

P

effectors Suppression of PTI P

Ub

P

AvrPtoB

Fig. 2. Suppression of PAMP-triggered immunity at the receptor level. PAMPtriggered defense responses are suppressed by Pseudomonas syringae pv. tomato effectors AvrPto and AvrPtoB that are injected into the plant cell via a TTSS. (A) The bacterial effector protein AvrPto is a kinase inhibitor that suppresses downstream responses by the inhibition of PRR complex formation. Interactions of AvrPto with the protein kinase domains of both FLS2 and its co-receptor BAK1 were shown. However, in planta AvrPto appears to target BAK1 preferentially. This results in suppression of all PRR activities that are regulated by the co-receptor BAK1. (B) Interference with FLS2 activity is also mediated by the bacterial effector AvrPtoB that harbors intrinsic E3-ligase activity. Ubiquitinylation of FLS2 promotes degradation of FLS2 by the 26S proteasome, and thereby interferes with PTI.

Arabidopsis RIN4 is a target for proteolytic degradation by the cysteine protease, AvrRpt2 (Coaker et al., 2005; Mackey et al., 2003), while AvrRpm1 mediates phosphorylation-dependent inactivation of this protein (Mackey et al., 2002). Strikingly, resistant Arabidopsis ecotypes that harbor the resistance (R) proteins RPS2 or RPM1, develop AvrRpt2/AvrRpm1mediated ETI, that is based upon R-protein-mediated sensing (‘‘guarding’’) of the manipulation of PTI by these effectors (Mackey et al., 2002, 2003). This is an impressive case for a mechanistic link between both PTI and ETI, the two major evolutionary forms of plant immunity. Recently, it was demonstrated that abscisic acid-dependent stomatal closure is observed upon bacterial infection and is due to PPR-mediated perception of bacterial patterns (Melotto et al., 2006). Infection of Arabidopsis with TTSS-deficient Pst DC3000 resulted in FLS2-dependent stomatal


27

closure. Initial closure of stomata was also observed upon infection by Pst, but was reversed at later times of infection, which suggested that virulent pathogens have evolved effectors to reopen closed stomata. Indeed, stomatal reopening was accounted for by the TTSS-independent activity of the bacterial toxin, coronatine (Melotto et al., 2006). Taken together, the mere fact that several types of effectors (secreted toxins and TTSS effectors delivered into the plant cell) have evolved to act in concert to suppress PTI documents the importance of basal defenses for plant immunity and indicates that evasion of host immunity is an inevitable requirement for microbial proliferation on host plants. Further details on microbial effector-mediated suppression as a general virulence strategy of microbial phytopathogens is provided in the following chapter of this volume (Me´traux et al., 2009).

VI. CONCLUDING REMARKS There is now ample knowledge that plants and animals deploy innate immune (PTI) systems that share a similar logic (Ausubel, 2005; Chisholm et al., 2006; Jones and Dangl, 2006; Nu¨rnberger et al., 2004; Zipfel and Felix, 2005). Self/ nonself discrimination is based on the detection of invariant, microbe-specific patterns through host PRRs. As a result, nonspecific antimicrobial defenses are triggered that are supposed to arrest and terminate microbial ingress. Moreover, both plants and animals appear to possess sensor systems that facilitate recognition of host-derived determinants of host damage (nonintact self). Common principles in the organization of innate immunity in different kingdoms are also documented by similar microbial patterns that are recognized, by similar PRR types and by similar signaling cascades. However, significant differences in the molecular organization of immunity in plants and animals remain. Plant cells respond to microbial infection in a cell-autonomous manner, whereas in animals specialized cell types protect host tissues against microbial invasion (Jones and Dangl, 2006; Medzhitov, 2007). Moreover, adaptive immunity, which evolved in jawed vertebrates most likely as a result of insufficient innate immunity (Medzhitov, 2007), is missing in plants (Ausubel, 2005). In plants, insufficiency of PTI brought about by suppressive activities of microbial effectors might rather have driven the evolution of ETI (Alfano and Collmer, 2004; Chisholm et al., 2006). Although there is currently no experimental evidence that supports the latter assumption, there is evidence that PTI is an important element of basal immunity against adapted pathogens and of species immunity against nonadapted pathogens (Bittel and Robatzek, 2007; Nu¨rnberger and Lipka,

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2005). Moreover, resistance protein-mediated ETI has recently been shown to operate through derepression and potentiation of PAMP-inducible gene expression, thus demonstrating a functional interdependence between the two types of plant immunity (Shen et al., 2007).

ACKNOWLEDGMENTS We thank Jen Sheen and Georg Felix for critical discussions and comments. Research in the lab of T.N. and B.K. is supported by the Deutsche Forschungsgemeinschaft (AFGN, SFB 446, SFB 766), the European Community and the German Ministry of Education and Research (BMBF).

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Plant Pathogens as Suppressors of Host Defense

´ TRAUX,*,1 ROBERT WILSON JACKSON,{ JEAN-PIERRE ME ESTHER SCHNETTLER{ AND ROB W. GOLDBACH{,w

*Department of Biology, University of Fribourg, CH-1700 Fribourg, Switzerland { School of Biological Sciences, University of Reading, Reading RG6 6AJ, Berks, United Kingdom { Laboratory of Virology, Wageningen University, Binnenhaven 11, 6709 PD Wageningen, The Netherlands

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Suppressors Produced by Fungal and Oomycete Pathogens . . . . . . . . . . . . . . . A. Suppressors Comprise a Wide Group of Metabolites ................... B. Race-Specific Elicitors Turn Out to Suppress Defenses ................. C. Concluding Remarks.......................................................... III. Suppressors Produced by Bacterial Pathogens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Bacterial Evolution to Overcome Plant Resistance ...................... B. Bacterial Suppression of PTI ................................................ C. Type III Protein Secreted Effectors are Used to Suppress PTI......... D. Multifunctional Effectors .................................................... E. RNA and RNA-Binding Protein Targeting ............................... F. Attack of Negative Regulators of PTI ..................................... G. Targeting Hormone Signaling? .............................................. H. Disruption of Vesicle Trafficking ........................................... I. Targeting MAP Kinase Signaling........................................... J. Other Effectors Involved in PTI Suppression for Which Targets are Unknown.........................................................

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Corresponding author:Email: [email protected] Prof. R. W. Goldbach tragically died in India on 7 April 2009

w

Advances in Botanical Research, Vol. 51 Copyright 2009, Elsevier Ltd. All rights reserved.

0065-2296/09 $35.00 DOI: 10.1016/S0065-2296(09)51002-6

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´ TRAUX ET AL. J.-P. ME

K. Other Effectors Involved in PTI Suppression, but Lacking Functional Information..................................................................... L. Other Potential Mechanisms—Type VI Secretion........................ M. Complexity and Evolution of PTI Suppression by Bacterial Pathogens IV. RNA Silencing, the Plant’s Innate Immune System Against Viruses. . . . . . . A. The Discovery of RNA Silencing as the Plant’s Innate Immune System Against Viruses ................................................................ B. Current Views of RNA Silencing as Antiviral Mechanism in Plants .. C. Viral Suppressors of RNA Silencing ....................................... D. Possible Interactions Between Plant Viruses and the miRNA Pathway .............................................................. E. Is Antiviral RNAi Restricted to Plants and Insects? ..................... Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ABSTRACT This chapter reviews our current knowledge about mechanisms of suppression developed by pathogens to avoid host defense responses. In general, plants perceive pathogens by diverse pathogen- or microbe- or even damage-associated molecular patterns (PAMPs, MAMPs, DAMPs) and induce a variety of defense mechanisms referred to as horizontal or basal resistance, nowadays designated PAMP-triggered immunity (PTI). In addition, plants can also recognize specific pathogen-derived effectors and have derived a highly specific defense response termed effector-triggered immunity (ETI), classically called R gene-mediated, specific or vertical resistance. Both PTI and ETI are responses to potential dangers and have common components. Fungal, oomycete, and bacterial pathogens have evolved various effector-based mechanisms of suppression that interfere with such components. Plants strongly depend on RNA gene silencing to interfere with viral pathogens. Plant viruses counteract this response by encoding suppressor proteins of RNA silencing.

I. INTRODUCTION The notion that chemical interactions take place between plants and pathogens goes back to the early days of plant pathology when De Bary studied the disease development caused by Sclerotinia sclerotiorum. The first concept that emerged from such studies is that plant pathogens use various factors to invade their hosts. These factors were proposed to comprise cell wall-degrading enzymes, the presence of which was first reported in bacterial soft rot of potato by Jones (1909). At about the same period, Hitchinson (reviewed in Dimond, 1955) proposed that wilt-inducing fungi release toxins, thus opening another area in the field of pathogenicity factors. Research on toxins has followed a vigorous course and numerous chemical structures have been identified (Walton, 1996). The introduction of genetic analyses has greatly helped to determine the biological relevance for several toxins (Scheffer, 1991;

PLANT PATHOGENS AS SUPPRESSORS OF HOST DEFENSE

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Yoder, 1980). Besides cell wall-degrading enzymes and toxins, yet another group of substances has made its appearance. The discovery of pathogeninduced antimicrobial compounds in plants, the phytoalexins, by Mu¨ller and Bo¨rger (1940) opened the way to a vigorous research effort in induced resistance. Later, besides pathogens, chemicals from fungal cultures were shown to induce phytoalexins. A first example was provided by monilicolin A, a partly characterized peptide factor first isolated from the mycelia of Monilinia fructicola, which induces phaseollin, a phytoalexin from bean (Cruickshank and Perrin, 1968). In the early 1970s, Noel Keen proposed the term elicitor for metabolites from pathogens that induce phytoalexins (Keen et al., 1972). This concept was later expanded, and elicitors were proposed to induce various resistance mechanisms (De Wit, 1986). A number of reviews relate the progress of what became a very busy and fruitful area of molecular plant pathology (Boller, 1995; Ebel and Mitho¨fer, 1998; Hahn, 1996; Yoshikawa et al., 1993). In fact, elicitors were shown to include common microbial molecules, such as bacterial lipopolysaccharide (LPS), flagellin, elongation factor Tu, cold-shock protein and peptidoglycan (Aslam et al., 2009; Nu¨rnberger and Kemmerling, 2009), as well as products associated with damage of plant components by pathogen virulence factors, for example, oligogalacturonides and cellulose byproducts. These general elicitors are nowadays named using acronyms such as PAMPs (pathogen-associated molecular patterns), MAMPs (microbe-associated molecular patterns) or DAMPs (damage-associated molecular patterns; see Boller and Felix, 2009). The elicitor concept has received strong support with the characterization of the structure and biological relevance of several receptors for elicitors. These receptors are part of a pathogen surveillance system of proteins coupled to a set of antipathogen defense mechanisms via mitogen-activated protein kinase (MAPK) and various hormone signaling pathways. This defense system has the hallmarks of the innate immunity known in animals and serves as the basic frontline defense against potential pathogens, regardless of whether the interacting organism is a pathogen or not. Traditionally, this has been called basal resistance, but it has recently been designated as PAMP-triggered immunity (PTI) (Chisholm et al., 2006; Jones and Dangl, 2006). The second pathway is a more advanced and specific form of resistance that detects pathogen effectors leading to sacrificial programmed cell death, known as the hypersensitive response (HR), at sites of infection—and is known as effector-triggered immunity (ETI). This relates to what was formerly called R gene-based or vertical resistance. Typical defense responses in innate immunity include the closure of stomata (Melotto et al., 2006), the strengthening of the plant cell wall (thickening of the wall occurs by formation of papilla, an apposition composed of phenolics, lignin, and callose among others) (Keshavarzi et al., 2004), and the release of antimicrobial products, for example,

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reactive oxygen species (ROS), phytoalexins, glucosinolates (Clay et al., 2009), and enzymes such as glucanases and chitinases belonging to a wide variety of so-called pathogenesis-related (PR) proteins (Sels et al., 2008; Van Loon et al., 2006). While MAMPs provide a convenient explanation for the induction of resistance in response to avirulent pathogens, the scenario of a compatible interaction remained unanswered. This is especially puzzling as virulent pathogens produce PAMPS similar to the MAMPs of avirulent microbes. How do virulent pathogens avoid the detrimental effects of being recognized by their PAMPs? An older concept already hinted at by Gaümann (1946), proposed that virulent pathogens, besides their ability to tolerate or detoxify phytoalexins, also produce suppressors of phytoalexin accumulation (see also Heath, 1981). Suppressors interfere with the induced defenses of the infected plant and promote virulence of the pathogen. Suppressor activities from virulent pathogens had been described earlier on by Japanese colleagues (Doke, 1975; Oku et al., 1977). This initial spurt of activity was soon to be followed up by other laboratories worldwide as reviewed by Shiraishi et al. (1994). This functional definition of suppressors has been the conceptual basis for further research and has gained considerable support in recent years. Nowadays, the notion of suppressor is often used in alternation with effectors of virulence (Da Cunha et al., 2007; Ma and Guttman, 2008). This chapter will review the current state of research on suppressors of plant defenses in interactions of plants with fungi, bacteria, or viruses.

II. SUPPRESSORS PRODUCED BY FUNGAL AND OOMYCETE PATHOGENS A. SUPPRESSORS COMPRISE A WIDE GROUP OF METABOLITES

Preliminary infection of plants with a virulent pathogen can lead to an increased susceptibility to subsequent inoculation with avirulent pathogens. This was observed in the potato/Phytophthora infestans, barley/Blumeria graminis and oat/Bl. graminis, and Puccinia coronata interactions (as reviewed in Staples and Mayer, 2003). These observations provided experimental support for the hypothesis that virulent pathogens release suppressors of plant defenses, and led to an active search for such suppressors (initially coined as impedins (Ouchi and Oku, 1986), then supprescins (Shiraishi et al., 1992)) in fungal culture filtrates (Shiraishi et al., 1997). Supprescins A and B secreted by the phytopathogenic fungus Mycosphaerella pinodes are good examples of such compounds. Application of these small glycoproteins to the


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epicotyls of peas delayed the transcription and activity of elicitor-induced phenylalanine ammonia-lyase (PAL), as well as the accumulation of pisatin, the pea phytoalexin (Yamada et al., 1989). Similarly, the expression of the BvPAL and cinnamic acid 4-hydroxylase (BvC4H) genes in sugar beet was repressed at both the transcript and the enzyme activity levels upon infection by virulent Cercospora beticola. This fungal repression was shown to reside at the core promoter of PAL (Schmidt et al., 2004). The nature of the fungal suppressor responsible for this repression remains to be determined but a possible control by the phytohormone abscisic acid (ABA) was suggested for the infection of sugar beets by Ce. beticola (Schmidt et al., 2008). In fact, several examples illustrate how ABA might be a virulence factor that suppresses defense responses (Asselbergh et al., 2008; De Torres-Zabala et al., 2007). Fungi of the genera Botrytis, Ceratocystis, Fusarium, and Rhizoctonia can produce ABA, making it likely that this hormone is involved in pathogen virulence (Do¨rffling et al., 1984; Siewers et al., 2006, reviewed in Tudzynski and Sharon, 2002). The precise chemical structure of suppressors was mostly unknown, making it difficult to study their interaction with a potential binding site, and their mode of action as well as their biological relevance has remained elusive. A first example of a chemically characterized suppressor came with the study of a yeast elicitor. Cleavage of yeast invertase by -chymotrypsin leads to highly active glycopeptide elicitors that stimulate the biosynthesis of ethylene (ET) and the activity of PAL in tomato cells. Release of the mannosecontaining side chain of one purified elicitor-active glycopeptide by an endo -N-acetylglucosaminidase yielded a mannose-rich oligosaccharide that acts as a specific suppressor of the glycopeptide-induced ET biosynthesis and PAL activity. Based on the structural requirements of the glycopeptides mannose side-chains for elicitor activity and mannose oligomers for suppression, it was concluded that elicitor and suppressor compete for the same binding site (Basse et al., 1993). This opens up the possibility of determining the relevance of this suppressor in the larger context of a plant–fungus interaction. A study on the oomycete pathogen Phytophthora sojae has led to the cloning of a glucanase-inhibiting protein (GIP) that was shown to form a complex in vitro and in vivo with a soybean endoglucanase (PR-2 protein) and to inhibit its activity. This interaction also inhibits the release of a glucan elicitor from Ph. sojae cell walls in vitro. Thus, GIP1 is an example of a suppressor of a plant defense response produced by a virulent pathogen of soybean (Rose et al., 2002). Suppression of disease resistance in the form of a two-step process was reported for the soft-rot fungus Septoria lycopersici, a pathogen of tomato. Tomato accumulates an antifungal glycoalkaloid, the saponin -tomatine that can be hydrolyzed to -tomatine by a detoxifying enzyme, tomatinase,

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secreted by Se. lycopersici. Mutants of Se. lycopersici deficient in tomatinase induced plant defense genes and plant cell death in contrast to wild types. They were not otherwise impaired in their ability to cause disease on tomato leaves. This led to a model, in which -tomatine produced after hydrolysis of -tomatine by tomatinase in wild-type strains, acts as a suppressor of plant defenses (Martin-Hernandez et al., 2000). Support for this hypothesis was obtained by studying the interaction between Se. lycopersici and Nicotiana benthamiana. When defense reactions of the host were suppressed by virusinduced gene silencing (VIGS), the tomatinase-deficient mutant caused disease compared to plants with intact defenses. Infiltration of leaves with purified tomatinase or -tomatine, but not -tomatine, led to susceptibility to tomatinase-deficient mutants of Se. lycopersici. Infiltration of tomatinase or -tomatine reduced the HR induced by transient expression of the bacterial effector gene AvrPto (AVIRULENCE GENE OF PS. SYRINGAE PV. TOMATO) in N. benthamiana expressing the matching resistance (R) gene Pto of tomato. Together these results show a dual effect of tomatinase in saponin detoxification and in suppression of the resistance response (Bouarab et al., 2002). Since it is not known if N. benthamiana accumulates -tomatine, the products of tomatinase activity in N. benthamiana remain to be determined. It will be interesting to learn about the existence of other cases of suppressors formed by the action of fungal detoxifying enzymes. Botrytis cinerea and Sc. sclerotiorum produce oxalic acid during infection (Germeier et al., 1994; Godoy et al., 1990). Oxalic acid is a pathogenicity factor necessary for the infection of various plant species by Sc. sclerotiorum (Godoy et al., 1990) and to a certain extent for Bo. cinerea (Schoonbeek et al., 2007). Oxalate deserves special attention since this molecule was shown to suppress the elicitor-induced oxidative burst in soybean and tobacco cultured cells (Cessna et al., 2000). An oxidative burst was shown to be effective in defense against Bo. cinerea in ABA-deficient tomato lines (Asselbergh et al., 2007). It appears therefore reasonable to ask if oxalate could act in planta as a non-proteinaceous suppressor of the oxidative burst. B. RACE-SPECIFIC ELICITORS TURN OUT TO SUPPRESS DEFENSES

Our knowledge about the structure and biological relevance of fungal suppressors has leaped forward as a result of studies on elicitors and their role in the molecular basis of host specificity. Gene-for-gene interactions as studied in tomato infected with the leaf mold fungus Cladosporium fulvum turned out to be particularly rewarding since they allowed the chemical characterization of so-called race-specific elicitors. Cl. fulvum avoids breaching the cell wall,


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enters through stomata and dwells exclusively in the intercellular spaces of the plant tissue. In this interaction, elicitors released from the fungus are located in the apoplast. Very ingeniously, Pierre De Wit realized the unique opportunity offered by the lifestyle of this fungus, and centrifuged vacuuminfiltrated leaf tissue to extract race-specific elicitors from Cl. fulvum-inoculated tomato leaves. A number of small-sized proteins were isolated that induced a HR race-specifically when injected into leaves of tomato plants containing the cognate R genes (De Wit and Spikman, 1982; De Wit et al., 1985). The elicitors were referred to as products of fungal Avr genes, in agreement with pathogen avirulence (Lauge´ and De Wit, 1998). For quite some time, researchers pondered about the real function of the evolutionarily conserved avirulence gene products. Their sole function is very unlikely to make its bearer recognized by plants, and in the late 1990s a concept emerged whereby pathogen avirulence gene products are in fact effectors that target plant proteins to promote disease. Products of R genes, if present, guard these targets and when interacting with the cognate Avr gene product, initiate a defense reaction often in the form of a HR (Dangl and Jones, 2001; Dixon et al., 2000; Mackey et al., 2003). Detailed studies were mainly carried out on specific interactions between plants and bacterial pathogens notoriously known for their large number of secreted effectors (see Section III). Among the main reasons for the strong breakthroughs in plant–bacteria interactions, is that bacteria were far more amenable to molecular studies with the advent of a plethora of molecular techniques. Eventually, tools such as whole genome sequences and transformation protocols also became available for fungal and oomycete pathogens. The host target systems perturbed by various bacterial effectors include protein ubiquitination, MAPK signaling, vesicle trafficking, hormone signaling and transcriptional regulation (Da Cunha et al., 2007). Several effectors that target plant defenses have now also been identified in pathogenic fungi and oomycetes. A well-studied case is the Cl. fulvum AVR2 protein. AVR2 is a cysteine proteinase inhibitor that binds to, and inhibits RCR3, an extracellular papainlike Cys protease (PLCP) that is induced and secreted by tomato as a defense response against pathogens (Kru¨ger et al., 2002). Such secreted proteases can be involved in various ways in plant defenses: for example, by acting directly on the invading pathogen, or by being part of signaling cascades for the induction of the HR (Van der Hoorn and Jones, 2004). AVR2 is a pathogen effector with a suppressor activity: in the absence of the tomato resistance gene Cf2, AVR2 suppresses the action of a PLCP; but in the presence of Cf2 the AVR2 /RCR3 complex triggers ETI-dependent HR (Rooney et al., 2005). AVR2 expression in tomato causes susceptibility to races of Cl. fulvum defective in AVR2 and also towards Bo. cinerea and Verticillium dahliae. In addition, heterologous

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expression of AVR2 in Arabidopsis thaliana leads to enhanced susceptibility against Bo. cinerea and Ve. dahliae (Van Esse et al., 2008). It turns out that RCR3 is not the major PLCP targeted by AVR2. The tomato apoplast was shown to contain a diversity of PLCPs with up to seven proteins clustering into four different subfamilies. Sequencing of PLCP alleles showed that only two relatives, RCR3 and a PLCP produced by tomato leaves infected with Ph. infestans named Phytophthora-inhibited protease (PIP1), undergo selection leading to diversified residues around the binding site of the substrate at the protein surface. This variance might help the plant to prevent inhibition of its defenses by pathogen-derived proteinase inhibitors. The higher number of variants observed at the surface of AVR2 tends to show that its gene is under stronger selection. Inhibition by AVR2 is mainly targeted at RCR3 and PIP1. As PIP1 is more abundant in the apoplast than RCR3, PIP1 was proposed to be the main target of AVR2 and to contribute to the fitness of the pathogen in the absence of Cf2. The major contribution of the less abundant RCR3 (referred to as a decoy) is to bind AVR2 and lure it into a recognition event with subsequent activation of ETIdependent HR in Cf2-bearing tomato (Shabab et al., 2008). This example is one of the few cases illustrating the so-called decoy model, where the binding of an effector with its target is not the main contributor to pathogen fitness in the absence of a corresponding R gene (Van der Hoorn and Kamoun, 2008). Effectors with protease inhibitor activity are also secreted by the oomycete Ph. infestans, a pathogen of potato and tomato. A family of Kazal-like Ser protease inhibitors (extracellular proteinase inhibitors, EPIs) of at least 35 members was found among five different Phytophthora species (Tian et al., 2004). Two of these, EPI1 and EPI10, inhibit the tomato serine protease P69B (PR-7) (Tian et al., 2004, 2005). Another family includes four EPIs with cystatin-like domains (EPICs), EPIC1-4. Of these, EPIC2B was shown to interact with and inhibit PIP1 (Tian et al., 2007). A recent study shows that EPIC1 and EPIC2B of Ph. infestans can also target the RCR3 protease from tomato, like AVR2 from Cl. fulvum. However, in contrast to AVR2, the interaction of EPIC1 and EPIC2B with RCR3 does not induce a HR in tomato harboring the resistance gene Cf2. These results demonstrate that effectors from phylogenetically unrelated pathogens target the same host defense protein (Song et al., 2009). The importance of EPICs in the virulence of Ph. infestans remains yet to be documented. Another apoplastic effector of Cl. fulvum is AVR4, which is characterized by a chitin-binding motif. AVR4 was shown to bind specifically to fungal chitin but not to plant cell-wall preparations in vitro. AVR4 can protect fungal walls against chitin hydrolysis by plant chitinases (PR-3 type proteins) (Van den Burg et al., 2006). When Avr4 was expressed in Arabidopsis,


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increased virulence of several fungal pathogens containing chitin in their cell wall can be observed. Similarly, expression of Avr4 in tomato increased virulence of Fusarium solani pv. lycopersici. To complete this study, Avr4 was silenced in Cl. fulvum leading to decreased virulence on tomato. These results demonstrate the requirement of AVR4 as an effector for pathogenicity. AVR4 antagonizes the defensive function of plant chitinases and illustrates another form of suppression of the action of the plant against an invading fungus (Van Esse et al., 2007). The smut fungus Ustilago maydis was shown to produce PEP1, a protein secreted into the apoplast that is required for the establishment of the fungal haustorium and for further biotrophic development of the fungus within its host, maize. Deletion of the PEP1 gene in U. maydis prevents fungal penetration and unleashes a combination of defense responses by the plant. A PEP1 orthologue with similar function was also observed in Ustilago hordei, the smut pathogen of barley. PEP1 from barley can substitute for PEP1 in U. maydis, as evidenced by the restoration of full virulence to pep1 mutants. Unlike AVR2 of Cl. fulvum, the central domain essential for the function of PEP1 is highly conserved, making it reasonable to speculate that it is an enzyme inhibitor with little specificity or that it affects the deployment of plant defenses in some other ways (Doehlemann et al., 2009). It will be very interesting to learn more about the action of this newly identified fungal suppressor. Fusarium oxysporum f.sp. lycopersici, a fungus invading the xylem, secretes the small cysteine-rich protein SIX1 during the colonization of tomato (Rep et al., 2005). Tomato harboring the I-3 resistance gene develops an incompatible interaction with Fu. oxysporum containing the SIX1 gene. SIX1 can be equated to Avr3, given its gene-for-gene relation with I-3. Fu. oxysporum also produces the avirulence protein AVR1, which matches the product of the tomato resistance gene I-1. However, Avr1 has the additional virtue of suppressing the protective effect of resistance genes I-2 and I-3. As I-2 is cytoplasmic, and AVR2 is apoplastic, AVR1 has been tentatively proposed to interfere with the uptake of AVR2 or AVR3. It might also possibly interfere with the signaling downstream of I-2 or I-3 (Houterman et al., 2008). In addition to apoplastic effectors, a number of cytoplasmic effectors defined by a conserved RXLR amino acid motif have been identified in oomycetes. These molecules are delivered into the cytoplasm of the host cell where they suppress cell death and contribute to the virulence of the pathogen. A well-studied example is the AVR3aKI protein of Ph. infestans. The interaction between R3a, the product of the cognate R gene of Avr3aKI, and AVR3aKI was studied in N. benthamiana using concurrent agro-infiltration. Interaction of AVR3aKI with R3a triggers cell death, but AVR3aKI also

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suppresses cell death induced by INF1, a major elicitin of Ph. infestans (Bos et al., 2006). Elicitins are small, highly conserved proteins secreted by phytopathogenic oomycetes (Phytophthora and Pythium); they induce necrosis in infected plants and elicit an incompatible HR-like reaction. INF1 is a conserved PAMP in oomycetes (Vleeshouwers et al., 2006). Mutants of Avr3a were identified that activate R3a but do not suppress INF-induced cell death. Distinct amino acids in AVR3a determine recognition by R3a but not suppression of INF-induced cell death (Bos et al., 2009). AVR3a can therefore be considered a suppressor of immunity triggered by PAMPs. The importance of the amino acid residues identified in this study will eventually allow for resolving the three-dimensional structure of AVR3a. C. CONCLUDING REMARKS

The weight of the evidence accumulated in the past years demonstrates the validity of the concept of suppressors of disease resistance. As far as fungi and oomycetes are concerned, molecular insights have been obtained by studies on pathogens like Cl. fulvum or Ph. infestans with partly biotrophic lifestyles. In a majority of cases described so far, the interplay of suppressors with their targets is mainly taking place in the apoplasm, as illustrated in Fig. 1. It will be particularly interesting to follow up these studies in the future as we gain more and more information on the molecular targets of suppressors. Only a limited number of cases have been studied so far and it will be exciting to learn more about suppressors produced by other types of fungal pathogens, for example necrotrophs or basidiomycetes.

III. SUPPRESSORS PRODUCED BY BACTERIAL PATHOGENS A. BACTERIAL EVOLUTION TO OVERCOME PLANT RESISTANCE

Bacteria have evolved to employ a variety of virulence factors including toxins, enzymes, hormones, polysaccharides, and most famously, type three protein secretion effectors (T3SE). Many of them are used for the suppression of plant resistance and the release of nutrients; these physiological conditions are thus conducive to the establishment and replication of bacterial cells. The pathogen we know today, however, would have been a different beast in the distant past. A co-evolutionary arms race has seen the development of increasingly sophisticated strategies by pathogens to infect plants and for plants to prevent infection. By considering the ZigZag model defined by

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Pathogen

Pathogen

Pathogen

Chitinbinding protein

AVR3a, SIX1 Protease Glucanase inhibitor inhibitor

Apoplasm

ABA

Elicitin

Chitin or other PAMPS

Oxalate

?

?

ROS MAPK cascade

HR HR

?

Chitinase

Extracellular protease Glucanase

PR-Proteins PAL,... Activation of gene expression Nucleus

Effector targets Plant cell defense mechanisms Plant cell cytosol

Fig. 1. General overview of the mechanisms of defense suppression by fungal and oomycete pathogens. The suppressors and their targets are represented by gray symbols and connected by dotted lines. The targets include interference with various aspects of PAMP-triggered immunity (PTI) (reactive oxygen species (ROS), expression of defense-related genes, or the action of defense proteins) or effector-triggered immunity (ETI). Plant defense mechanisms, PTI and ETI, are in black (symbols and lines). See text for details.

Jones and Dangl (2006), it can be envisaged that a nonpathogenic bacterium interacts with a plant. Changes in the bacterial genotype, through mutation, recombination and/or gene acquisition that allow the bacterium to manipulate the plant to obtain nutrients then occurs. The detrimental effect on the health of the plant (disease) induced by this emerging pathogen imposes strong selection pressure on the plant host for the selection of varieties that are resistant—the varieties develop sensing systems to detect conserved molecules of the bacterium. Consequently, the resistant plant imposes selection for bacteria that can overcome the resistance system and so on. McCann and Guttman (2008) defined this arms race as analogous to the Red Queen principle in Lewis Caroll’s Through the Looking Glass—both pathogen and host are evolving as fast as possible just to maintain the equilibrium. Over a period of time pathogens have evolved to first overcome PTI and then ETI. As an example of the paradox that bacteria face, the flagellum chokepoint is considered here.

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Plants deploy cell-surface pathogen recognition receptors (PRR) that can detect specific, conserved patterns in proteins expressed by bacteria, that is PAMPs. The function of the bacterial proteins encoding the PAMP is often important for bacterial cellular function or for ecological success (fitness). For example, the flg22 peptide is part of the flagellin protein that is the main unit of the bacterial flagellum filament. The flagellum plays a critical role in bacterial motility, enabling the bacterium to move towards nutrients and away from antagonists; in several cases the flagellum has been implicated as important for bacterial virulence in plants (e.g., Antuńez-Lamas et al., 2009; Jahn et al., 2008; Naito et al., 2008), probably for entry into the intercellular areas of plant tissue. Therefore, many bacteria, both pathogens and nonpathogens, are under strong selective pressure to maintain flagellum function. If the bacterium maintains flagellum function, then it inevitably runs the risk of being detected by plant PRR sensors and exposure to host resistance. One consequence of this plant-induced stress is selection for strains that do not trigger resistance (Arnold et al., 2007; Pitman et al., 2005). At least three outcomes are envisaged: (1) The bacterium downregulates flagellum expression inside the plant; (2) Mutations in the PAMP occur so that the PAMP is no longer recognized by the PRR; (3) The bacterium obtains a mechanism that acts downstream of PRR-PAMP detection, thereby removing PAMP recognition as an issue. Little is known about the expression status of the flagellum system in planta or the role the flagellum might play. However, it is clear that mutations to the PAMP will be limited to a select number of sites so that the function of the PAMP-encoded protein is not lost (Naito et al., 2008). Over a period of time, it is likely that PRRs will evolve to recognize PAMP variants. PAMP mutations that have a significant detrimental effect on bacterial function will not be maintained within the population. This strong selective pressure imposed on the bacteria has inevitably led to the evolution of new gene systems or the acquisition of other genes by horizontal transfer. These potentially include polysaccharides and host-specific toxins, which appear to have limited effects and are only partially effective. Undoubtedly, the single most important evolutionary leap made by bacteria is the acquisition of the type III protein secretion system (TTSS). B. BACTERIAL SUPPRESSION OF PTI

When bacteria enter the vicinity of plant cells, either on the plant’s external surface or within the plant tissue, they express a wide variety of proteins including various PAMPs that betray the bacterial presence in the plant. Plant cells deploy cell-surface PRRs, such as FLS2 (Flagellin sensitive 2), EFR (Elongation factor-Tu receptor) and CERK1 (Chitin elicitor receptor


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kinase 1, a PRR receptor-like kinase (RLK) that detects the fungal elicitor chitin to trigger PTI), to detect distinct PAMPs. Some studies, described below, have now shown that detection of PAMPs leads to association of PRRs with the RLK BAK1 (BRI1 (Brassinosteroid-insensitive 1)-associated receptor kinase 1), subsequent activation of MAPK signaling and WRKY transcription factors and the expression of a wide range of genes, many of which contribute to the rapid defense response—of note here are NHO1 (NONHOST RESISTANCE TO PS. SYRINGAE PV. PHASEOLICOLA 1) and FRK1 (FLG22-INDUCED RECEPTOR-LIKE KINASE 1), which are commonly used markers for PTI. NHO1 encodes a glycerol kinase that is necessary for Arabidopsis resistance to non-host pathogens, but it is not effective against the pathogen Pseudomonas syringae pv. tomato strain DC3000 (Pst); FRK1 is a flg22-induced RLK. The signaling pathways are accompanied by ion channel openings leading to calcium influx from the apoplast to the plant cytosol. It is immediately apparent that a number of plant mechanisms are potential targets for bacterial suppression. These are described in detail below. 1. Calcium signaling suppression by extracellular polysaccharides (EPS) Calcium is a key secondary messenger that binds to calcium-binding proteins such as calmodulin, leading to triggering of signaling mechanisms for activation of PTI and ETI (Ma and Berkowitz, 2007). Thus, calcium signaling naturally poses one potential target for suppression by pathogens. Recent evidence by Aslam et al. (2008) showed that the polyanionic EPS produced by bacterial symbionts and pathogens of plant and animals were able to specifically bind calcium ions. It has long been known that EPS is an important virulence factor, for example in the production of bacterial biofilms and for protection against antimicrobials, but a role in calcium sequestration is relatively new. Aslam et al. (2008) found mutations in Xanthomonas and Pseudomonas xanthan and alginate genes, respectively, which resulted in loss of virulence. Concomitantly, there was an increase of callose deposition, production of ROS and upregulation of the defense-related PR-1 and PDF1.2 (PLANT DEFENSIN 1.2) genes. Critically, there was also an increase in cytosolic calcium levels. These calcium surges were shown to be PAMP-inducible and suppressible by purified polyanionic EPS from various symbiotic and pathogenic bacteria, but not by the neutral EPS, levan. Examination of the ultrastructure of infected sites showed that pathogen cells are embedded within high quantities of EPS, which also appears to be interacting with cell wall fibrils and therefore, is in direct contact with this calcium store. The implication of this discovery is that bacteria have evolved to use EPS for sequestration of apoplastic calcium to interrupt signaling and douse PTI.

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Considering the context of EPS suppression with that of T3SE-mediated suppression of PTI and the more advanced ETI, this may therefore be considered to be one of the pathogens’ most primitive PTI suppression systems. That the expression and function of EPS is maintained may reflect its constant role in PTI suppression or its multifaceted protective and structural roles (e.g., biofilms, UV and desiccation resistance). 2. Coronatine toxin suppression of stomatal closure A recent study by Melotto et al. (2006) showed that in Arabidopsis a variety of plant and animal pathogenic bacteria triggered stomatal closure whereas Pst has evolved a mechanism to suppress the closure. Stomatal closure is clearly an organelle-scale PTI mechanism to stop invasion of the internal plant tissue by bacteria. Further investigation found that the PAMPs flg22 and LPS were triggering stomatal closure and clearly indicated that Pst carried a suppressor mechanism. A detailed analysis revealed that the polyketide toxin coronatine was responsible for the inhibition of closure. Coronatine toxin is composed of coronamic acid and coronafacic acid and is structurally similar to jasmonic acid (JA), a plant hormone that activates plant defenses. Coronatine toxin inhibited ABA-induced stomatal closure, but not in coronatine-insensitive (coi1) mutant plants, indicating that the action of coronatine is downstream of ABA signaling. Thus, coronatine is used to open stomata to allow ingress of the bacteria into the plant tissue. A recent paper has identified that Xanthomonas also produces a diffusible signal factor-dependent molecule to suppress stomatal closure and that the MAPK MPK3 is involved in the closure response (Gudesblat et al., 2009). The gene clusters containing the coronatine biosynthetic genes have only been found in a handful of plant-pathogenic bacteria (Ps. syringae pvs alisaliensis, atropurpurea, glycinea, maculicola, morsprunorum, and tomato; Xanthomonas campestris pv. phormiicola; Pectobacterium atrosepticum) (Bender et al., 1999; Cintas et al., 2002; Mitchell, 1991; Toth and Birch, 2005) including the model pathogen, Pst. Why the toxin cluster is not more widely distributed is unclear—possibly coronatine acts as a PAMP in other plants, its usefulness has been superseded by other virulence factors (e.g., toxins), or there is an element of plant or environment specificity associated with its action. C. TYPE III PROTEIN SECRETED EFFECTORS ARE USED TO SUPPRESS PTI

The TTSS gene system is comprised of 20–30 hrp/hrc (HYPERSENSITIVE REACTION AND PATHOGENICITY/HYPERSENSITIVE RESPONSE CONSERVED) genes clustered in several operons on a pathogenicity island in the chromosome or on plasmids (Jin et al., 2003). The expression of the


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genes is tightly regulated and occurs primarily inside the plant intercellular spaces, that is once a bacterium arrives inside plant tissue and makes contact with the plant cells. The proteins encoded by the TTSS genes are similar to the flagellum proteins, with evolutionary analyses showing that the two systems actually evolved from a common ancestor (Gophna et al., 2003). Like the flagellum, the TTSS forms a pore complex that spans the inner and outer membranes of the bacterium. A pilus is formed from the pore that extends away from the bacterial cell to neighboring plant cells. The function of the TTSS is to deliver type III secreted effector (T3SE) proteins into plant cells. Once inside the plant cells, the effectors target plant mechanisms to suppress plant defenses and implement release of nutrients to the bacterium. Current knowledge indicates that effectors target PTI pathways, but also ETI pathways as a consequence of plants evolving to recognize PTI-suppressing effectors. The following sections will review the various effectors found that suppress PTI; because effectors can often have multiple roles, effectors will be considered individually.

D. MULTIFUNCTIONAL EFFECTORS

1. avrPto avrPto was first identified in Ps. syringae pv. tomato as an avirulence gene detected by varieties of tomato expressing the resistance gene Pto (Ronald et al., 1992); an avirulence gene is an effector that has become recognized by a plant host as a consequence of plant evolution to detect bacterial virulence factors. Although AvrPto is recognized by the resistance protein Pto in tomato, AvrPto suppresses basal defenses in Arabidopsis and N. benthamiana. Several important studies demonstrated this role: firstly, expression of avrPto in transgenic Arabidopsis plants prevented the formation of papillae and deposition of callose (a PTI response) when the plants were challenged with a nonpathogenic (TTSS-minus) Ps. syringae that still expresses PAMPs (Hauck et al., 2003). In N. benthamiana, AvrPto can suppress flagellin-induced cell death and callose deposition (Hann and Rathjen, 2007). Secondly, AvrPto suppresses both expression of Arabidopsis NHO1 and FRK1, genes that are induced by the PAMP, flg22 (He et al., 2006; Li et al., 2005a), and activation of the MAPKs, MPK3 and MPK6. These kinases lie at the bottom of a signaling pathway that follows: FLS2 activation leads to activation of MAPKKK activating MAPKK activating MAPK. Further analysis showed that overexpression of the MAPKKK, MEKK1, could block AvrPto suppression of MPK3 and MPK6—this indicates that AvrPto must act at some stage between the FLS2 activation and MEKK1.

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Two recent studies indicate that AvrPto acts at the level of the receptor kinases (Shan et al., 2008; Xiang et al., 2008). Xiang et al. (2008) showed that AvrPto can bind to FLS2 and EFR PRRs and Shan et al. (2008) showed that AvrPto targets BAK1, a RLK that associates with FLS2 and EFR after PAMP recognition. BAK1 was originally found because of its role in brassinosteroid signaling. Although Xiang et al. (2008) showed that the FLS2–AvrPto interaction still occurred in bak1-1 protoplasts, Shan et al. (2008) provided convincing evidence that AvrPto makes a stronger association with BAK1. Indeed, AvrPto disrupts FLS2-BAK1 association and BAK1 is important for multiple PAMP-triggered FRK1 expression. Taken together, these data show that AvrPto binds to protein kinases, potentially acting as a kinase inhibitor. AvrPto has also recently been found to interfere with microRNA (miRNA) accumulation, which plays a key role in RNA silencing defense responses against Pst (Navarro et al., 2008). Taken together, Ps. syringae uses AvrPto to target the BAK1 RLK and deactivate PAMP signaling at the top tier. One interesting consequence of AvrPto function is increased ET production, as discussed below for avrPtoB. 2. avrPtoB (hopAB2) avrPtoB (hopAB2, hrp outer protein AB2) has a number of overlapping functions with avrPto including the targeting of RLK PRRs and miRNA interference (He et al., 2006). However, one intriguing difference between AvrPto and AvrPtoB defense suppression is that, unlike AvrPto suppression, AvrPtoB suppression is dependent upon RAR1 (Required for Mla resistance 1), SGT1 (Suppressor of G2 allele of Skp1), both chaperone proteins that stabilize NBARC-LRR (Nucleotide binding domain shared by Apaf-1, certain R gene products and CED-4, fused to C-terminal leucine-rich repeats), R-proteins, and EDS1 (Enhanced disease susceptibility 1), indicating AvrPtoB targets different defense pathways (Hann and Rathjen, 2007). The 553-amino acid AvrPtoB was first identified in Pst AvrPto as an interactor with Pto and demonstrated to be the product of an avirulence gene recognized by tomato plants expressing the Pto and Prf kinase genes (Kim et al., 2002); it shares 52% identity to the Ps. syringae pv. phaseolicola effector HopAB1 (VirPphA) (Jackson et al., 1999, 2002). Like AvrPto, AvrPtoB also plays a role in interfering with miRNA levels (Navarro et al., 2008). Several seminal studies by Greg Martin and coworkers have shown that AvrPtoB contains domains that contribute to both virulence and avirulence (e.g., Abramovitch et al., 2003, 2006; Kim et al., 2002; Rosebrock et al., 2007) and that it shares many of the same roles and functions as AvrPto, described above. The C-terminus encodes an E3 ubiquitin ligase that is involved in suppression of ETI (Abramovitch et al., 2006; Janjusevic et al., 2006; Rosebrock et al., 2007)


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by directing host proteins to the proteasome. The N-terminal amino acid residues 1–307 are sufficient for eliciting Pto/Prf-dependent immunity. Upon delivery into the plant cell, AvrPtoB is phosphorylated on a key serine residue in the N-terminus (Xiao et al., 2007a); alanine substitution of this residue abolishes the virulence function of AvrPtoB. Phosphorylation is independent of the Pto and Prf protein kinases, indicating the activity of a novel kinase interaction. The AvrPtoB N-terminus also promotes virulence by two distinct mechanisms: residues 1–307 contribute to virulence in tomato lines lacking Pto/Prf by increasing ET production via upregulation of ET biosynthetic proteins (Cohn and Martin, 2005; Xiao et al., 2007b); residues 308–387 are functionally distinct in being involved in a second virulence function to suppress tomato PTI. AvrPtoB can also suppress PTI in Arabidopsis and N. benthamiana (De Torres et al., 2006; Hann and Rathjen, 2007) including suppression of callose deposition and PTI-induced genes, for example, NHO1 and FRK1. In a manner similar to AvrPto, AvrPtoB targets BAK1 and FLS2 RLKs (Go¨hre et al., 2008; Shan et al., 2008), with the kinase domains being essential for the interaction. Two recent reports have shown that the E3 ubiquitin ligase domain also contributes to PTI by ubiquitinating FLS2 and CERK1, thus directing them for degradation (Gimenez-Ibanez et al., 2009; Go¨hre et al., 2008); interestingly, CERK1 appears to operate independently of BAK1. Two studies have now shown that AvrPtoB manipulates plant hormone biosynthesis pathways to promote virulence. Cohn and Martin (2005) found that AvrPtoB (and also AvrPto) upregulates ET-related genes during the interaction of Pst with its host, tomato. ET production enhanced the severity of necrotic symptoms in infected leaves. Pst also modulates the ABA signaling pathway in Arabidopsis, driving production of ABA and subsequent enhanced growth of the pathogen in the plant tissue (De Torres-Zabala et al., 2007). Since plant hormones, including ET and ABA, are important for plant stress response and disease resistance, the pathogens have evolved a strategy to alter plant hormone levels to their benefit. Clearly, there are a number of interesting mechanisms that need to be elucidated for this remarkable, multifunctional effector. 3. avrRpt2 avrRpt2 was isolated as an avirulence gene recognized by the RPS2 gene in resistant Arabidopsis plants (Dong et al., 1991; Innes et al., 1993; Whalen et al., 1991). Pst expressing avrRpt2 grows to higher cell densities in susceptible Arabidopsis ecotypes lacking a functional RPS2 gene (Chen et al., 2000), indicating that AvrRpt2 enhances virulence probably by suppression of PTI. AvrRpt2 can also suppress hypersensitive resistance caused by avrRpm1/

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RPM1 indicating it can also suppress ETI (Ritter and Dangl, 1996). The AvrRpt2 protein is a cysteine protease that degrades the plant protein RIN4 (RPM1 interacting protein 4) (Axtell and Staskawicz, 2003; Axtell et al., 2003; Kim et al., 2005a,b), which is a negative regulator of PTI (overexpression of RIN4 led to inhibition of flg22-triggered callose deposition). The possibility exists that AvrRpt2 interferes with FLS2 signaling. However, RIN4 is not essential for AvrRpt2 virulence function (Belkhadir et al., 2004). AvrRpt2 also alters Arabidopsis auxin physiology which appears to promote virulence, although the mechanisms are yet to be determined (Chen et al., 2007). AvrRpt2 is also important for virulence of Pst in tomato (Lim and Kunkel, 2005); taken together, these findings indicate AvrRpt2 probably has other host targets. 4. xopD The X. campestris pv. vesicatoria TTSS effector XopD is a 545-amino acid cysteine protease that targets tomato small ubiquitin-related modifier (SUMO) protein precursors (Hotson et al., 2003). The N-terminus of XopD contains a nuclear localization signal that causes it to localize to the plant cell nucleus. A recent study has shown that XopD can suppress PTI by dampening salicylic acid (SA) synthesis, which inhibits X. campestris pv. vesicatoria growth in planta (Kim et al., 2008). XopD was also shown to be able to bind to the promoters and to suppress expression of the defense-related genes PR-1 and PDF1.2 that are upregulated by SA. Considering the multifunctional nature of this effector, it would appear to have the potential to interact with multiple DNA and protein targets. Intriguingly, this study also found that XopD appears to delay the onset of senescence, such that the pathogen is effectively ‘‘farming’’ the plant host to extend the period that nutrients are available. E. RNA AND RNA-BINDING PROTEIN TARGETING

1. hopU1 (hopPtoS2) hopU1, along with hopO1-1 (hopPtoS1) and hopO1-2 (hopPtoS3), encode proteins containing ADP-ribosyltransferase (ADP-RT) active sites (Fu et al., 2007). Compared to the wild type, a Pst hopU1 mutant was more effective at triggering a HR in the non-host N. benthamiana, indicating that HopU1 plays a role in suppressing non-host HR. Transgenic Arabidopsis Col-0 plants expressing HopU1 deposited less callose than wild-type plants when treated with flg22, and also exhibited a delayed HR to Pst expressing AvrRpt2. These observations indicate that HopU1 can interfere with both PTI and ETI pathways of innate immunity. Biochemical analysis demonstrated that HopU1 exhibits ADP-RT activity that ADP-ribosylates plant host proteins at arginine


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residues. In Arabidopsis, HopU1 targets at least two glycine-rich RNA-binding proteins (RBP), including GRP7. GRP7 regulates mRNA levels in plants at the posttranscriptional level, but was previously characterized in circadian rhythm functions. HopU1 specifically ADP-ribosylates GRP7 at one of the two arginine residues, both in vitro and in planta, which is believed to block binding of the protein to RNA. An Arabidopsis grp7-1 mutant line that does not produce GRP7 exhibited enhanced disease susceptibility to wild-type Pst and its hrcC mutant, confirming that GRP7 is important in plant PTI. Taken together, this elegant study demonstrated a novel target of bacterial effectors, namely RBP, for defense suppression. 2. hopT1-1 HopT1-1 has been shown to interfere with miRNA-dependent RNA silencing-based defense (Navarro et al., 2008). Small RNA’s, including miRNAs, guide Argonaute (AGO)-containing RNA-induced silencing complexes (RISCs) to suppress the expression of genes at either transcriptional or posttranscriptional levels, that is AGO-RISC targets the transcripts of genes and slices them. HopT1-1 appears to act in two ways: it suppresses AGO1mediated slicing and also miRNA-mediated translational inhibition. The exact mechanism of HopT1-1 action remains elusive. F. ATTACK OF NEGATIVE REGULATORS OF PTI

1. avrB avrB was the first avirulence gene isolated from a plant pathogen (Staskawicz et al., 1984). It was identified from Ps. syringae pv. glycinea race 6 and recognized by soybean R gene Rpg1-b (Ashfield et al., 1995). In susceptible soybean and Arabidopsis plants, AvrB enhances virulence (Ong and Innes, 2006) indicating that it plays a role in PTI suppression. AvrB, like AvrRpm1, can interact with and phosphorylate RIN4 (Desveaux et al., 2007; Mackey et al., 2002). From an elegant screen to identify Arabidopsis host proteins required for AvrB function, Shang et al. (2006) identified the chaperone RAR1, previously linked to ETI. By using wild-type and rar1 mutant lines encoding dexamethasone-inducible AvrB it was found that AvrB expression reduced callose deposition in wild-type plants treated with flg22, whereas there was no effect in the mutant line. RAR1 was found to be a negative regulator of PTI through the use of rar1 mutant lines expressing AvrB, which showed higher levels of callose deposition than the wild-type line expressing AvrB. Both AvrB and RAR1 proteins were found as a complex in immunoprecipitation experiments, although yeast two-hybrid analysis did not detect

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a direct physical interaction in vitro. Thus, AvrB appears to target RAR1 to enforce the accumulation of negative regulators of PTI. 2. avrRpm1 avrRpm1 was originally found as an avirulence gene in Ps. syringae pv. maculicola recognized by the resistance gene RPM1 in Arabidopsis (Dangl et al., 1992; Debener et al., 1991; Grant et al., 1995). It also has a virulence function (Ritter and Dangl, 1995; Rohmer et al., 2003). Expression in plants of avrRpm1 can suppress PTI and promote the growth of a Pst hrcC mutant (Kim et al., 2005a,b). Like AvrB, AvrRpm1 can interact with and phosphorylate RIN4, the negative regulator of PTI (Mackey et al., 2002). Two studies have shown that AvrRpm1 has a significant effect on the Arabidopsis proteome and can suppress accumulation of proteins associated with PTI (Jones et al., 2006; Kaffarnik et al., 2009). G. TARGETING HORMONE SIGNALING?

1. hopAN (avrE1/wtsE/dspA/dspE) Homologues of the hopAN family are well characterized in Ps. syringae (avrE), Erwinia amylovora (dspA/E) and Pantoea stewartii (wtsE). It was first identified as a Ps. syringae pv. tomato avirulence gene recognized by soybean to trigger a HR, but also as a virulence factor for strain PT23 on tomato (Lorang and Keen, 1995). The gene is found in the conserved effector locus (CEL) of the TTSS pathogenicity island, located close to hopM1 described below, and encodes a very large protein (ca. 1800 amino acids). The hopAN effector was later recognized as an important pathogenicity gene for Erwinia/Pantoea on apple, gypsophila, maize and pear (Bogdanove et al., 1998; DebRoy et al., 2004; Gaudriault et al., 1997; Mor et al., 2001) and acts synergistically with hopM1 in promoting Ps. syringae virulence in Arabidopsis (Badel et al., 2006) and suppressing PTI in tomato and N. benthamiana (DebRoy et al., 2004; Oh and Collmer, 2005). An elegant study by Debroy et al. (2004) showed that an E. amylovora dspA/E and Pst CEL mutant caused limited disease symptoms in apple and Arabidopsis, respectively, but caused increased callose deposition in the cell walls of the host plant indicating the mutants trigger PTI. Moreover, the Pst CEL mutant was able to grow better in Arabidopsis transgenic NahG and mutant eds5 lines compared to wild-type plants; NahG and eds5 plants are unable to accumulate SA, confirming the importance of SA in disease resistance of Arabidopsis against Pst. Further analysis showed that the Pst CEL mutant could not suppress SA-mediated PTI, while the wild type could do


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so—however, the action of the CEL effectors did not affect SA-responsive genes in the plant, implying that they operate by a different mechanism. Complementation of the Pst CEL mutant with avrE partially restored suppression of the SA-mediated PTI defense, observed as reduced callose deposition compared to the CEL mutant itself. More recently, it has been shown that dspA/E and wtsE are important for early growth of E. amylovora and Pa. stewartii in the non-host N. benthamiana (Ham et al., 2008; Oh et al., 2007). Inoculation of Arabidopsis Col-0 with Ps. syringae pv. phaseolicola expressing wtsE was sufficient to downregulate PR-1 gene expression and callose deposition compared to an empty vector control, further implicating the role of wtsE in PTI suppression; the function of this class of effectors is yet to be determined. 2. hopAM1 (avrPpiB) The hopAM1 effector was first identified in Ps. syringae pv. pisi as an avirulence gene recognized by the R3 resistance gene in certain cultivars of pea (Arnold et al., 2001; Cournoyer et al., 1995). During a screen of effectors that could enhance the virulence of the weak Arabidopsis pathogen Ps. syringae pv. maculicola M6CDE, HopAM1 was found to significantly improve bacterial growth (Goel et al., 2008). Furthermore, HopAM1 improved bacterial growth in plants growing in water-stressed conditions compared to an empty vector control and water-sufficient grown plants. Since ABA is well known for its role in plant responses to abiotic stresses, the use of the abi5-1 (aba insensitive 5) regulatory mutant line was used to show that HopAM1 virulence effects are enhanced by ABA. HopAM1 had a wide range of effects on Arabidopsis plants, including inducing chlorosis in newly emergent leaves, enhancing ABA sensitivity, reducing salt stress, stimulating stomatal closure and inhibiting seed germination. Furthermore, transgenic hopAM1 Arabidopsis plants enhanced proliferation of a Pst hrcC-minus mutant and showed suppressed papilla formation and callose deposition compared to wild-type plants. Taken together these findings indicate that HopAM1 can suppress PTI in Arabidopsis. Thus, like AvrPtob, HopAM1 can enhance the defense suppressive effects of ABA and appears to be active at various stages of bacterial infection. H. DISRUPTION OF VESICLE TRAFFICKING

1. hopM1 (hopPtoM) hopM1 is located close to avrE in the CEL of the TTSS pathogenicity island in Pst. Deletion of the CEL from Pst leads to loss of pathogenicity on tomato, which can be restored by complementation with hopM1 and

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its cognate chaperone, shcM (Badel et al., 2003). HopM1 can fully suppress SA-mediated callose deposition triggered by a Pst CEL mutant in Arabidopsis (DebRoy et al., 2004), unlike the partial suppression seen by AvrE (described above); HopM1-mediated PTI suppression was also observed in a vascular staining assay in which leaves detached from the plant 6 h after inoculation were placed with their petiole in Neutral Red solution. Basal resistance leads to less dye accumulation and this PTI-based reduced vascular flow into the leaf veins was suppressed by HopM1 (Oh and Collmer, 2005). In a seminal study by Nomura et al. (2006) it was found that HopM1 targets immunity-associated AtMIN7 (A. thaliana HopM1 interacting protein 7). Transgenic Arabidopsis plants expressing hopM1 allowed the Pst CEL mutant to grow to near wild-type levels—HopM1 localized to the endomembrane compartments. A series of transgenic plants expressing Nand C-terminally truncated versions of HopM1 were used to discover that the N-terminus is important for virulence. The N-terminal protein HopM11–300 was used to isolate 21 plant-interacting AtMIN proteins in a yeast twohybrid screen—none of these were identified from a screen with full-length HopM1 due to HopM1-dependent destabilization; whether this is a direct or indirect effect remains to be tested. AtMIN protein destabilization was observed in both Arabidopsis and N. benthamiana, where ubiquitination of the proteins was enhanced by HopM1. Inoculation of the Pst CEL mutant into T-DNA knockout lines of AtMIN genes showed that only mutation of AtMIN7 allowed the bacterial strain to grow to higher levels. AtMIN7 is one of eight members of an ADP-ribosylation guanine nucleotide exchange factor that are important in vesicle trafficking in eukaryotes. Increased vesicle trafficking is associated with cell-wall defenses—testing the AtMIN7 mutant line for callose deposition showed that it was not able to deposit as much callose as the wild-type plant when challenged with the Pst CEL mutant. Thus, HopM1 targets this vesicle trafficking system as part of PTI suppression. More recently, Ham et al. (2007) used the plant defense gene mutant lines sid2 (SA induction deficient 2), npr1 (nonexpressor of PR genes 1), pad4 (phytoalexin deficient 4), rar1 and pmr4 (powdery mildew resistant 4) in combination with Ps. syringae pv. phaseolicola with and without ectopically expressed hopM1 to unravel the basis of PTI in Arabidopsis. They showed that Ps. syringae pv. phaseolicola carrying HopAM1 suppresses non-host PTI in Arabidopsis, observed as a reduction in small and large callose deposits and also PR-1 expression, and concluded that a multilayered defense is expressed by Arabidopsis to resist Ps. syringae pv. phaseolicola infection.


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I. TARGETING MAP KINASE SIGNALING

1. HopAI1 The role of HopAI1 in suppression of PTI was first found during a screen for suppressors of NHO1 expression (Li et al., 2005a). The protein shares 35% identity to the Salmonella enterica serovar typhimurium effector VirA. Transgenic expression of HopAI1 in Arabidopsis plants enabled a TTSS mutant to grow in planta although knockout of hopAI1 in Pst had no discernible effect on virulence. Further analysis of other Ps. syringae pv. tomato strains indicated that knockout of hopAI1 in strain 0288-9 led to a reduction in virulence (Zhang et al., 2007). It was revealed that HopAI1 directly interacts with MPK3 and MPK6 MAPKs and uses a phosphothreonine lyase domain to dephosphorylate the proteins and prevent further phosphorylation of the protein. The consequence of this was the blocking of the MAPK-signaling pathway, callose deposition and suppression of FRK1 expression. Thus, HopAI1 acts downstream of the PRR action of effectors such as AvrPto and AvrPtoB.

J. OTHER EFFECTORS INVOLVED IN PTI SUPPRESSION FOR WHICH TARGETS ARE UNKNOWN

1. avrRps4 This effector was originally identified from Ps. syringae pv. pisi as an avirulence gene recognized by the RPS4 resistance gene in some Arabidopsis ecotypes (Hinsch and Staskawicz, 1996); AvrRps4 also triggers an HR in turnip cv. Just Right (Sohn et al., 2009). After secretion into plant cells via the TTSS, the 28 kDa full-length AvrRps4 is processed to a smaller 11 kDa form. Only the C-terminal 88 amino acids of the 221-amino acid AvrRps4 are required to trigger a HR in turnip but processing is not necessary to trigger the HR. A KRVY motif was identified that is located just downstream of the in planta processing site within the 88-amino acid processed peptide—the KRVY motif is required for avirulence in Arabidopsis, but was also found to be necessary for AvrRps4-dependent increased virulence of Ps. syringae pv. tabaci in N. benthamiana; expression of AvrRps4 in N. benthamiana does not trigger a HR. Creation of transgenic Arabidopsis ecotype RLD plants (that lack a functional RPS4 gene) expressing AvrRps4 led to increased growth of wild-type and hrcC-minus strains of Pst and suppression of flg22-induced callose deposition and production of ROS, that is suppression of PTI. Interestingly, an AvrRps4 orthologue, XopO, was identified in X. campestris pv. vesicatoria that shows 41% identity and has a KRVY motif—the protein is processed in planta, but does not elicit an HR in turnip. Taken together,

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these observations suggest this to be an important effector, with an important role in PTI suppression; however, the dual activities and processing phenotype may indicate that two distinct pathways are involved in promoting virulence and triggering resistance. 2. hopAO1 (hopPtoD2) HopAO1 is a protein tyrosine phosphatase and contributes to the virulence of Pst in Arabidopsis—deletion mutants lacking the gene exhibit reduced virulence (Bretz et al., 2003; Espinosa et al., 2003). Underwood et al. (2007) showed that the TTSS hrpA mutant of Pst was able to multiply to higher levels in transgenic plants expressing HopAO1 compared to wild-type plants. Concomitant with this phenotype, the HopAO1 transgenic plants suppressed PTI immunity, as observed by reduced callose deposition upon challenge with the Pst hrpA mutant, compared to wild-type plants. This was further confirmed when using flg22 versus water treatment on wild-type and HopAO1 transgenic plants—the HopAO1 plants suppressed flg22-induced immunity compared to controls. In both experiments, the phosphatase activity of HopAO1 was shown to be essential for the suppression phenotypes. The site of action of HopAO1 is likely to be the plant cell cytosol due to the lack of transmembrane regions or myristoylation sites in the protein sequence. Indeed, HopAO1 protein was discovered to be present in the soluble fraction of plant cell extracts. The MAPKs MPK3 and MPK6 were ruled out as targets for dephosphorylation since transgenic plants expressing HopAO1 had no reductive effects on kinase activity. A microarray analysis of genes expressed in HopAO1 transgenic plants compared to wild-type plants, upon challenge with a Pst hrpA mutant, indicated that HopAO1 blocks only a subset of PAMP-induced genes, possibly by altering JA responses or signaling. K. OTHER EFFECTORS INVOLVED IN PTI SUPPRESSION, BUT LACKING FUNCTIONAL INFORMATION

In the screen carried out by Li et al. (2005a) that identified avrPto, hopAI1, hopM1 and hopT1-1 described above, seven other effectors were found that suppressed NHO1 expression: hopAA1-1, hopAF1, hopC1, hopF2, hopG1, hopS1, hopT1-2. Of these, HopF2 and HopG1 were separately shown to suppress PTI using the vascular staining assay described in Section III.H.1, but the precise function of these effectors remains unknown (Oh and Collmer, 2005) Metz et al. (2005) used a genetic screen to identify X. campestris pv. vesicatoria elicitors that trigger the non-host defense response in


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N. benthamiana. X. campestris pv. vesicatoria cosmid clones were expressed in X. campestris pv. campestris (that only causes mild chlorosis) and screened on N. benthamiana to identify clones that caused cell death—the effector gene xopX was identified from the screen. Although expression of the gene on its own in X. campestris pv. campestris was able to trigger cell death, Agrobacterium-mediated transient expression in N. benthamiana did not trigger cell death, indicating a synergistic effect of XopX and another X. campestris pv. campestris effector. Furthermore, transgenic expression of XopX in N. benthamiana allowed increased growth of non-XopX Xanthomonas and Pseudomonas strains; this increased disease susceptibility is likely due to the suppression of PTI although the precise mechanism remains unknown. Further experiments using xopX knockout strains demonstrated that XopX is important for X. campestris pv. vesicatoria virulence on host plants. L. OTHER POTENTIAL MECHANISMS—TYPE VI SECRETION

The VAS/vgrG type VI secretion system (T6SS) was discovered initially in Vibrio cholerae as an anti-amoeba system (Pukatzki et al., 2006). Since then it has also been described in Pseudomonas aeruginosa (Mougous et al., 2006), Ps. syringae (Arnold et al., 2009) and Pe. atrosepticum (Liu et al., 2008). In Pe. atrosepticum, the T6SS is regulated by quorum sensing and T6SS gene knockout mutants display a reduced virulence on potato tubers and in potato stems, indicating a role in plant pathogenicity. The exact function of the T6SS has yet to be defined, but a role in PTI suppression cannot be ruled out. M. COMPLEXITY AND EVOLUTION OF PTI SUPPRESSION BY BACTERIAL PATHOGENS

It is clear that bacteria target a number of components of plant PTI including: calcium stores in the apoplast; PRR-RLKs that detect PAMPs; the signaling pathways that are activated by the PRRs; hormone signaling pathways, including ABA, auxin, SA and JA; and interference with RNA and protein structure, function and activity including regulators of PTI. This vast array of targets illustrates the complexity of the evolution of plant-pathogenic bacteria and why they have such large arsenals of virulence factors. A general overview is depicted in Fig. 2. Without a deeper evolutionary analysis, it is unclear how the bacteria have evolved to overcome plant innate immunity other than the prediction that they first overcame PTI and then suppressed ETI. Based on the nearubiquitous spread of EPS gene clusters among most free-living bacteria, it is possible that EPS production was the first true PTI suppression system.


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Guard cells (in epidermis) Pathogen Apoplasm

Elongation factor

Flagellin

EPS Coronatine

BAK1

Ca

BAK1 FLS2

EFR

++

Cell wall fortification with callose

ions

RIN4

Vesicle trafficking ROS

Effectors Hormone signalling

MAPK cascade

WRKYs PR1 NH01 and FRK1 Effector targets

Activaion of gane expression Small RNAs and RNA-binding proteins Nucleus

Plant cell defense mechanisms

Plant cell cytosol

Fig. 2. General overview of the mechanisms for suppression of PAMP-triggered immunity (PTI) by bacterial pathogens. The effectors and their targets are represented by filled gray symbols and connected by dotted lines. Mechanisms for PTI suppression include calcium chelation by extracellular polysaccharides (EPS), opening of stomata by coronatine, hormone signaling, blockage of vesicle trafficking, subjugation of surveillance and defense systems by effectors injected into the cell by the Type III secretion system. Plant defense mechanisms, PTI and effector-triggered immunity (ETI), are in black (symbols and lines). See text for details.

As EPS suppression was overcome, possibly due to EPS acting as a PAMP (Aslam et al., 2008), a subset of pathogens may then have acquired the coronatine biosynthesis genes so that the pathogens could interfere with JA-dependent stomatal closure. Further rounds of evolution, either in parallel or after pathogens acquired toxins and EPS, would see the emergence of the TTSS to secrete effectors into the plant to douse resistance (Jones and Dangl, 2006). Pathogens would then have evolved further by the acquisition/ evolution of more effectors to overcome ETI. Clearly, there is still considerable effort required to understand the functions and targets of all the effectors and then how these integrate together to subjugate host defenses. However, understanding of the different mechanisms employed in suppression of PTI is making good progress.


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IV. RNA SILENCING, THE PLANT’S INNATE IMMUNE SYSTEM AGAINST VIRUSES A. THE DISCOVERY OF RNA SILENCING AS THE PLANT’S INNATE IMMUNE SYSTEM AGAINST VIRUSES

The plant’s innate immune system against viruses is very different from that against fungi and bacteria. The basal antiviral defense relies on the recognition and sequence-specific breakdown of (double-stranded, ds) viral RNA rather than targeting the pathogen’s proteins. In this section, this recently disclosed defense system, which is generally referred to as antiviral RNA silencing or RNA interference (RNAi), will be discussed, as well as the strategy how viruses may counteract this innate defense system. Unlike fungal and bacterial pathogens, viruses are exclusively intracellular parasites, multiplying in either the cytoplasm or the nucleus of plant cells. As a consequence, the interplay between host defense systems and the virus is strictly an intracellular event. Only since the mid 1990s, plant molecular biologists and virologists have become aware that the plant possesses a sequence-specific RNA breakdown mechanism, often referred to as posttranscriptional gene silencing (PTGS) or RNA silencing, and that this mechanism acts as the major innate immune system against viruses. The discovery of this defense system occurred accidentally, by encountering unexpected results during attempts to obtain virus-resistant plants through genetic engineering approaches. In the 1980s, several groups were investigating whether transgenic forms of virus resistance could be obtained according to the concept of ‘‘pathogen-derived resistance’’ (PDR). This concept was first described by Grumet et al. (1987), who proposed the possibility to exploit pathogenderived genes as a means to obtain resistance in a variety of host–parasite systems. It was suggested that deliberate expression of such genes in an altered form, level or developmental stage, could interfere with pathogen replication resulting in specific host resistance. Among possible targets for PDR-mediated virus resistance, the most broadly exploited viral genes were those coding for the coat protein (CP), replicase and movement protein (Baulcombe, 1996; Powell et al., 1990; Prins and Goldbach, 1996). Following the demonstration that expression of a viral CP confers a level of resistance to the pathogen (Abel et al., 1986; Powell et al., 1990), it was observed in control experiments that nontranslatable CP transgenes conferred similar levels of resistance as the functional gene. For years, this phenomenon was referred to as RNA-mediated resistance, and only in 1993, William Dougherty and coworkers (Lindbo et al., 1993) linked this phenomenon to ‘‘cosuppression’’ in plants and ‘‘quelling’’ in fungi (Cogoni and Macino, 1999a,b),

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which involve sequence-specific degradation of transcripts from both transgenes and their homologous endogenous counterparts. In turn, cosuppression was discovered when transgenic petunia plants with additional copies of endogenous genes involved in flower pigmentation, became completely white due to a dramatic decrease in expression level of the respective genes (Napoli et al., 1990; Van der Krol et al., 1990). Identification of (induced) RNA silencing as the principle mechanism of transgenic resistance to viruses has—in retrospective—been a major break-through. Rapidly, multiple publications appeared providing evidence that RNA silencing is a naturally occurring, ancient mechanism having a major function in regulating gene expression, transposon behavior, and viral infections (Carthew and Sontheimer, 2009). Moreover, RNA silencing occurs not only in plants and fungi, but has later been found also in invertebrate (Fire et al., 1998) and vertebrate animals, including humans (Carthew and Sontheimer, 2009; Elbashir et al., 2001; Hammond et al., 2000; Zamore et al., 2000) where this phenomenon is usually referred to as RNAi. A crucial discovery was the finding of short, virus-derived dsRNA molecules in infected host plants, explaining the sequence specificity of the RNA breakdown mechanism (Hamilton and Baulcombe, 1999). These short dsRNA species are commonly referred to as short interfering RNAs (siRNAs). Next to the discovery of virus-specific siRNAs, it was demonstrated that plants that are deficient in essential RNA silencing genes, show enhanced viral pathogenicity (Dalmay et al., 2001; Morel et al., 2002; Mourrain et al., 2000). These, and the fact that all tested plant viruses encode proteins that interfere with, and suppress the RNA silencing pathway, supported the idea that RNA silencing acts as innate antiviral defense system in plants. The viral proteins antagonizing RNA silencing, often already known as ‘‘virulence factors,’’ are commonly referred to as RNA silencing suppressor (RSS) proteins (Brigneti et al., 1998; Kasschau and Carrington, 1998). B. CURRENT VIEWS OF RNA SILENCING AS ANTIVIRAL MECHANISM IN PLANTS

With increasing insights, it was found that RNA silencing (RNAi) is not one single RNA breakdown pathway but encompasses two major ones, the siRNA pathway and the miRNAs. The former includes the antiviral defense branch of the system, while the miRNA pathway is primarily involved in regulating (host) gene expression. Figure 3 presents a simplified scheme of the RNA silencing pathways in the plant (most data have been obtained from Arabidopsis). As visualized in the scheme, RNA silencing starts with the recognition of long dsRNA by a type III endonuclease, called Dicer-like protein (DCL) in plants


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(the term Dicer was coined for a similar enzyme in the fruitfly Drosophila melanogaster (Bernstein et al., 2001)). It will be obvious that in particular RNA viruses are excellent targets to provoke (antiviral) RNA silencing: they replicate through (partially) dsRNA intermediates, while also the singlestranded (ss) genome contains extensive secondary structures. For viruses with a DNA genome, like the caulimo- and geminiviruses, the viral transcripts are the targets for RNA silencing, induced by secondary structures (e.g., the 35S RNA transcript of cauliflower mosaic virus, CaMV) and/or by overlapping sense–antisense transcripts (Chellappan et al., 2004; Du et al., 2007; Moissiard and Voinnet, 2006; Molna´r et al., 2005; Sharp and Zamore, 2000). 1. The siRNA pathway The siRNA pathway represents the antiviral branch of RNA silencing and this process takes place entirely in the cytoplasm (Covey et al., 1997; Ratcliff et al., 1997) (Fig. 3). It is known that plants encode different DCLs; in Arabidopsis DCL-4 is the most important one in the antiviral siRNA pathway while DCL-3 is needed for long-distance silencing. When DCL-4 is inactivated, its function is partly replaced by DCL-2 (Gasciolli et al., 2005). DCL-4 cleaves the viral dsRNA target molecules into short viral specific dsRNA molecules (siRNA) of 21–30 nucleotides (nt) in length with 2-nt overhangs at their 30 ends (Dunoyer et al., 2005; Gasciolli et al., 2005; Hamilton et al., 2002). After cleavage by DCL, the 21-nt siRNAs are incorporated into the RNA induced silencing complex (RISC) complex, which harbors a member of the Ago protein family, a key molecule of RISC (Tanaka Hall, 2005). After unwinding and degradation of the passenger siRNA strand (or siRNA*), the guide siRNA strand is used to identify complementary ss viral RNA sequences. After duplex formation between the guide siRNA strand and viral ssRNA, RISC (more specifically the Ago protein) facilitates target cleavage of the viral ssRNA molecule, resulting in sequence-specific RNA degradation of the viral RNA (Tomari and Zamore, 2005b). A special feature of the silencing pathway in plants is the possibility to amplify the silencing signal, in order to extend silencing along the target gene, using a host-encoded RNAdependent RNA polymerase (hRdRp). The hRdRp is able to produce new dsRNA molecules in an either primer-dependent or -independent manner; those can again enter the siRNA pathway, resulting in secondary siRNA molecules (Baulcombe, 2004; Sijen et al., 2001; Vaistij et al., 2002) (Fig. 3). RNA silencing is not only induced within the infected cell; plants are able to preprogram not yet infected cells by spreading the silencing signal beyond the site of initiation. This feature is called systemic silencing and can be divided in short-distance spread (10–15 cells) and phloem-dependent longdistance transport. It is believed that the short-distance silencing is performed

Virus

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Fig. 3. Schematic representation of the siRNA and miRNA pathways in plants and the inhibitory action (indicated ‘‘Stop’’) by some selected viral RNA silencing suppressor proteins (tombusviral p19, auriusviral p14, potyviral HC-Pro, cucumoviral 2b and tenuiviral NS3). RISC, RNA-induced silencing complex; DCL, Dicer-like protein; Ago, Argonaute protein; vRdRp, viral RNA-dependent RNA polymerase; hRdRp, host-encoded RNA-dependent RNA-polymerase.


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by 21-nt siRNAs and dependent on the activity of the hRdRp (Dunoyer et al., 2005; Himber et al., 2003). The long-distance silencing is suggested to rely on the activity of DCL-3, producing 24-nt siRNA molecules (Voinnet, 2005b; Yoo et al., 2004). However, the precise mechanism for both shortdistance and long-distance systemic silencing remains to be further resolved. 2. The miRNA pathway The miRNA pathway has no primary function in antiviral defense; it rather represents a gene expression regulation mechanism, shared with animals, to downregulate plant genes. Comparing the siRNA and miRNA pathway (Fig. 3), it is obvious that there is a high degree of parallelism: both start with the processing of longer dsRNA substrates into small dsRNA species, of which the guide strands are incorporated into RISC (often denoted miRISC) and searching for complementary ssRNA molecules. A fundamental difference is that while the siRNA pathway occurs entirely in the cytoplasm, the miRNA pathway starts in the nucleus: the miRNAs are endogenous RNA species, encoded by host genes. Chromosomal miRNA genes are transcribed mostly by RNA polymerase II to deliver the primary miRNA (pri-miRNA) that are folded into a partly double-stranded stem-loop structure, and become a substrate for DCL-1, thus producing precursor miRNA (pre-miRNA). Cleavage of premiRNA, again performed by DCL-1, generates mature, 21–22 nt miRNAs, which, unlike siRNA, are not completely double-stranded (Bartel, 2004; Voinnet, 2009). The miRNAs are then exported from the nucleus, by the nuclear export receptor HASTY (the product of the Arabidopsis orthologue of EXPORTIN5/MSN5). In the cytoplasm, the miRNAs are incorporated into RISC, unwound and used as guide to find perfectly or partly complementary ssRNA sequences, resulting in degradation or translational inhibition, respectively, of target mRNAs. These target mRNAs often encode transcription factors that, in turn, are in charge of regulating multiple genes (Chen, 2005). Most miRNAs are expressed in a tissue-specific manner and some are able to downregulate the expression of key RNA-silencing proteins, like DCL and Ago. The complementary miRNA target sequences in the host mRNAs can be present in the coding sequence or in the 30 untranslated regions (Voinnet, 2009). C. VIRAL SUPPRESSORS OF RNA SILENCING

During a compatible interaction between a virus and its host plant, infected plant tissues contain significant amounts of virus-derived siRNAs, indicating that the invading virus is actively targeted by the antiviral silencing machinery.

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Plant viruses would not exist if they had not generated an efficient strategy to counteract this antiviral RNA silencing. Indeed, they do so by encoding RSS proteins that are able to suppress RNA silencing. Among the first viral RSS proteins identified was HC-Pro of potyviruses, a multifunctional protein involved in aphid-mediated virus transmission, genome amplification, polyprotein processing, viral long-distance movement and RNA silencing suppression (Anandalakshmi et al., 1998; Bećlin et al., 1998; Brigneti et al., 1998; Kasschau and Carrington, 1998). This protein was already known as ‘‘virulence factor,’’ as there was a causal linkage between HC-Pro expression and severity of disease symptoms (Atreya and Pirone, 1993; Atreya et al., 1992). This effect of HC-Pro can now be readily explained in view of its function to suppress the host’s antiviral RNA silencing. Mutational analysis has revealed that the RSS and proteolytic activity are independent, separable properties within HC-Pro, in contrast to genome amplification and long-distance movement functions, which seem to be related to the RSS activity (Kasschau and Carrington, 2001). At least part of these activities can be explained by the affinity of HC-Pro for siRNA and its interference with their methylation, reducing siRNA stability (Ebhardt et al., 2005; Lakatos et al., 2006; Li et al., 2005b). To date, for an increasing number of plant viruses the encoded RSS protein has been identified mostly based on transgenic suppressor assays in Arabidopsis or Nicotiana spp. (Li and Ding, 2006; Roth et al., 2004). These include positive-, negative- and dsRNA viruses as well as the geminiviruses, which have a ssDNA genome. Most of these viruses encode only a single RSS protein, which acts on a single step in the siRNA pathway, resulting in partial suppression (Li and Ding, 2006). The situation for geminiviruses is more complicated though, as among different viral species the RSS activity appears to reside in different proteins (Bisaro, 2006; Voinnet et al., 1999). The closterovirus citrus tristeza virus is also a special case, as this virus encodes three proteins involved in RSS action (Lu et al., 2004; Satyanarayana et al., 2002). Since RNA silencing has been recognized more and more as an ancient cellular mechanism shared by most living organisms (Dıáz-Pendoń and Ding, 2008; Li and Ding, 2006), it is obvious that viruses and antiviral RNA silencing will have co-evolved over a very long period, and hence viral RSS proteins are expected to form one or more clusters of similar proteins containing conserved sequence motifs. This is, surprisingly, not the case. In genomic position, in molecular size and in amino acid sequence, the reverse is true: viral RSS proteins are extremely variable among viruses, which is most prominently illustrated by the situation within the family Tombusviridae: depending on the species, the RSS function may reside in the viral CP, the viral polymerase, or in a separate viral protein (Fig. 4, see also Me´rai et al., 2005, 2006; Takeda et al., 2005). The general picture, which


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Aureusvirus P84 (replicase)

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Fig. 4. Schematic representation of the genome organization of four different viral species belonging to the Tombusviridae. Open reading frames (ORFs) in the respective RNA genomes are indicated as open bars. ORFs and names in gray represent the identified RNA silencing suppressor (RSS) proteins and their functional activity. PoLV, Pothos latent virus (genus Aureusvirus); TCV, turnip crinkle virus (genus Carmovirus); RCNMV, red clover necrotic mosaic virus (genus Dianthovirus); CymRSV, cymbidium ringspot virus (genus Tombusvirus). (After Takeda et al., 2005).

emerges when comparing different plant viruses, is that their encoded RSS activity is often part of a multifunctional protein. Viral RSS proteins not only come in very different shapes, but their mode of action may also differ. Some RSS proteins act by sequestering dsRNA molecules, either size-specifically, like tombusviral P19 (exclusively binding siRNAs), or nonspecifically, like aureusviral P14 (also binding longer dsRNAs) (Lakatos et al., 2006; Me´rai et al., 2005, 2006). Others bind protein factors of the RNA silencing pathway, like cucumber mosaic virus (CMV) protein 2b. While most RSS proteins interfere with only a single step in the RNA silencing pathway, some are able to block at different points, including again CMV protein 2b, which is able to both sequester siRNAs and to interact with Ago within the RISC complex, as demonstrated in infected Arabidopsis (Goto et al., 2007; Zhang et al., 2006). RSS proteins encoded by different virus families often share no homology at the amino acid sequence level, even if they have a similar mode of action (Lakatos et al., 2006; Me´rai et al., 2006). So far not a single sequence motif characteristic for (a subclass of) RSS proteins has been identified. This is surprising as RNA silencing is generally regarded to be an ancient mechanism. One explanation for this could be that the long-lasting evolutionary interplay with the plant’s antiviral RNA silencing mechanism has driven

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viruses to continuously change and adapt their suppressor protein sequences (and their coding sequences) to keep ahead of the host defense system. The RNA silencing’s selective pressure would then act as a major evolutionary driving force resulting in extreme speciation. This would also explain the overwhelming excess of RNA virus species versus DNA virus species within the plant kingdom. A similar situation may have occurred in bacteria where the great majority of phages have DNA genomes, targets for the (DNA-based) restriction/modification system in bacteria. Another explanation might be that RSS genes have been introduced into viral genomes through multiple independent evolutionary events. An argument in favor of this alternative is the observation that RSS genes often overlap with another viral gene, including in some cases the polymerase gene. In evolutionary terms, it is believed that overlapping genes are created by overprinting, meaning that an existing coding sequence is translated in a different reading frame (Ding et al., 1995; Keese and Gibbs, 1992). According to this scenario, the lack of sequence homology between different RSS proteins would be explained by multiple independent introductions into viral genomes. Plant viruses also replicating in their insect vectors, such as the rhabdo-, tospo- and tenuiviruses, need to counteract RNA silencing in two very distinct types of organisms. It is to be expected that this may be achieved by specifying an RSS protein which blocks a step in the RNA silencing mechanism that is conserved between plants and insects. Indeed, both tospoviral NSs and tenuiviral NS3 proteins exert their RSS function by sequestering siRNAs (Hemmes et al., 2007, 2009), a key molecule shared by plant and insect. D. POSSIBLE INTERACTIONS BETWEEN PLANT VIRUSES AND THE miRNA PATHWAY

During the infection process the presence of the viral RSS protein may, in addition to blocking the antiviral siRNA pathway, interfere also with the miRNA pathway. Both pathways share similar key molecules, either RNA or proteins, and nearly all tested RSS proteins which act by sequestering siRNA molecules are equally able to bind miRNAs in vitro. Examples are tombusviral p19 and tenuiviral NS3 (Dunoyer et al., 2004; Hemmes et al., 2007; Silhavy et al., 2002). Further research confirmed the ability of RSS proteins to suppress the miRNA pathway in vivo, resulting in virus diseaselike symptoms. Drastic effects in phenotypes, reminiscent of virus disease symptoms, have also been observed in transgenic Arabidopsis plants expressing viral RSS proteins (Chapman et al., 2004; Dunoyer et al., 2004). These observations point to a prominent role of viral RSS proteins in the induction


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of disease symptoms upon infection. Whether this symptom induction is intended or happens accidentally due to the high similarities between the siRNA and miRNA pathways is not known yet. E. IS ANTIVIRAL RNAi RESTRICTED TO PLANTS AND INSECTS?

After the ground-breaking work of Andrew Fire and Craig Mello (Fire et al., 1998), who discovered RNAi in nematodes, increasing evidence indicated that RNAi is an ancient gene regulation mechanism occurring in almost every eukaryotic organism, from algae and plants to insects and humans (Sontheimer and Carthew, 2005; Tomari and Zamore, 2005a; Voinnet, 2005a). The mechanism shared between all these organisms is the miRNA pathway, whereas the siRNA pathway may be shared only among plants and invertebrates and probably not by mammals. For insects, the existence of a separate antiviral siRNA branch within RNAi has been well established, and insect-infecting viruses, in turn, have been shown to encode RSS proteins (e.g., protein B2 of Flock House virus (Li et al., 2002)) to combat this antiviral response. In infected mammalian cells, virus-derived siRNAs have so far not convincingly been detected (Pfeffer et al., 2004), but antiviral RNA can be readily induced upon transfection with dsRNA (hairpin RNA) containing viral sequences (Haasnoot et al., 2007; Lo´pezFraga et al., 2008; Marques and Carthew, 2007). While a separate antiviral siRNA branch may be absent in mammals there is increasing evidence that also mammalian, for example, human viruses encounter antiviral RNAi and this may exclusively occur through the miRNA pathway, resulting in the thought that human viruses encode RSS proteins too (Berkhout and Jeang, 2007; Grassmann and Jeang, 2008; Murakami et al., 2009; Triboulet et al., 2007). In mammalian cells, a Human Immunodeficiency Virus (HIV)-1 mutant lacking its proposed RSS, HIV-1 Tat (Transactivator of transcription) could be complemented in its viral production by transfection with a plant viral RSS. Thus, it was convincingly demonstrated that HIV-1 specifies an RSS protein, HIV-1 Tat, that is capable of suppressing the action of miRNA in vivo and that this suppression is essential for efficient virus production (Schnettler et al., 2009; Qian et al., 2009). PTGS, or shortly RNA silencing, not only represents a major regulatory mechanism in most, if not all, eukaryotes, in plants this sequence-specific RNA breakdown mechanism, through a separate siRNA branch, also represents the basal defense system against viruses. In turn, plant viruses have been able to counteract this host response by encoding antagonizing RSS

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proteins. Some of these RSS proteins also interact with the miRNA pathway, explaining some of the observed disease symptoms in infected plants. Whether this interaction is on purpose or represents a non-specific side effect, remains to be established.

ACKNOWLEDGMENTS The authors thank Jens Boch for his comments on the manuscript.

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From Nonhost Resistance to Lesion-Mimic Mutants: Useful for Studies of Defense Signaling

ANDREA LENK AND HANS THORDAL-CHRISTENSEN1

Department of Agriculture and Ecology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Defense Induction Mediated by PAMPS and Effectors. . . . . . . . . . . . . . . . . . . III. Signaling Downstream of Pathogen Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The SA-Signaling Pathway.................................................. IV. Commonalities in the Defense Response of Host and Nonhost Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Penetration Resistance of Arabidopsis..................................... B. Nonhost Resistance to Bacteria ............................................ V. What is the Explanation for Nonhost Resistance?. . . . . . . . . . . . . . . . . . . . . . . . VI. Lesion-Mimic Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Mutant Screens Without Pathogens for Finding Genes in Defense Signaling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. SSD Mutants .................................................................. B. SFD Mutants ................................................................. C. MOS Mutants................................................................. VIII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Corresponding author: Email: [email protected]


0065-2296/09 $35.00 DOI: 10.1016/S0065-2296(09)51003-8

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A. LENK AND H. THORDAL-CHRISTENSEN

ABSTRACT Nonhost and host resistance are very similar phenomena which employ the same defense mechanisms, spanning from passive defense, through PAMP-triggered immunity to responses toward pathogen eVectors. The diVerence between an unsuccessful and a successful pathogen lies in its eVector portfolio. It has become apparent that only if the pathogen’s eVectors are able to suppress defense responses, and do so without being recognized, can the attacked plant serve as a host for the pathogen. Thereby, the sum of eVectors determines the host range of a given pathogen. Another approach for investigating defense mechanisms, using lesion-mimic mutants, is attracting interest. These plants have enhanced resistance to biotrophic pathogens, and due to their constitutively active defense-signaling pathways, lesion-mimic mutants are valuable for identifying genes involved in defense signaling.

I. INTRODUCTION Plants are surprisingly healthy considering the vast number of potential pathogens in their surroundings. To a large extent, this is due to nonhost resistance, where all genotypes of a plant clade are resistant to all genotypes of a pathogen (Mysore and Ryu, 2004; Thordal-Christensen, 2003). Nevertheless, significant application of pesticides is required to minimize crop damages resulting from the few pathogens that are able to cause disease. Pathogenic microbes are an expense factor, causing immense harvest losses and quality reduction in agriculture every year. As such, there is good reason to investigate the nature of nonhost resistance to pathogens in order to understand and exploit this durable form of plant protection. Nonhost and host pathogen resistance appear to be based on a complex interplay involving the same plant and pathogen components. Therefore, we summarize the most essential elements of this interplay, and present what is currently known about how this results in nonhost resistance. In Arabidopsis thaliana, nonhost resistance toward the nonadapted barley powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh) is mediated to a large extent by ‘‘penetration resistance.’’ In this review, we will address how studies of this system have identified three PEN (PENETRATION) genes required for resistance to fungal penetration. The first of these to be identified, PEN1, encodes a syntaxin (Collins et al., 2003) which is required for vesicle fusion at the plasma membrane. Combining mutations in PEN1 and its closest homolog, SYP122 (SYNTAXIN IN PLANTS 122), has demonstrated an overlapping function of these genes as negative regulators of defenses, resulting in a severe lesionmimic phenotype of this syntaxin double mutant. Constitutive production of diVerent defense-signaling compounds, including salicylic acid (SA), makes

NONHOST RESISTANCE AND LESION-MIMIC MUTANTS

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these plants resistant to biotrophic pathogens. Such lesion-mimic mutants (LMMs) have visible hypersensitive response-like symptoms, and therefore can be considered to have genetic diseases with significant similarity to diseases caused by pathogens. In classical defense expression studies, many important mutants have been discovered with altered response to pathogens. This has led to the identification of numerous defense-signaling genes and has provided most of our current insight into defense mechanisms. However, we and others have found that screening for suppressor mutants in LMMs can circumvent pathogen-based mutant screens, which are often time-consuming and problematic. Thereby, it is possible to simplify our continuous eVorts to unravel new components in defense-signaling pathways in plants. Furthermore, combining defense-signaling mutations in LMM backgrounds permits investigation of signaling gene interrelationships and allows signaling networks to be proposed.

II. DEFENSE INDUCTION MEDIATED BY PAMPS AND EFFECTORS Protection against pathogen attack occurs in diVerent layers of defense and can be divided into passive and active defense mechanisms. Passive defense against attacking microorganisms does not require pathogen recognition, but instead consists of permanent physical barriers such as plant hairs, wax layers, and strong cell walls. Moreover, pathogens can be resisted by the constitutive presence of toxic secondary metabolites as well as antimicrobial proteins. All these are preformed mechanisms of defense. Importantly, plants can also actively mount resistance upon recognition of pathogens. Microbes possess highly conserved structures, called ‘‘pathogenassociated molecular patterns’’ (PAMPs) (Chisholm et al., 2006; Ingle et al., 2006). PAMPs are indispensable for microbial life, and their recognition by the plant predominantly occurs preinvasively via plant receptors in the plasma membrane, leading to activation of PAMP-triggered immunity (PTI). PTI is often referred to as ‘‘basal defense.’’ Through MAPK (mitogen-activated protein kinase) cascades, defense mechanisms are turned on, including responses such as callose deposition, ion fluxes, production of reactive oxygen species (ROS), and secretion of antimicrobial compounds (Altenbach and Robatzek, 2007). Receptors for the fungal PAMPs, chitin and xylanase, have been identified (Kaku et al., 2006; Ron and Avni, 2004; Wan et al., 2008). However, the best characterized PAMP receptors are FLS2 and ERF, two leucine-rich repeat receptor-like kinases (LRR-RLK) which

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recognize bacterial flagellin and translation elongation factor TU, respectively (Go´mez-Go´mez and Boller, 2000; Zipfel et al., 2006). It has been shown that FLS2 directly interacts with flagellin and that it accumulates in intracellular endocytotic vesicles upon ligand activation (Robatzek et al., 2006). During co‐evolution with their hosts, pathogens have been able to circumvent this PTI by avoiding recognition or by suppressing PAMP-mediated defense mechanisms using eVector proteins (Chisholm et al., 2006; Da Cunha et al., 2006). EVectors are small molecules that are released by the pathogen to their potential host to promote the process of infection. Pathogenic bacteria use the type III protein secretion system (T3SS) to translocate their eVectors into the plant cell (Schechter et al., 2006), whereas some fungal pathogens deliver eVectors into the apoplast or through their feeding structure, the haustorium, into the host cell (Catanzariti et al., 2006; Kemen et al., 2005). Oomycetes secrete eVectors containing a specific RXLR-dEER-motif necessary for their translocation into the plant cell (Dou et al., 2008; Morgan and Kamoun, 2007; Whisson et al., 2007). In addition to eVectors, some pathogens produce plant hormones or hormone analogs to aVect plant defense. As some signaling pathways in plants act antagonistically, these pathogens aim at producing hormones antagonizing the defense pathway directed against their own propagation. A well-documented example is the SA-signaling pathway antagonized by the jasmonic acid (JA)-signaling pathway. The SA-signaling pathway is eVective against biotrophic pathogens, relying on living host tissue. Therefore, some pseudomonads produce coronatine, a bacterial analog of the JA-derivative, JA-isoleucine, in order to suppress the SA-signaling pathway (Cui et al., 2005; Laurie-Berry et al., 2006; Zhao et al., 2003). In attempts to counteract pathogen eVectors, plants have evolved genotypespecific disease resistance (R) genes. Resistance proteins directly or indirectly recognize microbial eVectors, which were previously referred to as avirulence (Avr) proteins. Avr genes are pathogen-race specific. R gene-mediated resistance is usually accompanied by a localized programmed cell death (PCD) called the hypersensitive response (HR) (Lam, 2004). During the HR, hydrogen peroxide (H2O2) accumulates, acting a dual role such as in preventing pathogen growth, due to its toxicity, and a signal for promotion of the PCD. Although R genes mediate resistance to diVerent types of pathogens, they share conserved characteristics. The most prominent class of R genes, with approximately 125 genes in Arabidopsis, encode NB-LRR proteins with a central nucleotide binding site (NB) and a C-terminal leucine-rich repeat (LRR) domain (Martin et al., 2003). These NB-LRR proteins are localized in the cytoplasm and can be divided into two subclasses based on their N-terminal domain. The region either contains a coiled-coil structure


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(CC-type) or has homology to the Drosophila Toll and mammalian Interleukin-1 receptor (TIR-type). The LRR domain is responsible for specific protein recognition and the CC or TIR domains control downstream signaling (Martin et al., 2003). The NB domain is proposed to act as a molecular switch that hydrolyzes ATP. Thereby, the NB-LRR protein undergoes conformational changes, which activate regulatory elements further downstream and trigger the HR (Takken et al., 2006). Autoactive mutant versions of NBLRR proteins have been identified, in which ATP hydrolysis appears to be impaired. Plants expressing autoactive R-proteins are more resistant to pathogens, but on the other hand, suVer from spontaneous necrosis (Howles et al., 2005; Shirano et al., 2002; Zhang et al., 2003). Other eVectors capable of suppressing eVector-triggered immunity (ETI) and the accompanying cell death have evolved during the arms race between plants and pathogens. There are documented examples of eVectors suppressing both PTI and ETI by utilizing diVerent strategies, ranging from host-protein ubiquitination, phosphorylation, and dephosphorylation to alteration of the RNA metabolism (Block et al., 2008; Da Cunha et al., 2007). Throughout the past decades, diVerent models have been proposed to explain how R-proteins and their corresponding Avr-proteins interact. In 1956, Flor hypothesized the ‘‘gene-for-gene model,’’ with one R gene for every Avr gene (Flor, 1956). In a few cases direct interactions have been documented, thereby supporting a ‘‘receptor-ligand-model’’ (Deslandes et al., 2003; Dodds et al., 2006; Jia et al., 2000; Ueda et al., 2006). However, in most cases, no direct interaction between the R-protein and the Avrprotein occurs. An explanation for this is provided by the ‘‘guard model’’ in which R-proteins are monitoring the action of eVectors/Avr-proteins by surveying whether eVector targets are being perturbed (Dangl and Jones, 2001; Van der Biezen and Jones, 1998). Additionally, the guard model can explain how only a few R genes can monitor various unrelated eVectors at once. This can occur when a single R-protein guards multiple eVector targets, or when a single target is attacked by diVerent eVectors. For example, the Arabidopsis Avr-protein RPM1, which is activated upon RIN4 hyperphosphorylation, detects two unrelated Pseudomonas syringae eVector proteins which both target RIN4 (Bisgrove et al., 1994; Chisholm et al., 2006). In tomato, the R-protein Mi recognizes organisms which are as remotely related as nematodes and aphids (Rossi et al., 1998). A recently proposed ‘‘decoy model’’ suggests that many eVector targets mimic real targets and only serve to recognize the eVector, which then can be detected by the R-protein to activate defense (Nandi et al., 2003; Van der Hoorn and Kamoun, 2008; Zhou and Chai, 2008). Decoys may have evolved from eVector targets through gene duplication (Van der Hoorn and Kamoun, 2008).

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Plants successfully employ both PTI and ETI to repel microbial invaders. These two layers of immunity result in the activation of similar subsets of genes (Lin and Martin, 2007; Lindeberg et al., 2006). In a transcription profiling study, comparing tomato plants treated with a PAMP elicitor (flg22) and an eVector (Avr9) from Cladosporium fulvum, similar genes were induced (Navarro et al., 2004). Another example of how PAMP- and eVector-mediated resistance are linked is the barley MLA resistance protein-mediated response to the powdery mildew fungus (Shen et al., 2007). Through PAMP-mediated signaling, WRKY transcription factors are activated and PTI is triggered. However, at the same time, defense protein production is reduced by the activation of other WRKY factors that suppress transcription. After eVector recognition, the activated MLA protein acts positively on PTI by interfering with the repression mechanism of these additional WRKY factors, thereby amplifying PTI and killing the pathogen. Initially, PTI responses are weak, not to damage the host. As soon as MLA protein activation occurs, responses become much stronger (Shen et al., 2007). This fascinating set of interactions between several components in the plant and the pathogen must have a long co‐evolutionary history. Each time either the plant or the pathogen has managed to develop an eYcient tool in its favor, the other organism has evolved a method to overcome this obstacle. The final result as we see it today is likely to have evolved one step at the time, alternating between the two organisms.

III. SIGNALING DOWNSTREAM OF PATHOGEN DETECTION Most work investigating signaling pathways has been done in mutant studies, notably in Arabidopsis. Proteins such as EDS1 (ENHANCED DISEASE SUSCEPTIBILITY 1) and its interacting partners, PAD4 (PHYTOALEXIN DEFICIENT 4) and SAG101 (SENESCENCE-ASSOCIATED GENE 101), act downstream of TIR-NB-LRR-type R-proteins in ETI (Wiermer et al., 2005). However, EDS1 is also a positive regulator of PTI. The Arabidopsis eds1 mutant has, for example, a reduced penetration resistance against barley and wheat powdery mildew fungi (Lipka et al., 2005; Wiermer et al., 2005; Yun et al., 2003; Zimmerli et al., 2004). While EDS1, PAD4, and SAG101 are important for signal transduction from TIR-NB-LRR-type R-proteins, NDR1 (NONRACE-SPECIFIC DISEASE RESISTANCE 1) acts in concert with the CC-NB-LRR-type R-proteins (Aarts et al., 1998; Century et al., 1997). SGT1b (SUPPRESSOR OF G2 ALLELE OF SKP1), RAR1 (REQUIRED FOR MLA RESISTANCE 1), and HSP90 (HEAT SHOCK PROTEIN 90),


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act as chaperones and stabilize R-protein complexes rather than acting in the downstream signaling per se (Azevedo et al., 2002; Holt et al., 2005). A. THE SA-SIGNALING PATHWAY

SA is a key signaling molecule in R gene-mediated resistance and PTI that is activated in response to biotrophic pathogens (Glazebrook, 2005). Mutants with reduced SA levels, or attenuated SA signaling, show decreased resistance to biotrophic pathogens, such as the oomycete Hyaloperonospora arabidopsidis (formerly known as (Hyalo)peronospora parasitica). The phytohormone SA is synthesized in response to pathogen infection. In Arabidopsis, pathogen-induced SA synthesis requires the gene SID2 (SA INDUCTION DEFICIENT 2), encoding isochorismate synthase 1 (ICS1) (Nawrath and Me´traux, 1999; Wildermuth et al., 2001). SID2 is located in the chloroplast and catalyzes the conversion of chorismate to isochorismate. An unknown, probably chloroplast-located enzyme is needed for conversion of isochorismate to SA (Strawn et al., 2007). sid2 mutant plants are more susceptible to pathogens, but still contain a small amount of residual SA after stress stimulation. The only other isochorismate synthase in Arabidopsis, ICS2, is responsible for production of a small part of the stress-induced SA in the sid2 mutant background. Double-mutant analysis revealed the existence of an ICS-independent SA biosynthesis pathway. However, this pathway only plays a minor role (Garcion et al., 2008). Similarly, eds5 (enhanced disease susceptibility 5) mutant plants fail to accumulate SA. EDS5 has homology to members of the MATE (multidrug and toxin extrusion) transporter family, and is believed to be involved in translocation of intermediates for SA biosynthesis across the chloroplast membrane (Nawrath et al., 2002). The functional order of EDS5 and SID2 is not yet established. Altered SA levels are also observed upon mutation of ALD1 (AGD2LIKE DEFENSE RESPONSE PROTEIN 1) and its close homolog AGD2 (ABERRANT GROWTH AND DEATH 2). However, mutations in ALD1 and AGD2 cause opposite phenotypes (Song et al., 2004a). While a specific agd2 mutant produces elevated SA levels and is resistant to P. syringae pv. maculicola, ald1 mutants show reduced SA accumulation and are more susceptible (Song et al., 2004a,b). ALD1 gene expression is highly upregulated in response to infection by P. syringae. ALD1 is likely to be a part of an EDS1/PAD4-controlled SA-independent pathway, because its expression is dependent on PAD4, but not on SA (Song et al., 2004b). Both ALD1 and AGD2 are predicted to be located in the chloroplast and show aminotransferase activity on several amino acid substrates in vitro (Song et al., 2004a).

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AGD2 catalyzes a step in lysine biosynthesis, while recombinant ALD1 does not have this particular activity (Hudson et al., 2006). Another gene, believed to be part of the EDS1/PAD4 regulated SAindependent pathway, is FMO1 (FLAVIN-DEPENDENT MONOOXYGENASE 1). FMO1 transcription is upregulated after pathogen attack and in lesion-mimic mutants such as acd11 (accelerated cell death 11), lsd1 (lesions simulating disease resistance 1), and the pen1 syp122 double mutant. FMO1 was identified independently by several groups as a positive regulator of defense (Bartsch et al., 2006; Koch et al., 2006; Mishina and Zeier, 2006; Olszak et al., 2006; Zhang et al., 2008). FMO proteins catalyze the transfer of hydroxyl groups to nucleophilic heteroatom-containing substrates, and have been speculated to function in changing the cellular redox state by production of ROS. But the FMO1 substrate is still elusive (Schlaich, 2007). The enzymatic activity of FMO1 is required for plant defense (Bartsch et al., 2006). PBS3 (avrPphB SUSCEPTIBLE 3) is a member of the GH3-like family of acyl-adenylate/thioester-forming enzymes which are known as phytohormone-amino acid synthetases (Jagadeeswaran et al., 2007; Lee et al., 2007; Nobuta et al., 2007). pbs3 mutants have decreased transcript levels of PATHOGENESIS-RELATED (PR)-1, and are more susceptible to several virulent and avirulent P. syringae strains. After inoculation with bacterial pathogens, pbs3 mutant plants exhibit lower levels of glucoside-conjugated SA (SAG), which is the primary storage form of SA. Total SA levels are decreased, while contradictory results were reported for accumulation of free SA in response to various P. syringae strains carrying diVerent Avr genes. Nevertheless, external SA application can restore PR-1 expression and resistance toward pathogens (Jagadeeswaran et al., 2007; Lee et al., 2007; Nobuta et al., 2007). It is speculated that PBS3 has a role in the SA-signaling pathway, acting as a regulator on SA and SAG accumulation, or even upstream of SA synthesis (Nobuta et al., 2007). In Arabidopsis and tobacco, many studies have employed plants expressing the bacterial transgene NahG from Pseudomonas putida. NahG encodes a SAhydroxylase that converts SA to catechol. Phenotypical diVerences between NahG plants and SA-deficient mutant plants raised the question whether NahG plants have a distinct phenotype caused by the production of catechol (Van Wees and Glazebrook, 2003). Furthermore, data suggest that SA is unlikely to be the only substrate of the NahG SA-hydroxylase, and those particular aspects of the NahG phenotype are neither SA dependent nor due to catechol (Heck et al., 2003). In addition, based on LMMs, evidence has been provided that NahG inactivates potential defense‐signaling compounds other than SA (Brodersen et al., 2005; Zhang et al., 2008).


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A central downstream component of the SA-signaling pathway is NPR1 (NONEXPRESSOR OF PR GENES 1) (Dong, 2004). In the cytosol, NPR1 is present in an oligomeric form. Upon increase of SA levels, the redox state of the cell is altered and internal disulfide bridges of the NPR1 protein are reduced. This leads to monomeric NPR1, capable of entering the nucleus (Mou et al., 2003). Here, it interacts with TGA transcription factors, modulating their binding activity to as-1-like promoter elements of PR genes; thereby, PR gene expression is induced (Despre´s et al., 2000). PR-1, the function of which is still unknown, is a widely utilized marker for SA signaling. Another common DNA sequence motif in PR promoter regions is the W-box, which is recognized by WRKY transcription factors (Eulgem and Somssich, 2007; Eulgem et al., 2000). In fact, many PR genes are more likely to be regulated by WRKY transcription factors than by TGA transcription factors (Maleck et al., 2000). WRKY factors act both as negative and positive regulators in pathogen defense, and interference with the negatively functioning WRKY factors has been documented to promote plant defense (Shen et al., 2007). In addition to local defense responses at the site of pathogen attack, a systemic, long-lasting disease resistance can occur. This form of induced resistance, termed ‘‘systemic acquired resistance’’ (SAR), is functional against a broad spectrum of pathogens. In many plant species, SA-dependent SAR can be induced by necrotizing pathogens and various chemical compounds. PR gene expression in uninfected leaves functions as marker for SAR (Durrant and Dong, 2004). Identification of the mobile signal inducing SAR in distal leaves has challenged scientists in the field for many years. It has been suggested to be a lipid-based molecule (Maldonado et al., 2002) or JA (Truman et al., 2007). In tobacco, the mobile signal has been identified as SA-derived methyl salicylate (MeSA), not excluding a lipid-based signal in Arabidopsis (Park et al., 2007). Many mutants that are deficient in the SAsignaling pathway are also unable to activate SAR. This is true for the mutants sid2, eds1, eds5, pad4, ald1, fmo1, pbs3, and npr1, and for plants expressing the transgene NahG.

IV. COMMONALITIES IN THE DEFENSE RESPONSE OF HOST AND NONHOST RESISTANCE In an attempt to describe how nonhost resistance functions, we find it interesting that many examples are available, documenting similarities between how plants respond to host and nonhost pathogens. This has been shown convincingly using transcript expression profiling (Stein et al., 2006;

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Tao et al., 2003; Truman et al., 2006). Moreover, a number of examples described below have utilized functional studies to demonstrate that the mechanisms underlying these two types of resistance overlap. A. PENETRATION RESISTANCE OF ARABIDOPSIS

Plants have a distinct type of PTI against powdery mildew fungi, manifested as ‘‘penetration resistance.’’ The powdery mildew fungus, Golovinomyces cichoracearum (Gc), in general overcomes this defense on its host plant, Arabidopsis (Zhang et al., 2007). Meanwhile, when the barley powdery mildew fungus Bgh attacks Arabidopsis, it only overcomes the penetration resistance at 10–20% of the attack sites (Fig. 1). Powdery mildew fungi are obligate biotrophs. Their life cycle is dependent on living host tissue, and they are often specialized to survive only on a specific host plant clade. Bgh proliferates on plants from the genus Hordeum, and is not able to complete its life cycle on other plants. Nevertheless, Bgh spores germinate and develop normally on

% Bgh penetration

A

100 80 60 40 20 0

Col-0

pen1

B co sh co

HR h

pap sh 100 mm

Fig. 1. (A) Penetration frequency of the nonhost barley powdery mildew fungus, Blumeria graminis f.sp. hordei (Bgh), on Arabidopsis Columbia (Col-0) wild type and its pen1 mutant. (B) Growth of Bgh on Arabidopsis plants. Germinating conidiospores (co) stopped by papilla (pap) or by single epidermal cell death (HR) after generating haustorium (h) and secondary hyphae (sh). Stained with Trypan Blue 48 h postinoculation.


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Arabidopsis. When specialized infection hyphae, the appressoria, attempt to penetrate through the cuticle and cell wall of an epidermal cell of this nonhost plant, only a minority succeeds. In about 80–90% of the cases, the appressorium is stopped at this preinvasive stage by a reinforcement of the cell wall, the socalled ‘‘papilla’’. The few spores that manage to penetrate develop a haustorium, an invasive feeding structure, inside the epidermal cell. In some cases, they even develop secondary hyphae, which reflects that the haustorium is functioning and able to take up nutrients from the host cell. However, the growth of Bgh is eventually stopped by an epidermal single cell HR, serving as a second layer of defense (Fig. 1) (Collins et al., 2003). This backup defense layer is predicted to be mediated by a gene-for-gene interaction between fungal eVector/Avr-proteins and R-proteins in this nonhost plant. Interestingly, both Bgh penetration and secondary hyphal growth rates are significantly higher in Arabidopsis eds1 mutants (Zimmerli et al., 2004). The same EDS1-dependence has been observed for penetration resistance and epidermal HR in Arabidopsis after attack by the wheat powdery mildew fungus, B. graminis f.sp. tritici (Yun et al., 2003). The need for the EDS1-signaling step in the single cell HR has been confirmed by its requirement for the EDS1-interacting proteins, PAD4 and SAG101 (Lipka et al., 2005). In summary, it appears that two layers of defense, PTI and ETI, prevent these nonhost fungi from completing their life cycle on Arabidopsis. In a genetic screen for mutants that are impaired in stopping B. graminis at the first layer of defense, an ethyl methane sulfonate (EMS)-mutagenized Arabidopsis population was analyzed. Bgh entry into epidermal cells was analyzed microscopically. The first penetration resistance mutant to be identified was pen1 (Collins et al., 2003). The PEN1 gene codes for the syntaxin SYP121 (PEN1), necessary for exocytosis. Syntaxins are SNARE proteins (Soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor) involved in vesicle traYcking, where they are located in the target membrane. Together with two other SNARE proteins, SNAP (soluble NSF attachment protein) and VAMP (vesicle-associated membrane protein), they form SNARE complexes that mediate fusion of vesicles to their target membranes. In Arabidopsis, the SNARE complex members corresponding to PEN1 have been identified as SNAP33 and VAMP721/VAMP722 (Kwon et al., 2008). When inoculated with Bgh, the pen1 mutant allows about 80% of the germinating Bgh spores to penetrate the epidermal cells (Fig. 1) (Assaad et al., 2004; Collins et al., 2003; Kwon et al., 2008; Zhang et al., 2007). Subsequently, Bgh is stopped by HR. This loss of penetration resistance can be explained by a delay of approximately 2 h in papilla formation around 11 h after inoculation, which also is the approximate time of penetration (Assaad et al., 2004; Kwon et al., 2008). Silencing of the VAMP721/VAMP722 proteins also results in increased Bgh penetration rates (Kwon et al., 2008).

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The role of SNAP in penetration resistance was documented in barley by silencing the SNAP33 ortholog, HvSNAP34 (Collins et al., 2003; Douchkov et al., 2005). The barley ortholog of PEN1, ROR2 (REQUIRED FOR mloSPECIFIED RESISTANCE 2), has been identified in a mutant screen for loss of mlo (mildew resistance locus O) resistance against Bgh (Collins et al., 2003; Freialdenhoven et al., 1996). In barley, Bgh cannot penetrate the host successfully in the absence of the MLO protein. However, the mlo ror2 double mutant has an intermediate penetration rate, demonstrating the importance of ROR2 in penetration resistance in this host–pathogen interaction. In the same way, a mutation in the PEN1 gene in Arabidopsis attenuates penetration resistance against the host powdery mildew fungus, Gc, in specific longitudinal epidermal cells along the leaf midrib (Zhang et al., 2007). The ROR2 gene is also required for nonhost penetration resistance to the wheat powdery mildew fungus (Trujillo et al., 2004). This illustrates that nonhost and host resistance have shared mechanisms in this type of PTI. The mlo resistance is the most commonly used powdery mildew resistance in barley production. Although employed for several decades, this broad-spectrum resistance against powdery mildew has not been overcome until now. A serious disadvantage linked to mlo resistance is an early leaf senescence and subsequent yield reduction (Jørgensen, 1992; Panstruga, 2005). It has attracted considerable attention that the barley MLO and ROR2 proteins, and the Arabidopsis PEN1 protein, all focally accumulate at the site of attempted penetration by the powdery mildew fungus (Assaad et al., 2004; Bhat et al., 2005; Kwon et al., 2008; Lipka et al., 2008). However, since mutation of PEN1 causes a delay in papilla formation at around 11 h after inoculation, and the earliest documented focal accumulation of PEN1 is 10–14 h after inoculation (Bhat et al., 2005), the accumulation is likely to occur too late to play a role in penetration resistance. We predict that the plasma membranelocated nondynamic PEN1 can be important for this PTI mechanism. More interesting, perhaps, is the physical interaction between MLO, which is a calmodulin-binding seven-transmembrane protein, and ROR2 syntaxin (Bhat et al., 2005). Even though these two proteins play counteracting roles in penetration resistance, their interaction is likely to hide the explanation for cellular functions of MLO and ROR2. It is noteworthy that Arabidopsis also has MLO genes that when mutated confer penetration resistance. However, due to gene redundancy in Arabidopsis, mlo single mutants do not show much diVerence in penetration resistance. Only the triple mutant, mlo2 mlo6 mlo12, has a strong powdery mildew resistance (Consonni et al., 2006). In addition to PEN1, two other genes (PEN2 and PEN3) have been identified to play a role in nonhost penetration resistance (Lipka et al., 2005;


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Stein et al., 2006). pen2 mutants allow a significantly higher Bgh penetration rate than wild type, but the attenuation of this PTI is less severe than in pen1. PEN2 is not only necessary for resistance against Bgh, but also against other biotrophic pathogens, as well as hemibiotrophs and necrotrophs (Lipka et al., 2005). PEN2 encodes a family 1 glycoside hydrolase and may catalyze the hydrolysis of O- or S-glycosidic bonds of metabolites. Penetration resistance is dependent on this PEN2 enzymatic activity (Lipka et al., 2005). GFP-labeled PEN2 has been shown to localize to peroxisomes, which accumulate at fungal entry sides. A peroxisome-derived product of PEN2 is predicted to have antifungal activity (Lipka et al., 2005). Analysis of a pen1 pen2 double mutant revealed higher fungal entry rates into host cells than in the two single mutants. Therefore, PEN1 and PEN2 appear to be involved in diVerent defense pathways, both contributing to penetration resistance (Lipka et al., 2005). The third gene required for penetration resistance, PEN3, encodes an ABCtransporter (ATP-binding cassette), also called PDR8 (PLEIOTROPIC DRUG RESISTANCE 8). The PEN3 protein is located at the plasma membrane, and like PEN1, accumulates at sites of fungal penetration (Stein et al., 2006). It has been suggested that PEN3 mediates the export of a toxic compound to stop fungal growth. This compound could be the product of PEN2, which is also located closely to sites of fungal attack. Surprisingly, pen3 mutants have higher resistance to the adapted powdery mildew fungus, Gc, and become chlorotic upon infection. This resistance can be explained by a higher level of SA in pen3 and is abolished by introduction of mutant alleles of SA-signaling genes (Stein et al., 2006). B. NONHOST RESISTANCE TO BACTERIA

Mutant studies have shown that the glycerol kinase NHO1 (NONHOST RESISTANCE 1) is required for Arabidopsis nonhost resistance to P. syringae pv. phaseolicola (Pph) (Kang et al., 2003). NHO1 expression is flagellininduced and can be suppressed by at least nine Pseudomonas eVectors and partially by the phytotoxin coronatine (Li et al., 2005). During ETI, induction of NHO1 gene expression is reestablished, leading to resistance. NHO1 overexpression diminishes bacterial growth, highlighting the importance for host bacteria to suppress NHO1 expression (Kang et al., 2003). As discussed below, ETI can play a role in nonhost resistance to bacteria. Therefore, not only PAMP-triggered, but also eVector-triggered expression of NHO1 is likely to be essential in nonhost resistance. As has been known for viruses, it is now proven that bacteria counteract host plant defense by suppressing gene silencing through interfering with

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small RNA pathways. This mechanism plays a role in both host and nonhost resistance to bacteria. Very recent data suggest that bacterial eVectors suppress plant gene silencing by interfering with the microRNA (miRNA) pathway. Most miRNAs act as negative regulators and downregulate gene expression by degradation of complementary mRNA molecules or by inhibition of their translation. A single miRNA can target several mRNAs, often coding for transcription factors (Novina and Sharp, 2004). The first evidence that small RNAs are involved in defense against bacteria was provided in a study where P. syringae, carrying the AvrRpt2 eVector, induced a small interfering RNA (siRNA) in plants carrying the cognate host R gene RPS2. This specific siRNA was able to silence a negative regulator of the RPS2-mediated defense response, thereby enhancing ETI (Katiyar-Agarwal et al., 2006). In a similar manner, the flagellin-derived peptide flg22 induces expression of miRNA393, which represses auxin signaling, consequently enhancing PTI. This also illustrates the role of auxin as a negative regulator of defense responses (Navarro et al., 2006). In another study, miRNA-deficient mutants failed to prevent growth of T3SS-defective virulent P. syringae pv. tomato (Pst), nonhost Pph and even nonpathogenic P. fluorescens and Escherichia coli (Navarro et al., 2008). It was demonstrated that the eVector AvrPtoB decreases miRNA precursor accumulation and that several other eVectors suppress transcriptional activation of a number of PAMP-responsive miRNAs. Finally, it has been observed that attack by several pathogens at once has synergistic eVects on infection. This could be demonstrated by co‐inoculation using either T3SS-defective Pst or Pph together with turnip mosaic virus (TuMV). TuMV induced suppression of siRNAs and miRNAs, thereby reducing nonhost resistance and allowing the nonhost Pph to grow (Navarro et al., 2008). The plant RNA-silencing pathways are dependent on specific members of the ARGONAUTE (AGO) protein family. Studies of the DNA methylation mutant ago4 confirm the involvement of RNA silencing in defense since this mutant has increased susceptibility to host bacteria and decreased resistance against nonhost P. syringae pv. tabaci (Agorio and Vera, 2007). In summary, small RNA-mediated gene silencing appears to play a role in both host and nonhost resistance.

V. WHAT IS THE EXPLANATION FOR NONHOST RESISTANCE? Since downstream defenses are similar in host and nonhost resistance, the discrimination between a host and a nonhost pathogen appears to occur at the stage of recognition or during early signaling. Host pathogen eVectors are


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characterized by their eVectiveness in suppressing host defense, while there are indications in the literature suggesting that nonhost pathogens are unable to deliver appropriate eVectors into the plant in order to suppress defense responses eVectively. Defense signaling is induced immediately upon pathogen recognition by the plant. For example, Arabidopsis expression of the essential defense component NHO1 is rapidly induced upon perception of flagellin (Li et al., 2005). In this case, host and nonhost pathogens can be distinguished by their ability to suppress NHO1 induction by eVector molecules. Unlike host Pst bacteria, nonhost Pph bacteria are incapable of suppressing NHO1 expression (Kang et al., 2003; Li et al., 2005). The importance of eVectors for the suppression of plant defense mechanisms was also demonstrated in another study. It was shown that Pph was able to proliferate moderately when co‐inoculated with Pst on Arabidopsis, since Pst suppresses Pph-activated defense responses (Ham et al., 2007). Furthermore, it was shown that eVectors AvrRpm1 and HopM1 from Pst are able to suppress plant defense, and that Arabidopsis expressing AvrRpm1 allows moderate growth of Pph (Ham et al., 2007). This clearly illustrates that an appropriate set of eVectors can turn a nonhost pathogen into a host pathogen. It is known that a given pathogen might use a few dozen eVectors to evade or suppress defense signaling and to promote virulence. This, together with the fact that heterologous expression of a single eVector from Pst did not result in maximum growth of the nonhost bacterium Pph, implies that adaption of one eVector alone is not suYcient to turn a nonhost pathogen into a host pathogen when the evolutionary distance between host and nonhost pathogen is large. On the other hand, defense-signaling-deficient Arabidopsis mutants, such as sid2 and pad4, are still resistant to Pph, as no elevated growth can be observed (Ham et al., 2007; Mishina and Zeier, 2007; Van Wees and Glazebrook, 2003). However, heterologous expression of single Pst eVectors in Pph enables enhanced proliferation of this nonhost bacterium on defense-signaling-deficient mutants of Arabidopsis. Moreover, Arabidopsis becomes susceptible to Pph, with proliferation rates comparable to those observed for Pst on wild-type plants, when several diVerent signaling pathways are disrupted simultaneously (Ham et al., 2007). In theory, the expression of several heterologous eVectors in a nonhost pathogen would lead to the same results as the disruption of multiple defense-signaling pathways. Pph obviously holds potent eVectors against target proteins in its host, bean, but its inability to suppress defense responses in Arabidopsis renders Pph a nonhost bacterium for this plant species (Ham et al., 2007). A similar situation is seen for powdery mildew fungi, where disruption of PTI increases the susceptibility of Arabidopsis to nonhost fungi (Lipka et al., 2005;

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Wiermer et al., 2005; Yun et al., 2003; Zimmerli et al., 2004). The fact that the host powdery mildew fungus, Gc, is able to penetrate at a very high rate, most likely reflects that it expresses eVectors that eYciently suppress PTI in the form of PEN gene-mediated defenses. There is evidence in the literature that some nonhost pathogens fail to infect a plant because their eVectors are recognized by the plant’s R proteins, which results in HR (Thordal-Christensen, 2003). For instance, three diVerent Avr gene products from Pst are recognized in the nonhost soybean (Kobayashi et al., 1989) and an Avr gene product from Pph is recognized in the nonhost pea (Arnold et al., 2001). In each case, the products activate HR only in nonhost plants with the corresponding R genes. A study in tomato documents the importance of the R genes Pto and Prf for resistance against 10 diVerent nonhost pathovars of P. syringae. It showed that pto and prf mutant plants allow moderate bacterial proliferation and display disease symptoms upon bacterial infiltration (Lin and Martin, 2007). There are also indications from plant–powdery mildew fungus interactions that ETI plays a role in nonhost resistance. This may, for instance, explain the formae speciales concept. It has been possible to cross wheat and rye powdery mildew isolates, which showed that the rye isolate has six Avr genes corresponding to known R genes in wheat (Matsumara and Tosa, 1995). Lipka et al. (2005) also showed that attenuation of ETI is important for resistance against nonhost powdery mildew fungi in Arabidopsis. Combining mutant alleles of PAD4 and SAG101 with a pen2 mutation reduces single cell HR and allows proliferation of barley and pea nonhost powdery mildew fungi to an extent not seen with the pen2 mutation alone. This stresses the additional importance of multiple gene-for-gene interactions that simultaneously contribute to nonhost resistance. Activation of R gene-mediated resistance in nonhosts probably occurs due to homology between eVector molecules in related pathogens (Lin and Martin, 2007; Lindeberg et al., 2006). For example, Arabidopsis recognizes an eVector protein homologous to AvrRpt2 from the nonhost pathogen Erwinia amylovora, which normally causes disease on rosaceous plants such as apple and pear (Zhao et al., 2006). Similarly, the soybean R gene product Rpg1-b recognizes AvrB from Pst, which is a nonhost pathogen on this plant (Ashfield et al., 2004). In the Pst host plant, Arabidopsis, AvrB is recognized by the corresponding R protein RPM1. The respective R genes are not related, meaning that AvrB recognition has developed convergently in the host and in the nonhost plant (Ashfield et al., 2004). Since nonhost resistance is durable and diYcult to overcome, most plants remain healthy. The outcome of whether a pathogen will be successful in colonizing a plant relies on the interplay between plant receptors for both


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PAMPs and eVectors, and the eVectors’ ability to suppress or evade defense responses and to avoid recognition. Essentially, nonhost resistance is the consequence of co‐evolutionary specialization of a given pathogen on its host plant. During their co‐evolution with host plants, pathogens have been challenged with one obstacle at the time, which has made it possible for them to develop an eVector to overcome this obstacle. In this way, an array of obstacles has accumulated in the host, all of which have been overcome by their pathogens. The nonhost pathogen, on the other hand, is hopelessly behind in the race with the nonhost plant, in that it has not developed the necessary eVectors in pace with the evolving defense mechanisms in the plant. Meanwhile, having to develop more eVectors simultaneously is an insurmountable task, which stabilizes the nonhost plant-pathogen situation. ETI is apparently a diVerent molecular level that contributes to nonhost resistance. EVectors in nonhost pathogens appear to maintain an ability to trigger ETI. This may also be explained by evolution. Some eVectors are likely to be maintained due to their role in targeting the host plant. However, in host plants, pathogens are forced to modify certain characteristics in the individual eVector in order not to cause R-protein recognition, and at the same time maintain suppression of PTI. Since there is no selection pressure against R-protein recognition in nonhost plants, resistance can remain intact. This could be the way AvrRpt2EA from Erwinia amylovora has maintained the same ability to trigger Arabidopsis ETI, mediated by the R-protein RPS2, as its Pst homolog AvrRpt2 (Zhao et al., 2006). It will be interesting to see in the future whether the ‘‘decoy model’’ plays a role in these aspects of nonhost resistance.

VI. LESION-MIMIC MUTANTS LMMs are plants that develop spontaneous lesions without pathogen infection, stress, or injury. The first LMMs were found in maize and were named ‘‘disease mimic mutants’’ (NeuVer and Calvert, 1975), which illustrates the link between LMMs with a genetic cause, and diseases caused by pathogens. Most LMMs have an elevated resistance to pathogens with a biotrophic lifestyle. They have elevated SA levels, and defense markers are upregulated (Lorrain et al., 2003). This makes LMMs an excellent tool for analysis of pathogen defense mechanisms. The lesion-mimic phenotype of LMMs is the consequence of defects in genes controlling programmed cell death (PCD). PCD is an active form of cell death, orchestrated by an internal cellular program. Introducing mutations from the SA pathway can rescue the lesion-mimic phenotype of

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many LMMs. Therefore, LMMs are used by several research groups to uncover novel aspects in defense signaling. Examples of prominent LMMs include lsd1 (Dietrich et al., 1997), acd2 (Mach et al., 2001), acd6 (Lu et al., 2003), acd11 (Brodersen et al., 2002), cpr22 (constitutive expresser of PR genes 22) (Yoshioka et al., 2006), ssi2 (suppressor of salicylic acid insensitivity 2) (Shah et al., 2001), and pen1 syp122 (Zhang et al., 2007). Also other mutants with constitutively activated defense pathways are studied, which do not necessarily lead to cell death. These include dnd1 (defense, no death 1) (Clough et al., 2000), dnd2 (Balague´ et al., 2003; Jurkowski et al., 2004), mpk4 (map kinase 4) (Petersen et al., 2000), cpr1 (Bowling et al., 1994), and snc1 (suppressor of npr1-1, constitutive 1) (Zhang et al., 2003).

VII. MUTANT SCREENS WITHOUT PATHOGENS FOR FINDING GENES IN DEFENSE SIGNALING Many defense-signaling genes have been found by screening mutated populations of wild-type plants following pathogen inoculation, and searching for less resistant mutants. However, by remutating LMMs and other mutants with constitutively active defense signaling, mutants with deficient signaling can be found simply by searching for individuals with improved performance. This eliminates the need for laborious pathogen inoculations. Below, three such strategies are described. Some of the mutations that rescue LMM plants have occurred in genes previously known to be involved in pathogen defense. Such mutations validate the method of the suppressor screen. Others have not previously been connected to pathogen defense, which demonstrates the value of this alternative approach in enriching our understanding of pathogen defense signaling. A. SSD MUTANTS

The closest homolog of the Arabidopsis PEN1, required for penetration resistance to the nonhost powdery mildew, Bgh, is SYP122. syp122 mutant plants show no phenotype regarding penetration resistance. However, the pen1 syp122 double mutant has an LMM phenotype. It is dwarfed and necrotic, and it has elevated SA and PR-1 transcript levels (Assaad et al., 2004; Zhang et al., 2007). The pen1 syp122 double mutant also has the reduced penetration phenotype against Bgh, like the pen1 single mutant, but it is resistant against the host powdery mildew, Gc. By introducing the known mutations in the SA pathway, sid2, eds5, and npr1, or the bacterial gene NahG, the LMM phenotype was partially rescued (Zhang et al., 2007).


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Mutations in the JA ( jar1 ( jasmonate resistant 1), coi1 (coronatine insensitive 1)) and ethylene (ein2 (ethylene insensitive 2)) pathways could not rescue the dwarfed and necrotic phenotype (Zhang et al., 2008). pen1 syp122 double-mutant plants were remutagenized, and in a screen for rescued phenotype, genes contributing to the lesion-mimic phenotype were identified. These genes were named SUPPRESSORS OF SYNTAXINRELATED DEATH (SSD) (Fig. 2). Complementation analysis of the obtained pen1 syp122 ssd triple mutants identified novel alleles of SID2 and EDS5. Furthermore, map-based cloning has revealed the identity of four other SSD genes. These turned out to be the already known defense-signaling genes FMO1, ALD1, PAD4 (Zhang et al., 2008), and PBS3 (S. M. Mørch, C. Pedersen, A. Lenk, Z. Zhang and H. Thordal-Christensen, unpublished data). In all of these genes, we have identified several novel mutant alleles. In addition to the six defense-signaling genes mentioned, mutant alleles of six others have been demonstrated to rescue pen1 syp122. Of these, EDS1, NDR1, RAR1, and SGT1b are known (Zhang et al., 2008), while two are not hitherto implicated in defense signaling (C. Pedersen, M. X. Andersson, Z. Zhang, A. Lenk and H. Thordal-Christensen, unpublished data). It is presumed that the SSD genes in their active form contribute to lesion formation. By crossing the rescued triple mutants, and combining mutations in diVerent SSD genes, it is possible to study the relationship of these SSD genes and to draw statements relating to the signaling network. In the pen1 syp122 background, quadruple and quintuple mutants were obtained. When a quadruple mutant performs significantly better than the parent triple mutants, it indicates that the mutations in SSD genes contribute additively to the rescuing of pen1 syp122. This, in turn, suggests that these SSD genes are on diVerent signaling pathways leading to the lesion-mimic phenotype.

Col-0

pen1 syp122

pen1 syp122 ssd1

Fig. 2. Suppression of the pen1 syp122 lesion-mimic mutant (LMM) phenotype by ssd1. Morphology of Arabidopsis Columbia (Col-0) wild type, syntaxin double mutant pen1 syp122, and rescued triple mutant pen1 syp122 ssd1 plants after 4 weeks of growth.

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On the other hand, when the quadruple mutant performs as the triple mutants, then the genes are on the same signaling pathway. By this signaling-network analysis, plant size permits statements about the relationship of proteins on defense-signaling pathways to be made. As an example, a cross was made between pen1 syp122 eds5 and pen1 syp122 sid2, and the quadruple mutant pen1 syp122 eds5 sid2 was generated. Remarkably, this quadruple mutant performed significantly better than the parental triple mutants, even though the eds5 and sid2 mutations both largely prevent SA accumulation. This observation demonstrates that EDS5 and SID2 are involved in diVerent SA-independent processes, illustrating that a more complex network exists in association with SA signaling (Zhang et al., 2008). npr1 mutants are hypersensitive to exogenously applied SA and show a bleaching phenotype (Cao et al., 1997). When spraying older plants with SA, stem and leaves turn white and the npr1 genotype can be determined by eye. Because of the high level of SA in the syntaxin double mutant, the triple mutant pen1 syp122 npr1 has a spontaneous bleaching phenotype. Additional knockout of, for example, sid2 causes the bleaching to disappear, while in the quadruple mutant with npr1 and ndr1 bleaching still occurs. This confirms that NDR1-mediated signaling is SA independent. Therefore, the npr1 bleaching phenotype allows statements about the involvement of SA in signaling processes and can be used as a simple and uncomplicated tool (Zhang et al., 2008). B. SFD MUTANTS

The npr1 mutant is SA insensitive and has reduced defense responses. However, remutagenesis of npr1 plants led to the identification of the mutation ssi2, which reestablishes the defense responses. The npr1 ssi2 double mutant and ssi2 single mutant are lesion-mimics due to a constitutive NPR1-independent defense pathway (Shah et al., 2001). SSI2 encodes a stearoyl-acyl carrier protein desaturase that catalyzes the conversion of stearic acid to oleic acid in plastids. ssi2 mutant plants possess altered fatty acid compositions (Kachroo et al., 2001). This shows that fatty acid desaturation is involved in defense responses. In a suppressor screen in the ssi2 npr1 genetic background, a number of sfd (suppressor of fatty acid desaturase deficiency) mutants were found that suppress the ssi2 LMM phenotype (Nandi et al., 2004). SFD1 is involved in the synthesis of plastidial glycerolipids and the sfd1 mutant is impaired in SAR, but its local defense responses are not significantly diVerent from wild type (Nandi et al., 2004). sfd4 is a mutant allele of the FAD6 gene that encodes an !6-desaturase that is involved in the synthesis of plastidial lipids containing polyunsaturated fatty acids (Nandi et al., 2003). The SFD2 gene is not yet cloned, but it confers


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altered fatty acid composition as in the other sfd mutants. All three sfd mutant alleles suppress the LMM phenotype and the increased resistance of ssi2 toward P. syringae pv. maculicola (Nandi et al., 2003). C. MOS MUTANTS

A mutation in the TIR-NB-LRR-type R gene SNC1 renders it constitutively active, and snc1 mutant plants are resistant to virulent pathogens (Zhang et al., 2003). In a suppressor screen of snc1, genes were identified with functions downstream of R genes without the use of pathogens (Zhang and Li, 2005). Mutations in seven MOS (MODIFIER OF snc1) genes have been found to rescue the snc1 phenotype. Three of these genes encode components of the nuclear traYcking machinery. MOS6 is an importin 3, necessary for the import of specific proteins across the nuclear envelope (Palma et al., 2005). MOS3 and MOS7 are nucleoporins and are involved in the transport of RNA, proteins, ribonucleoprotein particles, and other cargo across the nuclear envelope (Wiermer et al., 2007; Zhang and Li, 2005). At the same time, other discoveries connecting nuclear traYcking and plant innate immunity have been made. There are a few examples showing that R-proteins are imported into the nucleus. It was shown that the tobacco TIR-NB-LRRtype R-protein N functions inside the nucleus, and also the barley CC-NBLRR-type R-protein MLA10 shuttles between the cytoplasm and the nucleus, where it turns on defense (Burch-Smith et al., 2007; Shen et al., 2007; Wiermer et al., 2007). Whether the MOS proteins are required for transport of autoactivated snc1 or downstream components into the nucleus, still remains to be clarified. Other MOS genes include MOS2, that encodes a protein showing RNA-binding activity and acting inside the nucleus, and MOS4, which encodes a protein of a DNA-binding complex, probably controlling transcription of defense regulators (Palma et al., 2007; Zhang et al., 2005). This DNA-binding complex consists of two other compounds, AtCDC5 (a transcription factor) and PRL1 (PLEIOTROPIC REGULATORY LOCUS 1). Both are involved in pathogen resistance in Arabidopsis. An Atcdc5 mutation rescues the snc1 phenotype, and MOS4, AtCDC5, and PRL1 all are required for R-protein-mediated resistance of both CC- and TIR-NB-LRR-types (Palma et al., 2007). MOS5 and MOS8 are involved in protein ubiquitination and farnesylation, respectively (Goritschnig et al., 2007, 2008). Nearly all mos mutant alleles suppress the constitutive defense responses in snc1 completely and confer enhanced disease susceptibility toward oomycete and bacterial pathogens. mos6 only partially rescues the snc1 phenotype and confers moderately reduced resistance toward an oomycete pathogen, but no altered resistance to bacterial pathogens.

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VIII. CONCLUSION We have summarized current data relating to nonhost resistance and believe that the diVerence between host and nonhost resistance lies in the ability of eVectors to suppress activated defenses, and in the extent to which eVectors are recognized by R-proteins. Mutations identified in screens for nonhost resistance have turned out to expose downstream-signaling events. Defense needs to be under tight control, not only to protect healthy cells from damage, but also to conserve plant resources. A number of mutant lines exhibit autoactivated and permanent defense responses. Some of these are LMMs which suVer from uncontrolled defense, leading to unintended cell death. However, studying these plant systems can deepen our understanding of plant cell death, responses to pathogens, and defense mechanisms, without the need of pathogens. Some LMM mutant screens identify genes in specific processes, others in more general mechanisms. Depending on the starting material, outputs of screens have varied and resulted in, for example, novel genes involved in lipid saturation or transport across the nuclear envelope, which have now been linked to pathogen defense. Others, such as the SSD genes, are more generally involved in signaling pathways. By using LMM as a tool, additional genes in defense signaling will reveal new insights into the large field of pathogen–plant interactions.

ACKNOWLEDGMENTS We are grateful to Drs. Dale Godfrey and Ingo Lenk for critically reading the manuscript. Andrea Lenk was supported by a grant from the Faculty of Life Sciences, University of Copenhagen.

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Action at a Distance: Long-Distance Signals in Induced Resistance

MARC J. CHAMPIGNY* AND ROBIN K. CAMERON{,1

*Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, B.C., Canada V5A 1S6 { Department of Biology, McMaster University, Hamilton, Ontario, Canada L8S 4K1

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Time to Flower—Signaling Events in the Vegetative to Flowering Transition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Flowering Time as a Model for Long-Distance Signaling ............. B. Control of Flowering Occurs in Distinct Stages ......................... C. The Long-Distance Flowering Signal is Phloem Mobile and Highly Conserved ....................................................... D. Candidates for the Floral Long-Distance Signal—The Identity of ‘‘Florigen’’ ..................................................................... E. Salicylic Acid and Flowering—Convergence of Signaling Mechanisms?................................................... III. Mechanisms of Signaling During the Wound Response . . . . . . . . . . . . . . . . . . A. Role of Systemin in Systemic Wound Signaling ......................... B. Wound-Response Mutants are Deficient in the Biosynthesis or Perception of JA, or in Systemin Functioning........................ C. Systemin and JA Production in Wounded Leaves and JA Perception in Distant Tissue.............................................................. D. JA Biosynthesis Occurs in the Sieve Element/Companion Cell Complex ..................................................................

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0065-2296/09 $35.00 DOI: 10.1016/S0065-2296(09)51004-X

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M. J. CHAMPIGNY AND R. K. CAMERON

E. JA-Mediated Wound Response is Modulated by Other Signals ...... F. Mechanism of JA Action on Effector Genes............................. Long-Distance Signaling in SAR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. SAR Develops in Distinct Stages .......................................... B. Role of SA and NPR1 in SAR ............................................. C. SAR Signal Transport ....................................................... D. Candidates for the SAR Long-Distance Signal .......................... E. Other Genes Involved in SAR Long-Distance Signaling............... F. Role of ET in SAR Long-Distance Signaling ............................ G. SAR Long-Distance Signaling Across Species ........................... Systemic Induced Susceptibility (SIS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Signaling During ISR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Induction of ISR.............................................................. B. Signal Perception and Priming During the Development of ISR ..... Techniques to Further Elucidate Long-Distance Signaling. . . . . . . . . . . . . . . Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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ABSTRACT Plants perceive environmental changes in one tissue and inform distant tissues of these changes using long-distance signals. These signals are implicated in developmental processes such as the transition to flowering as well as in responses to abiotic and biotic stresses, including pathogen infection and herbivory. In this review, we discuss research findings in the regulation of flowering time as well as in induced defense responses from the perspective that long-distance signaling progresses in a series of distinct stages: (1) initiation or induction, (2) synthesis and movement of a signal to distant tissues, (3) perception of the signal, and (4) establishment or manifestation of the appropriate response. We highlight recent studies that implicate DIR1 and lipids, methyl salicylate, and jasmonic acid (JA) as long-distance signals during systemic acquired resistance (SAR). Additionally, it appears that a requirement for JA is common to the SAR, induced systemic resistance, and wound response pathways. Finally, we discuss future avenues of research to further elucidate the mechanisms of long-distance signaling in plants.

I. INTRODUCTION Plants are rooted to the earth and therefore cannot escape from adverse environmental conditions. Instead, they have evolved sophisticated mechanisms that allow them to survive both biotic and abiotic environmental stress. Plants respond locally at the individual cell level and systemically in distant tissues. The environmental condition is perceived in one part of the plant followed by transport of this information to distant tissues to alert the entire plant. Long-distance signaling pathways are often divided into a number of stages. The first stage has been termed induction or initiation in response to the stress in the initially affected tissue and production of (a) long-distance signal(s). Secondly, the signal(s) move(s) from the induced or initial tissue to distant tissues via the vascular system, air, or cell-to-cell movement.

ACTION AT A DISTANCE

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Thirdly, the distant tissues must perceive or interpret the newly arrived longdistance signal(s) and set in motion the appropriate response. A number of plant long-distance signaling pathways are being studied, including perception of nutrient limitation (reviewed in Forde, 2002) and drought stress in the roots followed by signaling to the shoots to produce the appropriate response (reviewed in Schachtman and Goodger, 2008). In this review, the most thoroughly studied long-distance signaling pathway, the photoperiodic flowering pathway will be discussed in relation to long-distance signaling in four defense response pathways (wound response, induced systemic resistance (ISR), systemic acquired resistance (SAR), systemic induced susceptibility (SIS)).

II. TIME TO FLOWER—SIGNALING EVENTS IN THE VEGETATIVE TO FLOWERING TRANSITION A. FLOWERING TIME AS A MODEL FOR LONG-DISTANCE SIGNALING

The transition between vegetative and reproductive growth is a critical developmental switch that is tightly regulated in plants to ensure that production and dispersal of seeds occur in environmental conditions most favorable for reproductive success. Different plant species utilize a variety of autonomous and environmental cues to trigger this transition to flowering, including the developmental stage of the plant, amount of rainfall, temperature, and photoperiod—the relative duration of light in a single day (Hastings and Follett, 2001). The contribution of photoperiod to flowering time was first reported early in the twentieth century with the observation that flowering in spinach occurred when the leaves were in long-day (LD) conditions and the shoot apex in short-day (SD) conditions, but not vice versa (Knott, 1934). Based on grafting experiments, Chailakhyan (1936) proposed the existence of ‘‘florigen,’’ a photoperiod-dependent, graft-transmissible signal that travels from leaves to the shoot apex to promote flower development. More recent work has enriched the simple florigen concept to include multiple mobile signals that may be involved in inhibiting flowering as well as promoting floral initiation (Bernier et al., 1993; Pe´rilleux and Bernier, 2002). The mechanism of florigen translocation and investigation into its identity have become valuable research models to understand long-distance signaling in plants. B. CONTROL OF FLOWERING OCCURS IN DISTINCT STAGES

Control of flowering time has historically been divided into a series of discrete steps: (i) induction of a floral signal within the leaves, (ii) commitment to flowering at the shoot apical meristem (SAM), and (iii) reprogramming of

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the meristem for floral morphogenesis (Pe´rilleux and Bernier, 2002; Sua´rezLo´pez, 2005). The broad mechanisms involved in the control of flowering time clearly have much in common with the induced defense responses, which progress in a similar series of steps that we and others have highlighted— induction of a signal, translocation of the signal to distant tissues, and perception of the signal, culminating in manifestation of the defense response. The primary, or induction, stage in the transition to flowering occurs in leaves. Multiple experiments have demonstrated that applying the inductive photoperiod only to leaves can induce flowering (Hempel et al., 2000; Zeevaart, 1976). Most tellingly, grafting of a single induced leaf was sufficient to elicit flowering in Perilla (Zeevaart, 1985). In many species, sensing of photoperiod can occur in immature leaves. Defoliation experiments established that the floral signal was produced in cotyledons of Impatiens balsamina (Pouteau et al., 1997) and Chenopodium rubrum (King, 1972). In addition, Arabidopsis thaliana grown in LD conditions flowers before any rosette leaf reaches maturity (Bradley et al., 1997). There exists great species variability in the observed capacity of the SAM to commit to flowering. Lolium and Xanthium were induced to flower after exposure to a single inductive photoperiod (Zeevaart, 1976), but other species such as soybean and some cultivars of Impatiens reverse the flowering process if inductive conditions cease (Pouteau et al., 1997; Washburn and Thomas, 2000). In most cases, it has not yet been determined whether the observed variation in commitment to flowering is due to species differences in the dosage of the flowering signal, or a change in the ability of the SAM to perceive the signal. In one case, I. balsamina becomes irreversibly committed to flowering because its leaves constitutively produce the flowering signal after induction (Tooke et al., 1998). C. THE LONG-DISTANCE FLOWERING SIGNAL IS PHLOEM MOBILE AND HIGHLY CONSERVED

The physiological experiments described above clearly demonstrated that the flowering signal is graft-transmissible, suggesting that it travels through the vasculature. Further studies established that the signal is phloem mobile because the speed of signal movement out of induced leaves, as well as the pattern of signal movement, was similar to the transport of photoassimilates (Zeevaart, 1976). Clever intercultivar and interspecies grafting experiments led to the suggestion that flowering signals must be very similar or identical in higher plants (Zeevaart, 1976). For example, grafting of day-neutral tobacco scions onto rootstocks of SD tobacco or rootstocks of the LD Nicotiana sylvestris caused acceleration of flowering when the rootstocks were exposed


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to the appropriate inductive conditions (Lang et al., 1977). The same signal(s) produced in response to an inductive photoperiod may participate both in the transition to flowering at the shoot apex and in the transition to tuberization (Sua´rez-Lo´pez, 2005), as grafted tomato scions promoted tuberization of potato rootstocks only when the scions were induced to flower (Chailakhyan et al., 1981). D. CANDIDATES FOR THE FLORAL LONG-DISTANCE SIGNAL—THE IDENTITY OF ‘‘FLORIGEN’’

Two contrasting approaches have been employed to uncover the molecular nature of the flowering signal. The first approach involved the physiological study of compounds transmitted to the shoot apex in response to changing photoperiod. The second approach, largely carried out in the genetic model Arabidopsis, was to identify and characterize mutants that showed either accelerated or delayed flowering time in response to various environmental stimuli. 1. Sucrose, cytokinins, and gibberellins Several different plant hormones and small molecules have been proposed as candidates for the floral stimulus, largely on the basis of their ability to promote flowering at the SAM or to complement the deficiencies of various flowering mutants. Among cytokinins, gibberellins, and sucrose, there is widespread agreement that gibberellins promote flowering in several different species (Pe´rilleux and Bernier, 2002). Although cytokinins accumulated at the shoot apex of Arabidopsis plants induced to flower (Jacqmard et al., 2002), exogenous application of cytokinins did not induce flowering in Arabidopsis or in Sinapis alba (Bonhomme et al., 2000). Sucrose was also found to accumulate in Arabidopsis phloem exudates collected from photoperiod-induced leaves (Bernier et al., 1993; Corbesier et al., 1998) and this transient increase in phloem sucrose concentration preceded cell division at the SAM (Corbesier et al., 1998). However, high levels of sucrose delayed flowering in wild-type Arabidopsis and rescued the deficiency of several important late-flowering mutants but not others (Ohto et al., 2001), making the precise role of sucrose in flowering promotion difficult to define. The role of gibberellins as flowering signals has been best studied in the LD plant Lolium temulentum, in which gibberellins are synthesized in the leaves and large increases in gibberellin levels are observed at the shoot apex, suggesting the long-distance movement of gibberellins to promote flowering (King and Evans, 2003; King et al., 2006). Gibberellins are also involved in the transition to flowering that eventually occurs under SD conditions in the

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quantitative LD plant Arabidopsis (Eriksson et al., 2006). Gibberellins are not, however, a universal floral stimulus, as their activity was not required in several species studied such as S. alba (Corbesier et al., 2004), and can inhibit flowering in others (Bernier, 1988; Zeevaart, 1976).

2. Characterization of genes involved in the regulation of flowering time Much of our knowledge concerning the genetic control of flowering has come from analysis of Arabidopsis mutants exhibiting premature or delayed flowering in response to environmental stimuli such as vernalization and photoperiod. To date, the photoperiod-dependent pathway is the best understood. For interesting discussions of genes involved in other pathways and crosstalk between pathways, refer to several recent reviews (Corbesier and Coupland, 2006; Imaizumi and Kay, 2006; Zeevaart, 2008). Photoperiod is perceived in leaves and several genes have been identified that regulate the transition to flowering in terms of their ability to discriminate light quality or intensity. GIGANTEA (GI) encodes a nuclear protein of unknown biochemical function (Fowler et al., 1999; Park et al., 1999), but overexpression of GI promoted early flowering (Mizoguchi et al., 2005). GI regulates flowering time in part by regulating the expression of a zincfinger transcription factor called CONSTANS (CO) (Sua´rez-Lo´pez et al., 2001). Transcription of GI and CO is regulated by the circadian clock: mRNA abundance of each gene is highest at approximately 12 h after dawn when plants are grown in LD conditions (16 h light) (Park et al., 1999; Sua´rez-Lo´pez et al., 2001). In addition, CO is controlled at the posttranslational level, such that the protein is rapidly degraded by proteasome-targeted ubiquitination, but stabilized through a mechanism involving signaling through phytochrome A and cryptochrome photoreceptors (Valverde et al., 2004). These complex regulatory steps result in CO protein accumulation only during long days and its degradation in the absence of light, pointing toward a mechanism through which CO promotes flowering only in long days. CO and an additional important floral regulator, FLOWERING LOCUS T (FT), are expressed in phloem companion cells of leaves (An et al., 2004; Corbesier and Coupland, 2006; and see below). FT encodes a protein similar to animal RAF kinase inhibitors (Kobayashi et al., 1999). FT is thought to be the major target of CO in leaves because expression of only this gene was highly upregulated in wild-type plants compared to co mutants grown in long days (Wigge et al., 2005). Moreover, CO-dependent regulation of flowering time required FT because expression of FT by companion cell-specific promoters rescued the late-flowering co phenotype (An et al., 2004).


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A fascinating spatial difference between the site of CO and FT transcription and the site of FT action was observed in studies expressing these genes under tissue-specific promoters. Expression of CO in companion cells of leaf minor veins using the minor vein-specific GAS1 promoter (Ayre and Turgeon, 2004), or in major veins using the phloem-specific SUC2 promoter (An et al., 2004) complemented the co-1 mutation, but CO expression at the SAM had no effect on flowering. Conversely, FT induced flowering when expressed in epidermal cells, companion cells, or in meristematic tissue (An et al., 2004). These results collectively suggested that the physiological role of CO is to activate transcription of FT, whose activity is required at the shoot apex. FT interacts with the bZIP transcription factor FLOWERING LOCUS D (FD) to activate transcription of the floral meristem-identity gene APETALA1 (AP1) and of SUPPRESSION OF OVEREXPRESSION OF CO1 (SOC1), thereby reprogramming the shoot apex for floral initiation (Abe et al., 2005; Wigge et al., 2005). 3. FT protein is phloem-mobile Having established that phloem-specific expression of CO induced expression of FT (Takada and Goto, 2003) and that FT acts at the shoot apex to promote flowering (Wigge et al., 2005), many studies have been conducted to understand the spatial discrepancy between the site of FT synthesis and the site of FT action. An early report suggested that production of FT mRNA in transgenic Arabidopsis leaves by activation of a heat-shock promoter led to detection of FT mRNA at the shoot apex (Huang et al., 2005), implicating the mRNA as the long-distance signal. This was a timely hypothesis, as several mRNAs, small RNA molecules and viruses have been reported to travel long distances in the phloem (Lough and Lucas, 2006). However, detection of FT mRNA in the phloem could not be reproduced and the original report was retracted (Bo¨hlenius et al., 2007a,b). Additional grafting experiments failed to establish that rootstock-produced FT mRNA molecules crossed into the scion and message encoding a translational fusion of FT with a green fluorescent protein (GFP) tag was not detected either in grafted Arabidopsis ft-7 mutant shoots (Corbesier et al., 2007). Additionally, messages encoding SINGLE FLOWER TRUSS (SFT), the tomato orthologue of FT, did not cross a graft junction (Lifschitz et al., 2006). The presence of FLOWERING LOCUS T-LIKE mRNA was not detected by RT-PCR in phloem sap collected from Cucurbita moschata (Lin et al., 2007). Finally, in an elegant complementary experiment, tissue-specific reduction of FT using artificial microRNAs established that FT mRNA is dispensable at the SAM (Mathieu et al., 2007). These data firmly established that FT mRNA was not the phloem-mobile floral stimulus.

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Additional studies strongly supported a model in which FT protein translocates from its site of synthesis in leaf phloem cells to the SAM. For example, experiments in which a FT:GFP fusion protein was expressed in Arabidopsis companion cells under the control of the SUC2 promoter revealed that the GFP signal could be detected both in the phloem and at the shoot apex (Corbesier et al., 2007). Similar results were obtained using Myc–FT fusions in Arabidopsis (Jaeger and Wigge, 2007) and GFP fusions with a rice FT orthologue (Hd3a-GFP) (Tamaki et al., 2007). One could argue that these results are not physiologically relevant for several reasons. First, FT is a low abundance protein and exogenous expression of FT using phloem-specific promoters may not reflect its natural movement. Second, diffusion of small proteins from companion cells into sieve elements is widespread (Lough and Lucas, 2006) and may explain the observed results. These objections were partially resolved by the observation that the GFP fusions mentioned above were biologically active; that is, phloem-specific expression of FT–GFP fusions in leaves promoted early flowering. Furthermore, native FLOWERING LOCUS T, a cucurbit orthologue of FT, could be identified by mass spectrometry in phloem sap collected from Cu. moschata (Lin et al., 2007). Elegant experiments were performed to demonstrate that the physical movement of FT protein from leaves to the shoot apex is a necessary and sufficient condition for flowering. In these studies, FT or an FT–GFP fusion protein was expressed via the minor vein-specific GAS1 promoter and the ability of these constructs to rescue the late-flowering phenotype in ft mutant grafted scions was assessed. Surprisingly, only FT on its own resulted in early flowering, suggesting that the FT–GFP protein was too large to move into minor veins. However, the fact that FT could rescue the ft late-flowering phenotype in these experiments meant that FT traveled a considerable distance through a graft junction (Corbesier et al., 2007). Similar experiments employed an FT protein fused to tandem yellow fluorescent protein (YFP) molecules under the direction of the phloem-specific SUC2 promoter. In this case, FT:3xYFP did not affect flowering time, presumably because it was too large to translocate into sieve elements, but release of FT from the fusion protein using a viral endopeptidase initiated early flowering (Mathieu et al., 2007). 4. FT, a near universal flowering signal: ‘‘florigen’’ revealed In addition to its ability to move in the phloem from induced leaves to the shoot apex and to reprogram the SAM, FT fulfills another key requirement of the florigen hypothesis, namely that it acts as a near universal floral stimulus. In many species, notably Arabidopsis and monocots, grafting


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experiments are technically challenging or physically impossible, respectively. Therefore, recent studies have focused on the ability of overexpressed FT transgenes to promote flowering in many different species. To date, overexpression of Arabidopsis FT or various orthologues promoted accelerated flowering in poplar (Bo¨hlenius et al., 2007a; Hsu et al., 2006), tobacco (Lifschitz et al., 2006), winter wheat (Yan et al., 2006), Arabidopsis (Abe et al., 2005), and Pharbitis nil (Hayama et al., 2007). In similar experiments, FT orthologues from Cucurbita maxima (Lin et al., 2007), rice (Kojima et al., 2002), grapevine (Carmona et al., 2007; Sreekantan and Thomas, 2006), and tomato (Lifschitz et al., 2006) induced early flowering when overexpressed in Arabidopsis. The role of FT as a universal floral stimulus is further supported by reports demonstrating delayed flowering time in distantly related rice and Arabidopsis plants in which the FT orthologue was disrupted by mutation or knock-down methods (Kojima et al., 2002; Komiya et al., 2008; Yamaguchi et al., 2005). Unlike the so-called autonomous and vernalization pathways regulating flowering time (reviewed in Corbesier and Coupland, 2006), the gibberellin pathway does not seem to converge on FT as a floral stimulus. Recent work in Arabidopsis demonstrated that application of gibberellin to either leaves or the apical region of plants grown in SD conditions caused flowering by upregulating expression of LEAFY and SOC1 (Eriksson et al., 2006; Moon et al., 2003), but expression of FT was unaffected. Although LEAFY is highly conserved in plants, exogenously applied gibberellin seems to induce flowering only in the subset of SD plants (Maizel et al., 2005). Further research is required to explain this phenomenon and to explore the redundancy of gibberellin and FT as leaf-produced, phloem mobile floral signals (Zeevaart, 2008). E. SALICYLIC ACID AND FLOWERING—CONVERGENCE OF SIGNALING MECHANISMS?

Some plant species, such as duckweeds, flower in response to treatment with the pathogen defense hormone salicylic acid (SA) (Zeevaart, 1976). Fortuitously, Arabidopsis is an excellent model to study the role of SA in regulating the control of flowering time because flowering is accelerated upon exogenous application of SA and mutants deficient in accumulation of the hormone exhibit delayed flowering (Martinez et al., 2004). SA was reported to affect flowering time by negatively regulating the floral repressor FLC and by stimulating expression of the floral long-distance signal, FT (Martinez et al., 2004). A recent study has shown a convergence for the requirement of the small ubiquitin-like modifier (SUMO) E3 ligating protein AtSIZ1 in regulating

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plant innate immunity as well as flowering time. Arabidopsis siz1 mutants have a pleiotropic phenotype including low tolerance to drought stress (Catala et al., 2007), heightened innate resistance to bacterial pathogens (Lee et al., 2007a), and accelerated flowering (Jin et al., 2008). The accelerated flowering phenotype in siz1 mutants was shown to be SA-dependent as demonstrated by the absence of early flowering in SA-deficient and siz1 double mutants. SIZ1 was proposed to delay flowering by facilitating the sumoylation of FLD, resulting in increased transcription of the floral repressor FLC (Jin et al., 2008). Further research will identify the specific roles of sumoylation in regulating SA-dependent pathogen defense and flowering pathways. In the meantime, these intriguing findings hint at a relationship between the regulation of flowering time and defense against pathogens. This may reflect conservation in long-distance-signaling mechanisms shared between the two processes, but may also simply be an example of SA-mediated cross-talk during the induction stages of flowering and pathogen defense.

III. MECHANISMS OF SIGNALING DURING THE WOUND RESPONSE Plants combat wounding by herbivorous insects and foraging animals with an array of chemical defense strategies. Included among these are a variety of noninducible defenses such as the synthesis of bitter or unpalatable secondary metabolites, including the tannins and saponins found in potato, as well as more toxic compounds, for instance nicotine in tobacco and pyrethrum produced by members of the genus Chrysanthemum. Wounding of several agriculturally important plant species such as corn and cotton results in the induction and emission of volatile substances that attract insect predators. Volicitin, present in oral secretions of feeding beet armyworm caterpillars was found to induce a variety of complex secondary metabolites, including monoterpenes, sesquiterpenes, homoterpenes, lipoxygenase products, and indole (Alborn et al., 1997). Local and systemic production of these volatiles caused increased predation of caterpillars by the parasitic wasp Hyposoter exiguae (Ro¨se et al., 1996; Turlings and Tumlinson, 1992). Members of the Solanaceae exhibit an additional important inducible defense against herbivory. Wounded potato and tomato plants express proteinase inhibitors (PIs or PINs) that limit the ability of foraging insects to extract nutrients by reducing the activity of digestive enzymes in the insect midgut (Green and Ryan, 1972).


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Inducible responses against wounding share the notable feature of being expressed not only in local tissue but also in tissue distant from the site of attack, implicating mechanisms of long-distance communication that may be shared between various species’ wound responses. The discovery of robust PIN expression by wounded tomato plants (Green and Ryan, 1972) has made the tomato leaf a valuable model for the study of signaling events leading to the wound response. Wounding causes the systemic reprogramming of tomato plant tissue resulting in expression of over 20 defense-related proteins, including several classes of PINs, proteinases, and signaling molecules, as well as several mitogen-activated protein kinases (MAPKs) (Ryan, 2000; Stratmann and Ryan, 1997).

A. ROLE OF SYSTEMIN IN SYSTEMIC WOUND SIGNALING

An 18-amino acid peptide hormone, systemin, was purified from wounded tomato leaves and discovered to be a potent inducer of PIN formation (Pearce et al., 1991). This, and the observation that jasmonic acid (JA) biosynthesis via the octadecanoid pathway is required for PIN expression (Farmer and Ryan, 1992) established JA and systemin as key players in the wound response. What roles do these hormones play in the wound response and how do they interact to establish systemic protection against herbivory? Systemin is produced as a specific response to insect feeding or mechanical wounding by the proteolytic cleavage of a 200-amino acid precursor called prosystemin (McGurl et al., 1992). Additional prosystemin-like proteins have been identified in tomato (Pearce and Ryan, 2003) and tobacco (Pearce et al., 2001) and all are cleaved into small peptides capable of inducing PIN expression. Transformed tomato plants expressing an antisense prosystemin cDNA under the direction of the cauliflower mosaic virus 35S promoter were deficient in systemic induction of PIN proteins (McGurl et al., 1992) and were consumed to a greater extent by Manduca sexta larvae (Orozco-Cardenas et al., 1993). Conversely, plants overexpressing prosystemin in its sense orientation demonstrated constitutive production of defenserelated proteins even in the absence of wounding, presumably from ectopic overproduction of the systemin hormone (McGurl et al., 1994). Purified systemin or bacterially expressed recombinant prosystemin applied to freshly wounded tomato stems rescued the deficiency of prosystemin-antisense plants in that the wound response approached wild-type levels in distant tissues (Dombrowski et al., 1999). This discovery, along with the observation that radioactively labeled systemin applied to wounded tomato leaves translocated from the site of application to the leaf petiole, suggested that

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systemin itself may be a crucial long-distance signal to establish the systemic wound response (Narvaéz-Va´squez et al., 2005; Pearce et al., 1991). Biochemical purification of a transmembrane systemin receptor SR160, a leucine-rich-repeat (LRR) receptor kinase (Scheer and Ryan, 2002), provided an important clue as to the requirement for systemin in the wound response. Interestingly, SR160 plays dual roles in the perception of both brassinosteroid and systemin hormones (Montoya et al., 2002; Scheer et al., 2003). Interaction of systemin with its receptor results in a suite of events including depolarization of the plasma membrane (Moyen and Johannes, 1996) and an increase in intracellular Ca2þ (Moyen et al., 1998), as well as activation of MAPKs (Kandoth et al., 2007; Stratmann and Ryan, 1997; Usami et al., 1995) and a phospholipase A2 (Lee et al., 1997; NarvaézVa´squez et al., 1999). The culmination of these biological processes is the release of linolenic acid from membranes and its biochemical conversion via the octadecanoid pathway to the oxylipins 12-oxyphytodienoic acid (OPDA) and JA (Farmer and Ryan, 1992; Schilmiller and Howe, 2005). B. WOUND-RESPONSE MUTANTS ARE DEFICIENT IN THE BIOSYNTHESIS OR PERCEPTION OF JA, OR IN SYSTEMIN FUNCTIONING

To identify additional components of the wound signaling pathway, a number of forward genetic screens were undertaken, searching specifically for mutants that failed to express PINs in response to wounding (Lightner et al., 1993) or failed to respond to the constitutive signal provided by a 35S: PROSYSTEMIN transgene (Howe and Ryan, 1999). Phenotypic analysis of these mutants and cloning of some of their deficient alleles revealed that wound response mutants are defective in distinct classes of biological functions, chiefly the biosynthesis of JA (defenseless, def1, Li et al., 2002a; acylCoA oxidase, acx1, Li et al., 2005; allene oxide cyclase, aoc, Stenzel et al., 2003; !3-fatty acid desaturase, spr2, Howe and Ryan, 1999), perception of JA, (JA insensitive, jai1, Li et al., 2004), and systemin-specific functions (suppressed in prosystemin-mediated responses, spr1, Lee and Howe, 2003). JA is a plant hormone that regulates many processes during development and in response to a broad array of mainly necrotizing pathogens and herbivorous insects. The importance of JA in the wound response was clearly illustrated by the observations that JA-biosynthetic mutants were highly impaired in expression of defense-related genes in response to wounding and in their ability to combat herbivory (Howe et al., 1996; Stenzel et al., 2003). For detailed discussions of the diverse roles of jasmonates in plants, the reader is referred to several excellent recent reviews (Balbi and Devoto, 2008; Wasternack, 2007).


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C. SYSTEMIN AND JA PRODUCTION IN WOUNDED LEAVES AND JA PERCEPTION IN DISTANT TISSUE

Elegant grafting experiments were performed to shed light on the mechanism of long-distance signaling during the wound response, in particular how systemin and JA interact and in which tissues each activity is required. Two contrasting models were postulated. The first model assumes that systemin is in fact a crucial component of the long-distance wound signal. In this case, systemin produced at the site of insect attack is translocated through the vasculature and stimulates the expression of jasmonates and defense genes in distant tissues. The contrasting model assumes that systemin signaling is only required in locally wounded tissue, such that locally synthesized JA or another octadecanoid is transported to distant tissues to promote wound resistance. In either scenario, a functional JA-perception mechanism must be in place in systemic tissue and indeed, JA-insensitive jai mutant scions were unable to respond to graft-transmissible signals from rootstocks (Li et al., 2002b). Grafting experiments involving a suite of tomato JA-biosynthetic mutants established that production of JA was required in the wounded rootstock but not in the grafted scion, indicating that de novo synthesis of JA was dispensable in perceiving and responding to a systemic wound signal (Li et al., 2002b). Taken together, these results demonstrated that a member of the octadecanoid pathway is an essential component of the long-distance wound signal. Grafting experiments utilizing the OPDA-forming but JA-deficient mutant acx1 as rootstock were similarly deficient in the generation of the systemic wound signal, strongly implicating JA itself or one of its conjugates as the signal (Li et al., 2005). Similar experiments utilizing spr1, a mutant deficient in systemin function, established that SPR1 is required for generation or transmission of the longdistance signal, but not for its perception (Lee and Howe, 2003). Although systemin was detected in phloem, these results established that it is not a component of the mobile signal because its activity was not required in distant tissues. However, spr1 demonstrated a novel phenotype in that the wound response of undamaged leaves was severely affected but the response of attacked leaves was nearly normal, suggesting that signaling events leading to wound resistance in local and systemic tissue operate differently (Lee and Howe, 2003). In addition, expression of several defense-related genes was maintained in the spr1 mutant, pointing toward the existence of redundant wound signaling pathways. JA is modified in planta into a number of bioactive conjugates including methyl-JA (MeJA) and JA-isoleucine (JA-Ile). An Arabidopsis mutant defective in conjugating JA to isoleucine, jar1 (Staswick and Tiryaki, 2004), was

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compromised in its response both to necrotrophic fungi (Staswick et al., 1998) and to feeding insects (Kang et al., 2006), suggesting that this conjugation is important in manifesting the wound response. There is currently no evidence that JA-Ile is transported in the vasculature. Therefore, it is not a compelling candidate for the long-distance wound signal. However, isotopically labeled MeJA was detected both in xylem and in phloem upon application to tobacco leaves (Thorpe et al., 2007). Tantalizing recent results suggest that exogenously applied MeJA is converted to JA and JA-Ile within plant leaves (Tamogami et al., 2008). Some reports suggest that damaged leaves emit volatiles that alter the resistance of neighboring plants (Heil and Silva Bueno, 2007; Karban et al., 2006; Kessler and Baldwin, 2001). Highly volatile MeJA could conceivably be an interplant signal for heightened wound response. For example, tomato leaves accumulated PIN proteins when incubated in the same growth chamber with sagebrush, Artemisia tridentata, a plant that contains MeJA in leaf surface structures (Farmer and Ryan, 1990). However, contrary evidence has been reported in that exogenously applied MeJA failed to induce PIN expression or induce protection to M. sexta in wild tobacco, Nicotiana attenuata (Preston et al., 2004). The role of atmospheric MeJA in regulating various biotic and abiotic plant stresses is discussed in more detail elsewhere (Cheong and Choi, 2003; Heil and Ton, 2008).

D. JA BIOSYNTHESIS OCCURS IN THE SIEVE ELEMENT/COMPANION CELL COMPLEX

Studies on the tissue-specific localization of prosystemin function and the JAbiosynthetic complex have shed light on the transport mechanism of the longdistance wound signal. Transgenic tomato expressing PROSYSTEMIN: -GLUCURONIDASE (GUS) reporter constructs originally revealed that prosystemin was particularly enriched in vascular bundles within the center vein of a leaf (Jacinto et al., 1997). Further analysis with more sensitive methods showed prosystemin mRNA and protein accumulation solely within phloem parenchyma cells (Narvaéz-Va´squez and Ryan, 2004). Complementary research using specific antibodies directed against enzymes of the JA-biosynthetic pathway established that 13-lipoxygenase, allene oxide synthase, and allene oxide cyclase were all localized to phloem companion cells and sieve elements (Hause et al., 2003). These observations also describe an amplification loop for JA accumulation within the vasculature, on the basis of the observation that prosystemin expression itself is induced by JA accumulation (Stenzel et al., 2003).


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These results suggest a model in which systemin is processed from its prosystemin precursor in phloem parenchyma cells, followed by its binding to the SR160 receptor on companion cell plasma membranes to initiate production of JA via plastidial and peroxisomal enzymes of the octadecanoid pathway (Schilmiller and Howe, 2005). JA itself or a JA-conjugate moves into sieve elements via plasmodesmatal connections for long-distance transport. Although compelling, there are several important gaps in our knowledge that will be filled only after further research: (1) Enzymes involved in the proteolytic processing of prosystemin have not been described, nor do we understand how systemin might travel to companion cells, although the recent discovery that prosystemin is embedded within the plant cell wall (Narvaéz-Va´squez et al., 2005) suggests that it or systemin may reach companion cells through the apoplast. (2) A receptor for JA has only recently been described (see below). Therefore, we are only beginning to understand how the long-distance wound signal is perceived and future data must also explain the observation that PIN proteins are solely expressed in mesophyll cells (Orozco-Ca´rdenas et al., 2001). (3) The importance of JA in long-distance signaling is clear, but it is likely that long-distance translocation of the wound signal follows a more complex route than traveling from source to sink through the phloem. Although translocation of the wound signal to induce PIN expression was highly correlated with the strength of vascular connections within and between tomato leaves (Orians et al., 2000; Rhodes et al., 1999), phloem girdling experiments cannot rule out cell-to-cell movement of the signal (Malone, 1996; Nelson et al., 1983). Furthermore, studies on the systemic response to M. sexta larval regurgitant in wild tobacco showed bidirectional movement of a wounding signal through orthostichies, from sink to source leaves as well as from source to sink leaves (Schittko and Baldwin, 2003). (4) Finally, one report demonstrated that phloem transport could be inhibited in tomato without effect on the systemic accumulation of PIN proteins (Wildon et al., 1992). The authors provided evidence that chilling of cotyledons did not prevent the transmission of an electrical signal required for a systemic wound response. E. JA-MEDIATED WOUND RESPONSE IS MODULATED BY OTHER SIGNALS

Prosystemin orthologues are not found outside of the Solanaceae but several other groups, particularly the model crucifer Arabidopsis, exhibit robust and well-characterized JA-dependent responses, highlighting the importance of signals other than systemin in influencing the core JA wound response pathway. JA-regulated gene expression is induced by several plant-derived compounds such as H2O2 (Jih et al., 2003; Orozco-Ca´rdenas et al., 2001),

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ethylene (ET) (O’Donnell et al., 1996), and abscisic acid (Herde et al., 1996), environmental stimuli including ultraviolet light (Conconi et al., 1996), and substances such as the fatty acid conjugate volicitin present in the oral secretions of feeding insects (Alborn et al., 1997). SA is a potent negative regulator of the wound response, whose activity inhibits the local accumulation of JA during wounding (Doares et al., 1995). Combinations of these compounds are reported to act cooperatively (O’Donnell et al., 1996, 2003) but may also antagonize one another’s action (Orozco-Ca´rdenas and Ryan, 2002) in order to fine-tune the wound response. F. MECHANISM OF JA ACTION ON EFFECTOR GENES

Recent experiments have attempted to identify a receptor for jasmonates and to elucidate the downstream signaling events leading to activation of JAresponsive target genes. As it is outside the aims of this review, only a brief outline of recent progress will be outlined here, but for more comprehensive discussion of these topics please refer to several excellent reviews (Katsir et al., 2008; Kazan and Manners, 2008; Staswick, 2008). Our current understanding of JA-regulated expression of target genes involves a quartet of different molecular players: the intracellular JA signal, transcription factors that specifically upregulate transcription of target genes, JA ZIM-domain (JAZ) transcriptional repressor proteins, and the E3 ubiquitin ligase COI1. JA-responsive genes are repressed in cells with low JA levels and this repression occurs largely through the binding of JAZ family transcriptional regulators. External cues such as wounding or abiotic stress lead to the intracellular accumulation of bioactive jasmonates, particularly JA-Ile. JA-Ile binding to the E3 ubiquitin ligase COI1 recruits JAZ proteins to this complex, resulting in JAZ polyubiquitination and subsequent degradation via the 26S proteasome. Transcription factors such as MYC2/JIN1 are thus freed from JAZ-mediated repression and JA-responsive genes are highly transcribed. These molecular, genetic, and biochemical studies suggest that COI1 is a molecular receptor for jasmonates (reviewed in Katsir et al., 2008).

IV. LONG-DISTANCE SIGNALING IN SAR SAR has been described in the literature for over 100 years. An early review on the SAR phenomenon was published in 1933 by Kenneth Chester. This review described many observations and experiments suggesting that plants often display partial or complete immunity to reinfection after recovery from an initial pathogen attack. During this time plant scientists were trying to


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determine if plants had circulating antibodies and phagocytic cells that could explain the immunity to reinfection that was being observed (Chester, 1933). It was subsequently determined that plants do not possess antibodies or moving cells and that instead each plant cell is capable of responding to pathogen infection. Studies in the second half of the twentieth century carefully dissected the physiological aspects of SAR in tobacco and cucumber (reviewed in Kuc, 1982), providing the definition of SAR still in use today. The term SAR was first used by Ross (1961) to describe systemic resistance induced by necrosis-causing viruses in tobacco. SAR is defined as an initial infection in one part of a plant that leads to resistance in distant tissues to normally virulent pathogens. SAR also provides broad-spectrum resistance in that the initial infection, for example by a bacterial pathogen, will result in subsequent resistance to viruses, bacteria, fungi, and even nematodes (Kuc, 1982). A. SAR DEVELOPS IN DISTINCT STAGES

Like other long-distance-signaling pathways, SAR develops in distinct stages (Table I). Evidence from tobacco, cucumber, and Arabidopsis suggests that there are four stages in the SAR response. 1. Induction A SAR-inducing pathogen infects a leaf typically leading to the formation of localized necrotic lesions and local resistance (Hypersensitive Response, HR) or disease-induced necrosis (reviewed in Kuc, 1982). The SAR-inducing lesion elicits the expression of a set of genes encoding pathogenesis-related (PR) proteins in tobacco, cucumber, and Arabidopsis (Uknes et al., 1992, 1993; Van Loon, 1997; Ward et al., 1991). A study using ET-insensitive tobacco suggests that ET perception is required for production of the SAR long-distance signal(s) (Verberne et al., 2003). Accumulation of SA (10–50fold increase over background levels) is also associated with this stage (Delaney et al., 1994; Lawton et al., 1995; Malamy et al., 1990; Uknes TABLE I Long-Distance-Signaling Stages During Defense 1. Induction of response in local tissue (includes production of long-distance signal) 2. Movement of the long-distance signal 3. Molecular perception of signal in distant tissue immediate response in Wound Response and SIS plant becomes ‘‘primed’’ to withstand subsequent attack in SAR and ISR 4. Manifestation of response upon subsequent attack in SAR and ISR

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et al., 1992; Yalpani et al., 1991). A recent study by Mishina and Zeier (2007) suggests that cell death is not required to induce SAR in Arabidopsis as a number of nonhost Pseudomonas strains and infiltration with compounds acting as microbe-associated molecular patterns (MAMPs) did induce SAR. During the SAR induction stage, microscopic cell death levels were similar to cell death levels in mock-inoculated plants (no SAR induction) as measured by Trypan Blue staining. However, it is still possible that a few cells undergoing pathogen-induced cell death were enough to induce SAR. It is also possible that the MAMPs, bacterial flagellin, and lipopolysaccharide used in this study, are phloem mobile and moved from the infiltrated leaf to distant tissue and induced a basal defense response. This could also be the case for induction of SAR with high doses of nonhost flagellin-bearing Pseudomonas strains. 2. Movement of a long-distance signal(s) A signal or signals are produced and thought to move from the induced leaf through the phloem (Guedes et al., 1980; Jenns and Kuc, 1979; Kiefer and Slusarenko, 2003; Tuzun and Kuc´, 1985) or cell-to-cell via plasmodesmata to the rest of the plant to establish SAR. Shulaev et al. (1997) suggested that airborne methyl-SA (MeSA) may also participate in long-distance signaling during SAR in tobacco. A study using 18O-labeling of tobacco mosaic virus (TMV)-inoculated tobacco leaves is consistent with SA itself being the mobile SAR signal (Shulaev et al., 1995). However, other studies using cucumber (Rasmussen et al., 1991), or grafting experiments with transgenic tobacco with reduced levels of SA, have led to the suggestion that SA is not the mobile signal (Pallas et al., 1996; Vernooij et al., 1994). These results also suggest that SA accumulation may not be required in the induction phase of SAR, but that SA is required in distant tissues during the establishment stage of SAR in tobacco. 3. Establishment of the ‘‘primed’’ plant The establishment stage of SAR involves the arrival and perception of the long-distance signal in the distant leaves, the accumulation of SA in distant Arabidopsis (1- to 2-fold, Cameron et al., 1999; Delaney et al., 1994; Lawton et al., 1995) and tobacco leaves (10-fold, Yalpani et al., 1991) and in cucumber phloem sap (8- to 200-fold, Me´traux et al., 1990), as well as the expression of PR genes. A receptor is postulated to interact with the long-distance signal (s) to ‘‘prime’’ the plant to respond rapidly and effectively to future pathogen attacks. In one Arabidopsis study, establishment of SAR in distant tissues was associated with the occurrence of low frequency microscopic HRs (Alvarez et al., 1998).


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4. Manifestation The final phase is the manifestation stage which occurs when the plant is challenged with a second, normally virulent pathogen such that the plant responds with a rapid and effective resistance response (Kuc, 1982). Evidence for this final stage comes from a number of studies (Kuc, 1982, 1983). For example, PR-proteins accumulate earlier and more rapidly after challenge with Peronospora tabacina in tobacco induced with Pe. tabacina or TMV (Ye et al., 1989), or in Arabidopsis induced with avirulent Pseudomonas syringae pv. tomato (Pst) (Cameron et al., 1999). Additionally, Siegrist et al. (1994) observed increased deposition of phenolic compounds and increased chitinase activity at the site of secondary challenge with Colletotrichum lagenarium in induced cucumber hypocotyls. B. ROLE OF SA AND NPR1 IN SAR

As discussed above, SA was believed to be the long-distance signal in SAR because it is found in phloem sap and exudates collected from SAR-induced cucumber and tobacco leaves, respectively, and its presence is required in distant tobacco tissue for a successful SAR response. In other words, SA is in the right place at the right time to be a long-distance-signaling molecule. Although the elegant cucumber leaf detachment (Rasmussen et al., 1991) and SA-deficient transgenic plant studies (Gaffney et al., 1993; Pallas et al., 1996) strongly suggest that SA is not the signal moving from induced to distant leaves, these studies cannot entirely eliminate SA as the long-distance signal. SA-deficient transgenic tobacco lines still contain uninduced levels of SA (10–40 ng g fw 1) and it is possible that this is sufficient for long-distance signaling. Additionally, undetectable amounts of SA in cucumber phloem could be sufficient for long-distance signaling during SAR. More recent studies have unraveled the role of SA and NONEXPRESSOR OF PR GENES-1 (NPR1) during SAR in Arabidopsis. Studies of npr1 mutants indicate that NPR1 is a key regulator in SAR, basal resistance, and the ISR response in Arabidopsis (reviewed in Dong, 2004; Grant and Lamb, 2006). There is mounting evidence that SA acts as a local signaling molecule during defense by inducing the reduction of key intermolecular disulphide bonds in NPR1, allowing translocation of NPR1 into the nucleus, where NPR1 interacts with TGA transcription factors which subsequently upregulate defense genes like PR-1 (Fobert and Despre´s, 2005). In the Arabidopsis-Pst SAR model, SAR is often induced in the initial leaf by inoculation with Pst (avrRpt2) (Cameron et al., 1994). The AvrRpt2 bacterial effector is recognized by the RPS2 resistance receptor, initiating a signal cascade that results in the HR (Bent et al., 1994; Leister and Katagiri, 2000;

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Mindrinos et al., 1994). This RPS2-AvrRpt2 pathway is SA-dependent suggesting that SA accumulation is required during the HR and the induction stage of SAR in Arabidopsis. Moreover, the RPS2-avrRpt2 HR is NPR1independent in ecotype Columbia-0 suggesting that NPR1 is not required for the HR (Rairdan and Delaney, 2002; Zhang et al., 2004) or the induction stage of SAR in Arabidopsis. However, functional NPR1 is required for the establishment and manifestation of the SAR response in Arabidopsis (Cao et al., 1994) suggesting that NPR1 functions in the distant leaves to upregulate PR genes during SAR. Yet, in the Arabidopsis ecotype Nossen the RPS2-AvrRpt2 pathway is compromised in the npr1-5 mutant (Nandi et al., 2004), suggesting that NPR1 is required for the SAR induction stage in this ecotype. C. SAR SIGNAL TRANSPORT

Grafting experiments with cucumber provided evidence that a long-distance signal does move from induced rootstocks to distant scions (Jenns and Kuc, 1979). Girdling with hot cotton wool in cucumber (Guedes et al., 1980) or removing the stem sheath in tobacco (Tuzun and Kuc´, 1985) prevented signal transport to distant leaves, suggesting that the SAR long-distance signal moves via the phloem. However, these techniques reduce both phloem and cell-to-cell movement, indicating that the SAR long-distance signal could travel using either or both transportation routes. Source–sink relationships (orthostichies) in the Arabidopsis rosette were investigated in relation to SAR-competence (Kiefer and Slusarenko, 2003). Movement of the SAR signal from induced to distant leaves to establish and manifest SAR as measured by PR-1 expression and reduced growth of Pst, indicated that leaves outside the orthostichy of the induced leaf were also SAR-competent. These data suggest that the Arabidopsis long-distance SAR signal(s) moves via the phloem and other means, perhaps cell-to-cell. In the cucumber–Ps. syringae pv. syringae SAR model, leaf detachment experiments indicated that the long-distance signal moves out of the induced leaf by 4 h after inoculation (Rasmussen et al., 1991, 1995) and distant leaves become SAR-competent by 24 h after SAR induction. In contrast, Arabidopsis leaf detachment experiments demonstrated that it takes between 36 and 48 h for the SAR signal to move and render distant leaves SAR-competent (Cameron et al., 1994). In tobacco it takes between three and nine days for the signal to move and render distant leaves SAR‐competent (Ross, 1961; Tuzun and Kuc´, 1985; Vernooij et al., 1994). There is a large range in the time it takes the SAR signal to move and render distant leaves SAR-competent in these SAR model systems suggesting that in cucumber the rapid movement of the


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SAR signal occurs mainly in the phloem, while Arabidopsis and tobacco use a combination of phloem and cell-to-cell movement. D. CANDIDATES FOR THE SAR LONG-DISTANCE SIGNAL

Since the discovery of DIR1 (Defective in Induced Resistance), a putative lipid transfer protein (LTP) involved in long-distance signaling during SAR (Maldonado et al., 2002), a number of candidate SAR long-distance signals have been identified. Our work with the dir1-1 SAR-defective mutant indicated that dir1-1 petiole exudates (enriched for phloem sap) do not contain the SAR mobile signal, leading to the hypothesis that DIR1-LTP could be involved in production of the SAR signal or act as a chaperone for a lipid signal (Maldonado et al., 2002). Plant LTPs contain eight cysteine residues forming four disulphide bonds that participate in forming an internal hydrophobic tunnel that has been shown to bind long-chain fatty acids in vitro. Plant LTPs are associated with numerous plant developmental and defensive processes (reviewed in Yeats and Rose, 2008). Recently the structure of DIR1 has been solved and in vitro lipid binding studies indicate that DIR1 can bind two monoacylated phospholipids side-by-side in its large internal tunnel (Lascombe et al., 2008). These researchers identified two proline-rich regions on the DIR1 surface. These proline-rich regions are involved in protein‐ protein interactions in other signaling pathways leading to the idea that these regions of DIR1 interact with the long-distance signal receptor upon arrival in distant tissues (Lascombe et al., 2008). DIR1, which was identified as a putative LTP, was the first and, to date, only plant LTP that has a genetically defined function. This discovery led to the idea that lipid signals may be important in SAR long-distance signaling. Both EDS1 (Falk et al., 1999) and PAD4 (Jirage et al., 1999), putative lipases involved in both basal and R gene-mediated defense, were postulated to produce lipid-derived signals that might be chaperoned to distant tissues by DIR1 (Maldonado et al., 2002). Subsequent studies demonstrated that EDS1 and PAD4 are required for a successful SAR response (Truman et al., 2007; R. K. Cameron, unpublished data; J. E. Parker, personal communication) and therefore provide further evidence that EDS1 and PAD4 may be supplying an essential lipid for DIR1. Recent experiments in the group of Robin Cameron support the idea that DIR1 may be a SAR long-distance signal. Agrobacterium-mediated transient expression of DIR1 in one leaf is sufficient to rescue the dir1-1 SAR defect, but not the npr1-2 SAR defect, supporting the idea that NPR1 is required in the distant tissue during Pst (avrRpt2)-induced SAR. Moreover, DIR1 protein was found in petiole exudates collected from leaves transiently

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expressing DIR1 only after SAR induction, suggesting that DIR1 moves via the petiole to distant tissues during SAR (our unpublished data). Lipid involvement in SAR is further supported by the identification of the sfd1 (suppressor of fatty acid desaturase deficiency 1) mutant that, like dir1-1, displays normal local defense (basal and R gene-mediated) but is defective in SAR long-distance signaling (Maldonado et al., 2002; Nandi et al., 2004). The SFD1 gene encodes a dihydroxyacetone-phosphate reductase that functions in plastid glycerolipid metabolism, pointing yet again to the importance of lipids in SAR signaling (Nandi et al., 2004). In a recent follow-up article, Jyoti Shah and coworkers have gone on to demonstrate that SFD1 and FAD7 (FATTY ACID DESATURASE-7), genes involved in glycerolipid synthesis, are required to produce the SAR long-distance signal (Chaturvedi et al., 2008) along with DIR1. Petiole exudates collected from sfd1 and fad7 mutants did not contain a SAR-inducing activity. However, these mutants were responsive to exudates collected from wild-type SARinduced leaves, suggesting that they perceive the SAR long-distance signal. SAR-induced exudates from either the sfd1 or fad7 mutant combined with SAR-induced exudates from dir1-1 were infiltrated into lower untreated leaves such that resistance was restored in distant leaves challenged with virulent Pseudomonas (Chaturvedi et al., 2008). It is tempting to speculate that DIR1 may bind a glycerolipid supplied by SFD1 and FAD7 and chaperone this lipid signal to distant tissues to establish SAR. JA, another lipid-derived molecule, has also recently been implicated in SAR long-distance signaling. A study by Truman et al. (2007) demonstrated that the JA biosynthesis mutant opr3 and the JA signaling mutant jin1 are defective in SAR. Induction of SAR in wild-type plants using Pst (avrRpm1) led to an increase in JA levels in petiole exudates and distant leaves. Additionally, spraying JA on leaves led to increased basal resistance to virulent Pst. These data suggest that JA could be a long-distance signal during SAR as JA appears to move to distant tissues to establish SAR. Unfortunately, Arabidopsis SAR marker gene expression (PR-1, PR-2, PR-5) was not monitored to demonstrate that classical SAR was induced in these experiments. An additional problem with these experiments is that Pst produces coronatine that may act as a JA mimic (reviewed in Katsir et al., 2008) and therefore, Pst-produced coronatine could induce JA production that has nothing to do with the SAR response. It is interesting to note that Truman et al. (2007) observed that the JA signaling mutant jin1 was SAR-defective and upon attempted induction supported wild-type levels of virulent Pst growth. JIN1 (MYC2) has been shown to be a negative regulator of SAmediated resistance to Pst such that jin1 mutants are resistant to virulent Pst and accumulate wild-type levels of JA (Laurie-Berry et al., 2006; Lorenzo


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et al., 2004; Nickstadt et al., 2004). Experiments by Cui et al. (2005) also indicated that the JA pathway mutant coi1-1 is SAR-competent, suggesting that JA signaling is not required for SAR. These seemingly conflicting data suggest that the JA-mediated systemic resistance observed by Truman et al. (2007) requires further study before it can be called a classical SAR response. Chaturvedi et al. (2008) concluded that JA is not a long-distance signal during SAR because they observed that petiole exudates accumulating JA did not possess SAR-inducing activity, whereas exudates with SAR-inducing activity accumulated only basal levels of JA. However, it is possible that basal levels of JA are sufficient to participate in SAR long-distance signaling. JA has also been shown to bind to the tobacco LTP1 protein that is involved in disease resistance in tobacco to Phytophthora parasitica (Buhot et al., 2004), leading to the hypothesis that DIR1 may bind JA during SAR longdistance signaling (Buhot et al., 2004; Grant and Lamb, 2006; Truman et al., 2007). Volatile MeSA has been implicated in SAR signaling between induced and distant leaves and in plant-to-plant signaling in tobacco (Shulaev et al., 1997). As discussed by Shulaev et al. (1997), the extent of volatile signaling in field-grown plants is not known and has not been investigated further. However, in a recent study, MeSA accumulated to high levels in Arabidopsis, eliciting PR-1 expression in neighboring plants supporting a role for volatile MeSA in plant-to-plant signaling (Koo et al., 2007). Additionally, an elegant study by Park et al. (2007) provided evidence that SA‐binding protein 2 (SABP2) (Kumar and Klessig, 2003) is involved in the accumulation of MeSA in tobacco leaves and petiole exudates and long-distance signaling during SAR. SABP2 is a methyl esterase that converts MeSA to SA, while SA inhibits the esterase activity of SABP2 (Forouhar et al., 2005). Park et al. (2007) performed a number of grafting experiments in which wild-type and mutant versions of SABP2 were silenced either in tobacco rootstocks or scions, combined with MeSA determination in leaves and petiole exudates. Together these experiments indicated that the methyl esterase activity of SABP2 is inhibited in the induced leaf by the accumulation of SA that occurs during induction of SAR. Therefore, MeSA levels rise in the induced leaf, followed by MeSA increases in phloem exudates and distant leaves, suggesting that MeSA is a SAR long-distance signal. In the distant leaves of plants induced for SAR, SA levels are lower leading to activation of SABP2’s methyl esterase activity, such that MeSA is converted to SA. As discussed above, SA produced locally in the distant leaves activates NPR1 leading to PR gene expression.

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Park et al. (2007) speculate that SABP2 generates MeSA as a SAR longdistance signal, and also perceives MeSA in distant tissues. However, it is not known if SABP2 participates directly in the ‘‘priming’’ mechanism that allows the distant uninfected tissue to respond to the next pathogen challenge in a rapid and effective manner. It is possible that SA produced from MeSA via SABP2 in distant leaves acts as a local signal to activate the ‘‘primed’’ state in distant leaves. The nature of the ‘‘primed’’ state is totally unknown, but it seems reasonable to speculate that a long-distance signal receptor(s) becomes activated in some way and upon subsequent pathogen challenge, the distant uninfected leaves respond in a resistant manner. NPR1 function is required during SAR and NPR1 becomes activated by SA leading to PR gene expression in distant leaves (Cao et al., 1994; Fobert and Despre´s, 2005); therefore, it is possible that NPR1 may act as a long-distance signal receptor in distant leaves during SAR (Fig. 1). Vlot et al. (2008) have determined that a number of SABP2 family members in Arabidopsis also participate in SAR. The quest for SAR long-distance signal(s) has been very fruitful in the past few years. A number of small hydrophobic molecules including MeSA, JA, and lipids are potential candidates. It is tempting to speculate that DIR1 with its large hydrophobic tunnel could possibly form a complex with any or all of these small hydrophobic molecules to act as a chaperone or as part of a longdistance SAR signal complex (Fig. 1). E. OTHER GENES INVOLVED IN SAR LONG-DISTANCE SIGNALING

A number of other genes have been implicated in the SAR pathway in the last few years, including Arabidopsis CDR1 (CONSTITUTIVE IN DISEASE RESISTANCE‐1) (Xia et al., 2004). Intercellular washing fluids collected from SAR-induced leaves contain a CDR1-dependent elicitor that induces PR gene expression when infiltrated into untreated healthy leaves. Additionally, grafting experiments with an inducible CDR1 transgenic line suggest that expression of CDR1 in lower leaves leads to production of a signal that moves to the grafted scion to elicit PR gene expression. However, SAR competence in these leaves was not measured (Xia et al., 2004). Moreover, antisense-CDR1 plants were more susceptible to virulent and avirulent Pst and Ps. syringae pv. maculicola (Psm) compared to wild type. CDR1 encodes an aspartic protease leading Xia et al. (2004) to hypothesize that intercellular CDR1 (Suzuki et al., 2004) may generate a peptide involved in local and systemic defense responses. Moreover, given the evidence that supports phloem and cell-to-cell SAR signal movement in Arabidopsis (Kiefer and Slusarenko, 2003), it will be important to determine how an apoplastic

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4. Manifestation SA, NPR1, PRs

Pathogen

Other putative Stage 3 & 4 genes DTH9, EDR1 NDR1, FMO1 WIN3

3. Perception of signals/priming (SABP2, NPR1, ?) Volatile meSA

DIR1 Lipids CDR1 MeSA JA

SABP2 Me

SA

SA

SFD1, FAD7 Lipids

DIR1 CDR1

Lipids

2. Movement of signals

MeSA JA

1. Induction JA

Pathogen

Fig. 1. SAR long-distance-signaling model. Putative SAR long-distance signals are produced (MeSA, JA, lipids, CDR1) and may bind DIR1, a putative signal chaperone, in the induced leaf (Stage 1). The signals and/or DIR1þsignals move from the induced leaf to distant tissues (Stage 2, via the phloem and/or cell-to-cell), where they are perceived (Stage 3) by SAR long-distance signal receptors which could include SABP2, NPR1 and unknown receptors such that the plant becomes primed. Volatile MeSA could be perceived in distant tissues of induced or neighboring plants. Upon subsequent pathogen challenge the activated/primed SAR long distance receptor(s) allows the distant leaves to respond in a resistant manner (Stage 4). See SAR Signal Transport section for details. SA, salicylic acid; MeSA, methyl-SA; CDR1, Constitutive defense response-1; DIR1, Defective in induced resistance-1; SABP2, SA‐binding protein 2; NPR1, Nonexpressor of PR genes-1; SFD1, FAD7, glycerolipid biosynthesis enzymes; PRs, pathogenesis-related proteins.

CDR1-derived peptide moves to distant tissues as it is currently believed that proteins in the phloem arrive intracellularly via companion cell plasmodesmata (Lough and Lucas, 2006). It is interesting to note that DIR1 also has a signal sequence that directs it into the intercellular space (R. K. Cameron, unpublished data). Moreover, we have evidence that DIR1 with its signal sequence removed can complement the SAR defect in dir1-1. These data suggest that upon SAR induction, DIR1 remains in the cytoplasm and then moves cell-to-cell and/or through the phloem to distant leaves (our unpublished data). Another gene that implicates peptides in SAR

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signaling is ALD1 which encodes an aminotransferase that is required for SAR, as well as R gene-mediated and basal resistance, and may be involved in generating an amino acid-derived signal (Song et al., 2004). FMO1, a flavin-dependent monooxygenase, is required for a successful SAR response in Arabidopsis (Mishina and Zeier, 2006). An fmo1 mutant displayed a wild-type local response to SAR-inducing Psm (avrRpm1) in terms of SA accumulation, HR cell death, and defense gene expression; however, there was little accumulation of SA or expression of defense genes in distant leaves, suggesting that FMO1 function is required in distant leaves for the establishment of SAR. Additionally, the ndr1-1 (no disease resistance-1-1) (Century et al., 1995, 1997) mutant was shown to be SAR-defective and FMO1 expression was abolished in distant leaves of ndr1-1 plants induced with Psm (avrRpm1) (Mishina and Zeier, 2006). NDR1 is a plasma membrane associated protein required for RPS2‐ and RPM1-mediated resistance (Coppinger et al., 2004). Therefore, NDR1 function may be required during the induction stage of SAR elicited by Pst (avrRpt2) or Psm (avrRpm1). Moreover, plants overexpressing NDR1 display enhanced resistance to Pst in the absence of constitutive expression of PR-1 suggesting that NDR1 may be involved in the priming stage of SAR (Coppinger et al., 2004). Taken together, these results suggest that NDR1 function is required for FMO1 expression in distant leaves during SAR. It is interesting to note that in another report, the ndr1-1 mutant did display SAR in response to Pst (avrB) (Zhang and Shapiro, 2002). Additional genes have also been implicated in the priming response of SAR. The ENHANCED DISEASE RESISTANCE-1 (EDR1) gene encodes a putative MAP kinase kinase (Frye et al., 2001) that is hypothesized to negatively regulate the priming response in Arabidopsis (Conrath et al., 2006). The SARdefective dth9 (detachment 9) mutant displays wild-type levels of PR-1 gene expression in SAR-induced and distant leaves, suggesting that DTH9 function is required in distant leaves during priming or for the manifestation stage of SAR. Most recently, WIN3, a protein required downstream of RIN4 in the RPS2-avrRpt2 resistance pathway, was demonstrated to be required for the RPS2-avrRpt2-mediated HR and for SAR in Arabidopsis (Lee et al., 2007b). Lee et al. (2007b) speculate that WIN3 function is important in the induction as well as the establishment stages of SAR, as WIN3 expression was abolished in distant leaves of the win3-T mutant. The effect of the environment, a key component of the disease triangle, (Agrios, 2005) must be kept in mind when comparing data from various labs and among various species, and may explain why some mutants (ndr1-2, eds1-1) appear SAR‐defective in some labs and SAR-competent in others. Additionally, the environment may affect how well petioles exude phloem sap and may explain why Jyoti Shah and co-workers (Chaturvedi et al., 2008)


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observed that infiltration of Arabidopsis petiole exudates collected from SAR-induced leaves, did induce SAR in naı¨ve Arabidopsis leaves, while these types of experiments have not been successful in other labs (R. K. Cameron, unpublished data; L. C. van Loon, personal communication). F. ROLE OF ET IN SAR LONG-DISTANCE SIGNALING

ET is an important plant hormone involved in a number of developmental, as well as biotic and abiotic stress responses (Binder, 2008). Responsiveness to ET is required in the wound response (see above) and ISR (De Vleesschauwer and Ho¨fte, 2009). However, its role in the SAR response has not been studied as thoroughly. ET is synthesized prior to necrotic lesion formation and the development of SAR in TMV-infected tobacco (Knoester et al., 2001). Tobacco plants expressing the Arabidopsis etr1-1 dominant mutant ET receptor are insensitive to ET and when SAR was induced with TMV, PR gene expression in induced leaves was observed. However PR gene expression in distant tissue was abolished (Knoester et al., 1998), leading to the suggestion that ET signaling may be important for the expression of SAR in tobacco. Subsequently Verberne et al. (2003) performed grafting experiments using the same ET-insensitive tobacco plants demonstrating that the SAR induction stage requires a functional ET response to generate the long-distance signal, whereas ET responsiveness is not required in distant leaves to establish and manifest SAR. In contrast, Arabidopsis ET-insensitive mutants etr1-1 and ein2-1 were SAR-competent when induced for SAR using SA or the SA analog, 2,6dichloroisonicotinic acid (INA), followed by inoculation with virulent Hyaloperonospora arabidopsidis (formerly known as (Hyalo)peronospora parasitica), suggesting that ET signaling is not required for SAR in Arabidopsis (Lawton et al., 1995). In a subsequent study, SAR was induced with Pst (avrRpt2) and distant leaves were challenged with H. arabidopsidis. Mutant etr1-1 and wild-type plants were similarly competent for SAR. When other SAR-inducing molecules (vitamin B1, harpin, BTH (benzo(1,2,3)thiadiazole7-carbothioic acid S-methyl ester) were applied to Arabidopsis plants, SAR was induced in an ET-independent manner (Ahn et al., 2007b; Dong et al., 1999; Lawton et al., 1996). Taken together, these data strongly suggest that SAR in Arabidopsis does not require a functional ET-signaling pathway. G. SAR LONG-DISTANCE SIGNALING ACROSS SPECIES

SAR has been mechanistically studied in cucumber, tobacco, and Arabidopsis. In the last few years a number of studies in both tobacco and Arabidopsis have contributed to elucidating SAR long-distance signaling and to extend these

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findings to other plant species. Chaturvedi et al. (2008) were able to demonstrate that Arabidopsis petiole exudates collected from SAR-induced leaves, when infiltrated into wheat spikelets or tomato leaves, induced SAR to Fusarium graminearum or Pst, respectively. Additionally, Vlot et al. (2008) determined that like tobacco, Arabidopsis possesses a family of genes encoding SABP2 methyl esterases (AtMES). A number of these genes have methyl esterase activity and silencing of multiple AtMES genes reduced the SAR response. In our lab we have developed a cucumber–Arabidopsis model system to aid in our SAR studies. SAR-induced cucumber exudates can rescue the SAR defect in dir1-1 Arabidopsis and protein gel blot analysis indicates that these cucumber exudates contain a DIR1-like protein (R. K. Cameron, M. J. Champigny, and J. Faubert, unpublished data). These studies indicate that there appear to be shared components between tobacco and Arabidopsis and between cucumber, wheat, tomato, and Arabidopsis, hinting that longdistance-signaling mechanisms are conserved in diverse plant species.

V. SYSTEMIC INDUCED SUSCEPTIBILITY (SIS) Plants employ many defense responses to combat and withstand pathogen infection and in turn pathogens have evolved mechanisms to suppress or evade plant defense and manipulate plant metabolism (reviewed in Melotto et al., 2008; Speth et al., 2007). Recently it was discovered that in Arabidopsis, Psm not only suppresses plant defense in the infected leaf, but also in distant or systemic leaves, rendering the distant leaves more susceptible to subsequent infections (Cui et al., 2005). This SIS is dependent on the Psm phytotoxin coronatine as demonstrated by the inability of coronatinedeficient Psm to elicit SIS. A SIS long-distance signal has been detected in experiments in which petiole exudates enriched in phloem sap collected from Pst(avrRpt2)-induced wild-type leaves were infiltrated into the SAR-defective mutant plants sid2 and npr1-2. These SAR-defective mutants proved not only SAR-defective, but also exhibited SIS (Chaturvedi et al., 2008). Whether coronatine itself acts as the long-distance signal or instead stimulates JA long-distance signaling is not known (Cui et al., 2005). Coronatine is very similar in structure to JA and is known to act as a molecular mimic to activate downstream JA signaling and at the same time suppress SA-mediated resistance in infected leaves (reviewed in Katsir et al., 2008). The fact that JA is known to be a long-distance signal in the wound response leads to the idea that coronatine may also act as a long-distance signal in SIS.


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VI. SIGNALING DURING ISR Several soil-borne microorganisms are beneficial to plants in that they increase nutritional capacity and enhance resistance to biotic and abiotic stresses, including drought and pathogen infection. Colonization of roots by certain strains of nonpathogenic plant growth-promoting rhizobacteria (PGPR) results in enhanced resistance in aerial plant organs to normally virulent pathogens, a phenomenon known as rhizobacteria-induced systemic resistance (Van Loon et al., 1998). ISR has been observed for a variety of plant root/ microbe interactions, such as carnation/Fusarium oxysporum f. sp. dianthi (Van Peer et al., 1991), bean/Bacillus pumilis SE34 (Benhamou et al., 1996), Arabidopsis/Pseudomonas fluorescens WCS417r (Pieterse et al., 1996), as well as in the mycorrhizal symbiosis tomato/Glomus mosseae (Cordier et al., 1998). Signaling events during the development of ISR have features in common with other induced defense responses, particularly in that ISR occurs as a series of discrete steps temporally and spatially separated in plant tissues: First, during an induction step, beneficial microbes colonize and are recognized by plant roots. This stimulates the synthesis of a long-distance signal that is transported systemically and perceived in aerial tissues. Following perception, plants become ‘‘primed’’ to assert a more aggressive response to virulent pathogens and subsequent pathogen attack is met with a more abundant and/or earlier stimulation of defense-related genes (Conrath et al., 2006). The phenomenon of ISR has been reviewed recently (Conrath et al., 2006; De Vleesschauwer and Ho¨fte, 2009; Van Wees et al., 2008); therefore, we will highlight only issues relevant to long-distance signaling during ISR here. A. INDUCTION OF ISR

The initial, or induction stage in ISR involves the recognition of certain microbe-derived molecules by plant roots. Different classes of molecules, including bacterial flagellin and lipopolysaccharides (Bakker et al., 2007), antibiotics (Iavicoli et al., 2003), surfactants (Tran et al., 2007), and ironchelating siderophores (Meziane et al., 2005), have been implicated as inducers of ISR, largely on the basis of the inability of mutant microbes deficient in these compounds to promote systemic disease resistance in the host plant. It should be noted that many of these molecules are also recognized during plant immune responses against virulent pathogens suggesting that beneficial microbes and virulent pathogens are recognized in a similar way. Compared to SAR, little is known about plant-derived molecules required for the induction of ISR. Transcriptome analysis of Arabidopsis colonized by ISRinducing Ps. fluorescens WCS417r revealed significant changes in expression of

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97 genes in roots, but only subtle changes in gene expression within leaves (Verhagen et al., 2004). This observation suggested that, unlike SAR, ISR is not associated with massive plant-wide transcriptional reprogramming. The above-mentioned microarray study identified that the transcription factor MYB72 was upregulated in roots after induction of ISR (Verhagen et al., 2004). Null myb72 mutants, when treated with WCS417r, did not develop resistance against a variety of foliar pathogens, suggesting that MYB72 was required for an early ISR signaling step in plant roots (Van der Ent et al., 2008). Furthermore, MYB72 interacted with the Ethylene-insensitive3-like-3 (EIL3) protein in a yeast two-hybrid assay, associating the function of MYB72 with the ET-response pathway (Van der Ent et al., 2008). Although the importance of this interaction is not understood and the target genes of these transcription factors have not yet been identified, they may be required for production or transmission of an ISR long-distance signal. Currently there is little experimental evidence that implicates any molecule(s) as a long-distance signal(s) required for the development of ISR. In particular, grafting experiments or transgenic technologies have not yet been employed to specifically identify substances whose site of production, presumably in roots, is spatially separated from their site of action in aerial tissues. JA is a priori a compelling candidate because of its phloem mobility and the fact that ISR in Arabidopsis requires elements of the JA signaling pathway (see below). However, as is the case for SA in SAR, JA may be required to mount an effective ISR response in distant tissue but not itself be a long-distance signal. B. SIGNAL PERCEPTION AND PRIMING DURING THE DEVELOPMENT OF ISR

In Arabidopsis, ISR signaling events downstream of the initial induction step share features with both SAR and the wound response. Like the wound response, ISR is abolished in many JA- or ET-responsive mutants (Ahn et al., 2007a; Hossain et al., 2008; Pieterse et al., 1998) and manifestation of enhanced resistance to virulent pathogens during ISR was found to involve a variety of JA- and ET-responsive genes (Ahn et al., 2007a; Van der Ent et al., 2008; Van Oosten et al., 2008). Furthermore, SA is not required for most instances of ISR as demonstrated by the fact that ISR could be established in mutants deficient for SA synthesis or accumulation (Pieterse et al., 1996; Ton et al., 2002; Van Wees et al., 2008). A requirement for SA was observed in the Arabidopsis ISR response to Verticillium dahliae (Tjamos et al., 2005), demonstrating variation in ISR signaling requirements in different plant/microbe associations.


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Like SAR, ISR in Arabidopsis requires the master defense regulator NPR1 (Knoester et al., 1999; Pieterse et al., 1998; Van Wees et al., 2000). However, in ISR, NPR1-directed upregulation of PR genes is not observed (Pieterse et al., 1996; Van Wees et al., 1999). NPR1 can thus be considered an integral part of the molecular machinery involved in perceiving or ‘‘interpreting’’ both the SAR and ISR long-distance signals because NPR1 discriminates between signals originating from the action of SAR-inducing pathogens and soil-borne beneficial microbes in ISR and orchestrates a different suite of SA-dependent or JA/ET dependent defense genes for either case. Although the requirement for NPR1 was not examined, a recent report provided evidence that the transcription factor MYC2 is required for the priming step of ISR because MYC2 was upregulated in WCS417r-treated Arabidopsis plants and mutants deficient in MYC2 were unable to mount a systemic response to bacterial or fungal challenge (Pozo et al., 2008).

VII. TECHNIQUES TO FURTHER ELUCIDATE LONG-DISTANCE SIGNALING To date, a number of molecules appear to be in the right place at the right time to be candidates for the SAR long-distance signal (DIR1, MeSA, JA, lipids, CDR1-derived peptide). The evidence is convincing that JA is a long-distance signal during the wound response, although it is possible that additional signal molecules will be discovered in the future. The ISR and SIS long-distance signals are unknown; however, coronatine or JA appear to be reasonable candidates for a SIS signal and JA pathway genes are required for ISR. Although petiole exudates enriched for phloem sap collected from Arabidopsis are rather dilute, these exudates do contain both SAR-inducing (Chaturvedi et al., 2008; Maldonado et al., 2002) and SIS-inducing properties (Chaturvedi et al., 2008). Therefore, it should be possible to further characterize SAR and SIS long-distance signals by metabolomic and proteomic analysis of petiole exudates. For example, if DIR1 binds a lipid or other hydrophobic molecule in a covalent manner, as has been demonstrated for barley LTP1b (Bakan et al., 2006), then 2-D gel electrophoresis of SARinduced petiole exudates combined with protein-ligand identification via mass spectrometry analysis, may identify the DIR1 ligand. It may also be possible to detect the ISR signal by collecting petiole exudates from leaves that are receiving the ISR signal from the roots. Highly pure phloem exudates can be collected in abundance from species with robust SAR responses such as cucumbers. Mass peptide fingerprinting of uninduced

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and SAR-induced phloem exudates from cucumbers could potentially identify all proteins present in a sample and thereby discover new proteins acting as long-distance signals or chaperones. Another approach to identify the DIR1 ligand includes collection of petiole exudates from SAR-induced leaves and subsequent immunoprecipitation/mass spectrometry analysis of DIR1 plus its ligand. This has been attempted in the Cameron lab, but with little success probably because of the very low levels of DIR1 present in exudates even in DIR1 overexpressing lines and the use of an antiserum raised against unfolded DIR1 produced in transformed Escherichia coli cells. DIR1, like most LTPs is thought to enter the secretory system, obtain its four disulphide bonds in the endoplasmic reticulum and be secreted to the plant cell wall. Therefore, to enrich for DIR1-ligand complexes during immunoprecipitation, antibodies should be generated to correctly folded DIR1 produced in a eukaryotic system such as the methanotrophic yeast Pichia pastoris. DIR1 is expressed in all cells in Arabidopsis leaves (R. K. Cameron, M. J. Champigny, H. Shearer, J. Faubert, K. Haines, A. Mohammad and M. Neumann, unpublished data) making it challenging to design experiments to observe DIR1 move from induced to distant leaves during SAR. To address this question we have developed a transient Agrobacterium-SAR assay using the transient transformation method of Wroblewski et al. (2005). DIR1 fused to the gene encoding YFP is expressed in one dir1-1 leaf by Agrobacterium-mediated transient transformation, followed by SAR induction with avirulent Pst in the same leaf. Expression of DIR1:YFP in one leaf does rescue the SAR defect in dir1-1 and native DIR1 protein can be detected in petiole exudates of SAR-induced leaves using protein gel blot analysis. However, DIR1:YFP cannot be detected, suggesting that YFP is being cleaved from the DIR1:YFP fusion protein, making it impossible to follow DIR1 movement using fluorescence microscopy. In preliminary experiments using this transient Agrobacterium-SAR assay, we detected DIR1 protein in the petiole exudates of distant leaves suggesting that DIR1 does move to distant tissues during SAR (R. K. Cameron, M. J. Champigny and J. Faubert, unpublished data). To shed light on the long-distance signal perception mechanism in the wound response, SAR, ISR, or SIS, it should be possible to take advantage of mutants that are wild-type in the respective induction stages, but are defective in the establishment and manifestation stages. Comparing wildtype and mutant gene expressions (microarray or transcriptome sequencing) in distant leaves may identify important genes whose expression changes in the perception or establishment stage and give some clues as to the identity of the various long-distance signal receptors.

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VIII. CONCLUDING REMARKS The molecular receptor of FT in the SAM is FD, a bZIP transcription factor that interacts with FT, the long-distance signal, to initiate the gene expression cascade that induces expression of the floral meristem-identity genes resulting in the switch from vegetative to flowering meristems (reviewed in Kobayashi and Weigel, 2007). In the wound response pathway, emerging evidence suggests that COI1 is a molecular receptor for JA in distant leaves leading to COI1 interaction with the JAZ repressor proteins, their subsequent polyubiquitination and degradation, thus releasing the JIN1 (MYC2) transcription factor to upregulate the JA responsive genes (reviewed in Katsir et al., 2008). Thus, in both the photoperiodic flowering and wound response pathways, long-distance signals interact with intracellular receptors that are directly linked to upregulation of key response genes. This may also be occurring in SAR as NPR1 appears to be an intracellular receptor of SA leading to activation of TGA transcription factors that upregulate expression of the PR genes in distant leaves during SAR (Fobert and Despre´s, 2005). It is notable that the signaling molecule JA is a long-distance signal in the wound response and has been implicated in both SAR and ISR, while the JA mimic, coronatine, is required in SIS. This suggests that JA may be a common component of long-distance signaling during defense and that long-distance communication has been co‐opted to aid in bacterial pathogenesis (Table II). In the wound response pathway, systemin does accumulate in the phloem, as does SA during SAR; however, neither appears to be a long-distance signal. Are systemin and SA accumulation in the phloem necessary for the wound response and SAR or are they present in the phloem because of a high capacity to accumulate as has been demonstrated for SA (Rocher et al., 2006) and in fact serve no function during SAR or the wound response?

TABLE II Signaling Components in SAR, SIS, WR, and ISR Signaling components

SAR

SIS

Long distance signals

DIR1? JA? MeSA? CDR1 Peptide? SABP2? NPR1? ?

Long distance signal receptors

WR

ISR

JA? COR?

JA

JA?

COI?

COI?

COI?

COR, coronatine; WR, wound response; SA, salicylic acid; MeSA, methyl-SA; CDR1, Constitutive defense response-1; DIR1, Defective in induced resistance-1; SABP2, SA-binding protein 2; NPR1, Nonexpressor of PR genes-1; COI, Coronatine insensitive.

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Tobacco grafting experiments indicate that SA accumulation in the induced leaf is not required for production and movement of the long-distance signal to distant tissues (Pallas et al., 1996; Vernooij et al., 1994), However, SAR in Arabidopsis is most often induced via R gene-mediated resistance and this response does require SA accumulation (Delaney et al., 1994) suggesting that SA accumulation is required during the induction stage of SAR in Arabidopsis. Identification and characterization of mutants that cannot accumulate SA in the induced leaf, but still retain SAR-inducing ability could shed some light on this question. Many questions concerning long-distance signaling in SAR, ISR, SIS, and the wound response remain unanswered. Molecular perception and dissemination of the long-distance signal(s) in distant leaves is unknown. The signal arrives in the distant leaves via the phloem and/or cell-to-cell movement and thus in both cases the signal(s) will be present in the cytoplasm with the ability to move to neighboring cells using plasmodesmata. It is possible to speculate that some cells at the leaf base are exposed to the long-distance signal(s) eliciting production of a secondary local signal that can spread readily via the phloem and cell-to-cell. In the case of long-distance signal perception during the wound response, PIN expression is restricted to mesophyll cells (Orozco-Ca´rdenas et al., 2001), raising the question of how the phloem-mobile signal (presumably JA or a JA-conjugate) gets access to and exerts its effects in mesophyll cells in the leaf. Hydrogen peroxide could act as a secondary local signal in systemic leaves on the basis of the observation that H2O2 travels through the apoplastic space of wounded tissue and is itself a potent inducer of PIN expression (Orozco-Ca´rdenas et al., 2001). Further research is warranted to investigate the role of hydrogen peroxide in systemic as well as locally wounded tissue. During the perception or manifestation stages of SAR, SA is a possible secondary signal as it is phloem mobile (Rocher et al., 2006) and SA levels do increase substantially in distant leaves. Additionally, evidence from Arabidopsis indicates that SA may act in a feedback loop to increase its own accumulation (reviewed in Shah, 2003). Therefore, sufficient SA could accumulate and spread to neighboring cells in a reiterative fashion to disseminate the SA signal to cells in the distant leaf leading to the ‘‘primed’’ state which probably involves NPR1 and other unidentified players.

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Systemic Acquired Resistance

R. HAMMERSCHMIDT1

Department of Plant Pathology, 107 Center for Integrated Plant Systems Building, Michigan State University, East Lansing, MI 48824-1311, USA

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Systemic Acquired Resistance ................................................ B. Other Forms of Induced Resistance ......................................... II. The Biological Spectrum of SAR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. The Induction of SAR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Necrotizing Pathogens ......................................................... B. The Hypersensitive Response................................................. C. Is Pathogen-Induced Necrosis Needed for SAR Induction? ............. D. Pathogen-Produced Inducers of SAR ....................................... E. Chemical Induction of SAR .................................................. IV. Systemic Biochemical Changes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Pathogenesis-Related Proteins................................................ B. Other Proteins................................................................... C. SA Accumulation............................................................... V. How SAR Protects Plants Against Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Priming ........................................................................... B. Protection Against Fungi and Oomycetes .................................. C. Protection Against Bacteria................................................... D. Protection Against Viruses .................................................... E. Mechanisms of Defense in Summary........................................ B. What Don’t We Know? ....................................................... VI. Concluding Comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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0065-2296/09 $35.00 DOI: 10.1016/S0065-2296(09)51005-1

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ABSTRACT Systemic acquired resistance (SAR) is a form of induced resistance that is activated by pathogens that induce localized necrotic disease lesions or a hypersensitive response. A major characteristic of SAR is the broad spectrum nature of the protection it confers against a wide range of pathogens, although recent studies suggest that the resistance is most effective against biotrophic and hemibiotrophic pathogens and less effective against necrotrophs. SAR is dependent on salicylic acid signaling and is typically associated with systemic expression of pathogenesis-related protein genes and other putative defenses. Once induced, SAR-expressing plants are primed to respond to subsequent pathogen infection by induction of defenses that are localized at the site of attempted pathogen ingress. Finally, SAR typically does not provide full resistance to disease indicating that the practical application of this form of resistance will require the use of other disease management tools. On the basis of these types of observations, it is likely that SAR and other forms of induced resistance are based on the enhanced ability to express basal defenses.

I. INTRODUCTION It is probably safe to assume that all plants have the genes needed to mount an effective defense against pathogens. This is illustrated by the diversity of plant defenses and the fact that plants resist most pathogens (Hammerschmidt, 1999a,b; Hammerschmidt and Nicholson, 1999; Hu¨ckelhoven, 2007; Van Loon et al., 2006). Thus, one difference between a resistant and a susceptible plant may reside in the timely expression of these defenses. This is certainly true for resistance controlled by the interactions between pathogen effectors and host resistance (‘‘R’’) genes. In this case, plant R genes provide the plant with the ability to detect the pathogen and then rapidly express defenses after infection. This is also true for the nonhost resistance expressed in plants that are under attack by pathogens of other plant species. In these cases, the failure of the pathogen to invade and colonize the nonhost is associated with the expression of a number of defenses. The observations that even susceptible plants can mount some degree of defense against pathogens plays into the overall concept that plants come equipped with defense genes. This form of defense, known as basal disease resistance (Jones and Dangl, 2006), is induced in susceptible plants upon infection with compatible pathogens. Although not effective enough to stop the pathogen, basal defenses may help limit the spread of the disease in the infected tissue. These defenses are likely the same as those induced in other forms of resistance, though they may often be expressed too late or at too low a level to be totally effective (phytoalexin accumulation is one good example of this type of defense response) (e.g., Hammerschmidt, 1999b).

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The assumption that plants have all necessary defenses also provides support for the general phenomenon of induced resistance and forms the basis for the reason why induced resistance exists. In induced resistance, certain biotic or abiotic treatments of a susceptible plant alter its defensive abilities in such a way that resistance is enhanced to one or more pathogens (Hammerschmidt, 1999a; Kuc´, 1982). It is important to note that the induced plants may still become diseased, indicating that induced resistance does not provide the level of resistance mediated by major R genes. Depending on the type of inducing agent and the signaling pathways involved, induced resistance can be classified in different ways. The two forms of induced resistance that have been best characterized are systemic acquired resistance (SAR) and induced systemic resistance (ISR) (Van Loon et al., 1998). However, it is likely that other forms of induced resistance exist. There has been an explosion of information published on induced resistance and its many forms in the last few decades (e.g., see reviews by Durrant and Dong, 2004; Sticher et al., 1997; Vallad and Goodman, 2004; Van Loon et al., 1998; chapters in Walters et al., 2007). As such, it is impossible to cover all the aspects of this type of resistance in one chapter; here, the main focus will be on the various mechanistic and resistance characteristics of the SAR form of induced resistance. The reader is directed to other reviews in this volume and elsewhere for a more detailed discussion of the signaling pathways found in SAR and in other forms of induced disease resistance (e.g., Bostock, 2005; Durrant and Dong, 2004; Glazebrook, 2005; Pieterse and Van Loon, 2004, 2007; Van Loon et al., 1998). A. SYSTEMIC ACQUIRED RESISTANCE

SAR can be broadly defined as a form of induced resistance that is activated throughout a plant typically following infection by a pathogen that causes localized necrotic lesions. The necrosis can be the result of disease induced by a pathogen or a hypersensitive response (HR) (e.g., Kuc´, 1982; Kuc´ et al., 1975; Ross, 1961b). Multiple rounds of inducing inoculations (i.e., ‘‘booster’’ inoculations) can also increase the level of SAR (Kuc´, 1982). SAR is dependent on salicylic acid (SA) signaling (Gaffney et al., 1993). Although the role of SA as a mobile signal for SAR is still debatable (Rasmussen et al., 1991; Shulaev et al., 1995; Vernooij et al., 1994), there is little doubt that this simple phenol is essential for the expression of SAR (Delaney et al., 1994). The development of SAR throughout a plant takes several days and is accompanied by the systemic expression of genes encoding pathogenesisrelated (PR) proteins and the accumulation of their protein products (Hammerschmidt, 1999a; Van Loon, 1997; Van Loon and Van Strien, 1999;

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Van Loon et al., 2006). Another characteristic of SAR is the requirement of the NPR1 (Nonexpressor of PR genes 1) protein, functioning downstream of SA, as a major regulatory factor in the expression of SAR, PR-protein accumulation, and priming. SA accumulation in the cytoplasm affects the cellular redox state. This change is involved in the reduction of cytoplasmic NPR1 protein that then enters the nucleus and interacts with TGA transcription factors (Pieterse and Van Loon, 2004, 2007). Plants expressing SAR are ‘‘primed’’ to respond to subsequent infections by expression of additional defenses, such as the oxidative burst, cell wall alterations at the site of attempted infection, and phytoalexin production (Conrath et al., 2000, 2002, 2006). A final characteristic of SAR is that the resistance is effective against a broad range of pathogens that include bacteria, true fungi, oomycetes, and viruses (Deverall, 1995; Hammerschmidt and Kuc´, 1995; Kuc´, 1982). Within this range, recent studies with model systems (e.g., Arabidopsis thaliana) suggest that SAR, and SA-mediated resistance in general, may be most effective against biotrophic and hemibiotrophic pathogens and not against necrotrophs (Glazebrook, 2005; Oliver and Ipcho, 2004). However, as discussed later in this review, SA induces resistance to viruses by an NPR1-independent mechanism (e.g., Singh et al., 2004). B. OTHER FORMS OF INDUCED RESISTANCE

Another form of induced resistance is ISR. Like SAR, ISR is systemic and can be effective against a number of pathogens. Unlike SAR, ISR induction is associated with the interaction of roots with certain plant growth-promoting rhizobacteria that do not elicit a necrotic response or cause any type of visible symptoms. In addition, ISR is dependent on jasmonic acid (JA)- and ethylene (ET)-signaling pathways, and its induction does not result in systemic expression of PR genes (Van Loon et al., 1998). As a contrast to SAR, studies in Arabidopsis have suggested that ISR is most effective against necrotrophic pathogens (Glazebrook, 2005). Like SAR, plants expressing ISR are also primed to express additional defenses upon infection (Conrath et al., 2006) and also require NPR1 (Pieterse and Van Loon, 2004, 2007). More details on the ISR form of induced resistance can be found elsewhere in this volume (De Vleesschauwer and Ho¨fte, 2009). Induced resistance can also be localized to the tissue that is treated with an inducing agent. Some of the earliest studies on induced resistance were conducted by treating entire plants or parts of plants with an inducing agent, such as an avirulent race of a pathogen or a pathogen of another plant species, followed by inoculating the treated tissues one or more days later with a virulent pathogen (e.g., Hammerschmidt et al., 1976; Rahe et al.,


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1969; Ross, 1961a; Skipp and Deverall, 1973). In these cases, it is difficult to determine what types of mechanisms are involved in the resistance. It is possible that defenses induced by the initial inoculation may be sufficient to block infection by a subsequent inoculation with a pathogen. This is probably what occurs in the resistance activated by the herbicide lactofen in soybean to Sclerotinia sclerotiorum (Dann et al., 1999). Lactofen induces the accumulation of the glyceollin phytoalexins, and upon subsequent inoculation leaves that have accumulated glyceollin are more resistant to infection. No resistance is detected several weeks later when the content of glyceollin returns to baseline levels (Dann et al., 1999). However, prior localized infections may protect through the activation of resistance potential in surrounding host cells. For example, the resistance of cucumber to Cladosporium cucumerinum, the cause of scab, is associated with the rapid deposition of lignin at the site of attempted infection (Hammerschmidt et al., 1984). Preinoculation of scab-resistant cucumber seedlings with Cl. cucumerinum induces local resistance to Colletotrichum orbiculare (syn.: Colletotrichum lagenarium). A histochemical analysis of the tissues revealed that an active defense against Co. orbiculare was expressed in cucumber epidermal cells from seedlings preinfected with Cl. cucumerinum (Hammerschmidt and Kuc´, 1982). The host cells that Co. orbiculare appressoria were attempting to invade were several cells away from any lignification induced by Cl. cucumerinum. This suggested that the resistance was not merely the result of physical blocking of infection by the lignin induced by the inducing inoculation.

II. THE BIOLOGICAL SPECTRUM OF SAR Systemic resistance induced by necrotizing pathogens or the HR has been observed in many plant species (e.g., Deverall, 1995; Hammerschmidt and Kuc´, 1995; Kuc´, 1982; Sticher et al., 1997; Walters et al., 2007). Thus, it is likely that SAR is a general phenomenon throughout the plant kingdom. However, detailed studies on SAR have been limited to only a handful of plant species and some species, especially non‐angiosperm plants such as the conifers (Bonello et al., 2006), have received only cursory examination.

III. THE INDUCTION OF SAR A. NECROTIZING PATHOGENS

After inoculation with a resistance-inducing microbe, SAR takes several days to develop and the time of appearance is associated with the development of necrosis produced by the inducing organism (Kuc´, 1982). For example, early

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work on the tobacco-Peronospora tabacina interaction demonstrated that necrotic stem lesions caused by Pe. tabacina would systemically protect the foliage from infection by Pe. tabacina (Cruickshank and Mandryk, 1960; Pont, 1959). Cohen and Kuc´ (1981) later confirmed the observations with tobacco and Pe. tabacina. Using cucumber, Kuc´ and coworkers demonstrated that localized necrotic lesions produced on the first true leaf of cucumber plants following inoculation with Co. orbiculare or Pseudomonas syringae pv. lachrymans resulted in systemic resistance to these and other pathogens of cucumber (Caruso and Kuc´, 1979; Kuc´ et al., 1975). Increasing the number of necrotic lesions on the leaf used for induction also increased the expression of SAR (Caruso and Kuc´, 1979; Kuc´ and Richmond, 1977).

B. THE HYPERSENSITIVE RESPONSE

The necrotic local lesions typical of the HR induced by tobacco mosaic virus (TMV) on N gene tobacco are also effective in inducing resistance to TMV (Ross, 1961a,b) and other tobacco pathogens (e.g., Friedrich et al., 1996). Increasing the number of TMV lesions on the leaf used for induction also increased the level of systemic resistance (Ross, 1961b). Similarly, Jenns and Kuc´ (1977, 1980), reported that inoculation of cucumber plants with tobacco necrosis virus (TNV) induced systemic resistance to Co. orbiculare, Ps. syringae pv. lachrymans and to itself. In addition to viruses, the HR elicited by bacteria can also induce SAR. Cameron et al. (1994) successfully induced a SAR response in Arabidopsis by inoculating one leaf with Ps. syringae pv. tomato carrying avrRpt. This interaction resulted in a HR and, by 2 days, the expression of resistance in other leaves to inoculation with a strain of Ps. syringae pv. tomato lacking avrRpt and of Ps. syringae pv. maculicola. Smith et al. (1991) were able to induce SAR in cucumber with a wheat isolate of Ps. syringae pv. syringae that caused a rapid HR in cucumber leaves. Using Co. orbiculare as the challenge pathogen, the resistance induced by Ps. syringae pv. syringae was reported to be fully expressed within 24 h of the inducing inoculation. These authors also confirmed the observations of Caruso and Kuc´ (1979) that the cucumber pathogen Ps. syringae pv. lachrymans also induced SAR. Ps. syringae pv. lachrymans took several days longer to induce systemic resistance as compared to Ps. syringae pv. syringae. This may be because the necrotizing response to the pathogen was much slower.


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C. IS PATHOGEN-INDUCED NECROSIS NEEDED FOR SAR INDUCTION?

Although the formation of necrotic lesions has been correlated with the induction of SAR by pathogens, is necrosis really necessary for induction by these microbes? Some early indication of the need for pathogen-induced necrosis was the observation of Jenns and Kuc´ (1980) who found that nonnecrotic, yet localized, ‘‘starch lesions’’ induced by TMV in cucumber cotyledons did not induce SAR in this plant against Co. orbiculare (although it was later reported by Roberts (1982) that TMV would induce systemic resistance in cucumber to TNV). Neither heat- or formalin-killed cells of Ps. syringae pv. lachrymans nor cell-free sonicates of this bacterium induced necrosis or SAR in cucumber (Caruso and Kuc´, 1979). Furthermore, necrotic areas induced by dry ice damage were ineffective or SAR in inducing SAR in cucumber (Hammerschmidt et al., 1982). Dean and Kuc´ (1986a) indirectly addressed this question by inoculating the first true leaf of cucumber plants with Co. orbiculare and then excising the inoculated leaf at intervals over the next 6 days. The second (true) leaf of all plants was challenge inoculated with the same pathogen 7 days after the inoculation of the first leaf. They found that the first leaf needed to be on the plant for at least 72 h for any expression of SAR in the second leaf. This time is when necrotic lesions caused by Co. orbiculare are first visible. The longer the first leaf remained on the plant (and thus time for additional necrosis development as lesions expanded), the stronger was the observed SAR response in leaf two. Using the HR induced by Ps. syringae pv. syringae as the inducing treatment, Smith et al. (1991) found that resistance could be induced in cucumber within 24 h. Taking advantage of this rapid response, Jennifer Smith and colleagues essentially repeated the experiments of Dean and Kuc´ (1986a), but removed the Ps. syringae-inoculated leaf at 0, 3, 6, 9 and 24 h after inoculation. The second leaf of all plants was challenged with Co. orbiculare at 24 h after the induction inoculation. Smith et al. (1991) reported that the first leaf only needed to be on the plant for as little as 6 h for a measurable amount of SAR to be expressed. Visual observations of the Ps. syringae-inoculated tissue showed initial signs of tissue collapse by 6 h after inoculation, but no clearly visible necrosis until several hours later (unpublished observation). Inoculation of cucumber plants with hrp (hypersensitive response and pathogenicity)-minus mutants of Ps. syringae pv. syringae did not result in the expression of the HR or SAR. Restoration of HR-inducing ability in the mutants by complementation restored the ability to induce SAR (Smith et al., 1991). Further studies on this system by Rasmussen et al. (1991) demonstrated that systemic induction of SA by Ps. syringae pv. syringae

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required that the inoculated first leaf only be on the plant for a few hours. Thus, confirming that the induction of SAR may occur prior to full expression of host necrosis. Further supporting a role for HR-like necrosis as part of the induction phase was the observation of Strobel et al. (1996) who used Ps. syringae 61 (Pss61) and HrpZPss protein from this bacterium to induce SAR in cucumber. Both the pathogen and the protein induced SAR that was effective against Co. orbiculare, Ps. syringae pv. lachrymans and TNV with the degree of protection comparable to that induced by Co. orbiculare. A hrpH mutant of Pss61 was unable to elicit SAR (possibly because it was unable to secrete the HrpZPss protein). Another Hrp protein, harpin (HrpNEa) was also reported to induce the HR and SAR response when sprayed onto tobacco or Arabidopsis leaves (Peng et al., 2003). Although no visible necrosis was observed, microscopic observations demonstrated that micro‐HRs were induced in both plant species, thus further supporting some association of pathogenesis-induced necrosis with the induction of SAR. However, is pathogen-induced necrosis really necessary to induce SAR? The experiments described thus far show possible correlations but little proof. In fact, the research by Smith et al. (1991) suggests that perhaps events leading up to necrosis (i.e., part of the initial phase of plant–microbe interaction) are what trigger SAR and that host cell death is not needed. Recently, the need for pathogen-induced necrosis for SAR induction has been questioned by Mishina and Zeier (2007). They found that Ps. syringae pv. glycinea and pv. phaseolicola do not elicit a HR in Arabidopsis but do induce SAR at a level similar to the HR-inducing Ps. syringae pv. tomato. Interestingly, hrp mutants of Ps. syringae pv. tomato and pv. phaseolicola also induced SAR in Arabidopsis. The authors showed that lipopolysaccharides (LPS) and flagellin protein, two pathogen-associated molecular patterns (PAMPs) (Nu¨rnberger and Kemmerling, 2009), were able to induce SAR in Arabidopsis. These results differ from other reports in which hrp mutants are unable to induce SAR (e.g., Smith et al., 1991). However, it may be that Arabidopsis may respond differently than other plant species as also reflected by the observation that necrosis induced in Arabidopsis by Botrytis cinerea did not result in SAR expression (Govrin and Levine, 2002). Another line of evidence that suggests that necrosis is not needed for the induction of SAR comes from the effect of systemic mosaic virus infection on disease resistance. Mu¨ller and Munro (1951) reported that infection of potato with potato virus X (PVX) or potato virus Y (PVY) increased resistance of the plants to infection by Phytophthora infestans. Both viruses were systemic and produced mosaic patterns in the potato plants.


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Van Loon (1975) provided further evidence that systemic infection with mosaic-inducing viruses would induce resistance. Inoculation of tobacco with PVYº or cucumber mosaic virus (CMV) did not induce necrosis, yet both viruses induced resistance and the accumulation of PR-proteins. It would be of great interest to determine if infection by these viruses also induces the accumulation of SA. The question still remains: Is necrosis needed for the induction of SAR? The answer is probably ‘‘no.’’ However, the events involved in the early stages of pathogenesis that lead to necrosis might be. It is also possible that what is true in one plant system may not be valid in another. This is shown by the differences in induction observed in the work of Smith et al. (1991) with cucumber and that of Mishina and Zeier (2007) with Arabidopsis. Application of molecular tools derived from studies on Arabidopsis SAR may prove very useful asking the same questions in other plant species. D. PATHOGEN-PRODUCED INDUCERS OF SAR

As described above, there are pathogen-derived molecules, such as LPS (Newman et al., 2001), that can induce systemic resistance and possibly SAR. Other known inducers are the unsaturated fatty acids arachidonic acid and eicosapentaenoic acid from Phy. infestans that were first identified as an elicitor of sesquiterpenoid phytoalexins in potato tuber tissue (Bostock et al., 1981). These fatty acids were later shown to induce systemic resistance to Phy. infestans in potato plants (Cohen et al., 1991). Other SAR-activating compounds that could be released from oomycetes and true fungi include cell wall fragments composed of chitosan, chitin, and -1,3-glucan (Lyon, 2007; Reglinski et al., 2007). These elicitors could be released from invading pathogens by action of host chitinases and -1,3-glucanases and act as activators of local, if not systemic, resistance. Other examples can be found in the reviews by Lyon (2007) and Reglinski et al. (2007). E. CHEMICAL INDUCTION OF SAR

In addition to pathogens, induced resistance can also be activated by chemicals. Many naturally occurring and synthetic chemicals have been shown to increase disease resistance, and the reader is directed to Lyon (2007) and Reignault and Walters (2007) for more comprehensive lists. Kessmann et al. (1994) provide an interesting background perspective on the use of chemicals as SAR activators. Whether or not all chemicals induce a SAR type of response is not known, and this section/chapter will focus on a few chemistries that appear to induce SAR.

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1. Salicylic acid One of the first chemicals shown to induce resistance is SA (2-hydroxybenzoic acid). Well before this simple hydroxybenzoic acid was demonstrated to be an endogenous signal for SAR (Malamy et al., 1990; Me´traux et al., 1990), White (1979) reported that the application of SA or acetyl salicylic acid (ASA) induced resistance to TMV in N gene tobacco. Further studies showed that application to tobacco would also induce the formation of PR-proteins (another characteristic of SAR) (Van Loon and Antoniw, 1982). Mills and Wood (1984) demonstrated that SA induced resistance in cucumber to Co. orbiculare, and Narusaka et al. (1999) reported that SA induced resistance in this species to Cl. cucumerinum. 2. 2,6-Dichloroisonicotinic acid DCINA (2,6-dichloroisonicotinic acid and its methyl ester) was the first synthetic compound produced as a resistance inducer (Me´traux et al., 1991). This compound induced resistance in cucumber, pear, pepper and rice (Me´traux et al., 1991). Ward et al. (1991) reported that DCINA induced resistance in tobacco to TMV and also elicited the same spectrum of PR genes induced by TMV and SA. These results provided very good evidence that DCINA was an inducer of SAR. Treatment of Arabidopsis with DCINA induced resistance to both Ps. syringae pv. maculicola and Hyaloperonospora arabidopsidis (formerly known as (Hyalo)peronospora parasitica) (Uknes et al., 1992). Based on these observations, DCINA appears to induce SAR by mimicking the activity of SA. Treatment of the unifoliate leaves of bean (Phaseolus vulgaris) with DCINA or inoculation with Colletotrichum lindemuthianum (the cause of bean anthracnose) induced resistance in the trifoliate leaves to Co. lindemuthianum and the bean rust pathogen, Uromyces appendiculatus (Dann and Deverall, 1995). DCINA also induced resistance to Ps. syringae pv. phaseolicola but not to root rotting Fusarium solani or Rhizoctonia (Dann and Deverall, 1995). Further studies showed that DCINA would also protect field grown beans against rust (Dann and Deverall, 1996). Treatment of unifoliate bean leaves with DCINA or Co. lindemuthianum induced an increase in activity of chitinase and -1,3-glucanase in upper, trifoliate leaves, thus further demonstrating the SAR-inducing ability of DCINA (Dann et al., 1996). Dann et al. (1998) tested the efficacy of DCINA for inducing resistance in soybean to the white mold pathogen, Sc. sclerotiorum in both the greenhouse and in the field. Multiple applications of DCINA were needed to result in a reduction of the lesions’ size in greenhouse studies. Three years of field trials showed some efficacy of DCINA in reducing white mold severity after multiple applications of the compound.


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3. Acibenzolar-S-methyl Acibenzolar-S-methyl (S-methyl benzo [1,2,3]thiadiazole-7-carbothioate, ASM; synonym: benzothiadiazole-7-carbothioic acid S-methyl ester, BTH) is another synthetic compound that induces SAR (Kunz et al., 1997; Tally et al., 1999). This material is commercially available as a plant activator and sold under the names of BionÒ , ActigardÒ , and BoostÒ (Leadbeater and Staub, 2007). Like DCINA, ASM is thought to be a functional analog of SA as it induces resistance to multiple pathogens and elicits the expression of PR genes associated with SAR (Oostendorp et al., 2001). In 1996, several papers demonstrating the SAR-inducing ability of ASM were published. Lawton et al. (1996) showed that treating Arabidopsis plants with ASM induced resistance to turnip crinkle virus, Ps. syringae pv. tomato and Hy. arabidopsis. ASM was also capable of inducing expression of PR-1, PR-2 and PR-5, thus further demonstrating that ASM induced a SAR-like response. Through the use of NahG plants that are unable to accumulate SA (Gaffney et al., 1993), these authors were able to show that ASM action did not require SA and thus likely functioned downstream of, or at the same site as SA. Using tobacco, Friedrich et al. (1996) found that both TMV and ASM induced SAR against TMV, Cercospora nicotianae, Erwinia carotovora, Ps. syringae pv. tabaci and Phytophthora parasitica. They also reported that ASM induced resistance to Pe. tabacina. The ability of ASM to induce SAR was further demonstrated by Friedrich et al. (1996) by the ability of this compound to induce expression of PR genes as well as genes for peroxidase and other defense-associated enzymes. Recent work by LaMondia (2008) noted that ASM, especially when combined with traditional fungicides, was an effective management tool for Pe. tabacina under field conditions. Go¨rlach et al. (1996) further expanded the diversity of plants induced by ASM by testing its effect on wheat. ASM induced resistance to the powdery mildew pathogen Blumeria graminis and induced the expression of a novel set of genes in wheat (WCI genes). Interestingly, two PR-1 genes from wheat, PR1.1 and PR1.2, were induced by both compatible and incompatible isolates of Bl. graminis but not by ASM, DCINA or SA. This suggested that ASM-modulated defense in wheat may differ from dicots. Smith-Becker et al. (2003) induced resistance in melons (Cucumis melo) to both CMV and Co. orbiculare. ASM greatly reduced the spread of CMV from infected leaves to young developing leaves and nearly completely eliminated the lesion development after challenge inoculation with the fungus. Huang et al. (2000) were able to protect the fruit of Hami and rock melons from postharvest fungal diseases. The ability of ASM to induce resistance is not restricted to herbaceous plants. Brisset et al. (2000) and Maxson-Stein et al. (2002) treated

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orchard-grown apple trees with ASM and were able to suppress the fire blight disease caused by Erwinia amylovora. More than one application was needed to reduce fire blight severity, but the control was similar to that observed with streptomycin. Local and systemic accumulation of peroxidase and -1,3-glucanase activity (Brisset et al., 2000) and induction of PR-1, PR-2, and PR-8 gene expression (Maxson-Stein et al., 2002) following ASM treatment accompanied the induction of resistance. ASM also induced resistance in Japanese pear trees against the scab pathogen Venturia nashicola (Ishii et al., 1999). They reported that the ASM-induced resistance to scab was similar to control by polycarbamate fungicides. There are many other examples of ASMinduced resistance in the literature, and reviews by Reglinski et al. (2007) and Reignault and Walters (2007) provide much of this information. 4. Tiadinil A new plant activator, Tiadinil (TDL, N-(3-chloro-4-methylphenyl)-4-methyl1,2,3-thiadiazole-5-carboxamide), was developed for rice blast management (Yasuda et al., 2004, 2006). TDL also induces resistance in tobacco to Ps. syringae pv. tabaci and TMV as well as expression of PR genes. TDL is metabolized in rice to 4-methyl-1,2,3-thiadiazole-5-carboxylic acid which is also capable of inducing resistance and PR gene expression in tobacco. This metabolite is also effective in tobacco expressing NahG suggesting that these compounds, like DCINA and ASM, act downstream of SA. 5. Other chemical inducers There are many other synthetic and natural compounds that have been reported as activators of disease resistance, and there are several reviews that provide information on these materials (Deverall, 1995; Kessmann et al., 1994; Leadbeater and Staub, 2007; Lyon, 2007). In many cases, the mode of action of the compounds or materials that have been reported to induce resistance is not known. Some, like the nonprotein amino acid -aminobutyric acid (BABA) have been reported to induce resistance in a number of plant species (Cohen, 2002). The mode of action of this chemical suggests that it may function through both SA-mediated resistance (Siegrist et al., 2000) as well as abscisic acid signaling (Ton and Mauch-Mani, 2004). Thus, BABA may induce SAR (albeit via SA induction) and certainly other defense signaling pathways. The harpin protein from Erw. amylovora induces resistance in Arabidopsis to Hy. arabidopsidis and Ps. syringae pv. tomato and also induces expression of PR-1 and PR-2 genes (Dong et al., 1999). Because of PR gene expression and the observation that harpin did not induce resistance in NahG plants, harpin is likely an inducer of SAR through the induction of SA biosynthesis.


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The ability of harpin to induce microlesions which trigger the SAR response also suggests that this protein induces SAR (Peng et al., 2003). Another compound that activates resistance through induction of SA production is the fungicide probenazole (Leadbeater and Staub, 2007). Probenazole and its metabolite 1,2-benzoisothiazole-1,1-dioxide (saccharin) were shown to induce SAR by their ability to induce PR gene expression (Yoshioka et al., 2001). Unlike ASM or DCINA, these activators appear to act upstream of SA as both induce the accumulation of SA (Nakashita et al., 2002). Probenazole or saccharin treatment of NahG tobacco plants did not result in the induction of SAR, thus demonstrating the need for SA accumulation as part of the mode of action of these compounds (Yoshioka et al., 2001). Saccharin is also able to induce resistance in barley to powdery mildew (Boyle and Walters, 2006) and in broad bean to rust (Boyle and Walters, 2005). The strobilurin fungicide pyraclostrobin has also been reported to induce resistance. Injection of tobacco leaves with this compound induced resistance to TMV and Ps. syringae pv. tabaci (Herms et al., 2002). Pyraclostrobin by itself did not induce PR-1 protein accumulation. However, the fungicide did prime the tissue to more rapidly express PR-1 and accumulate PR-1 protein after inoculation with TMV. This suggests that pyraclostrobin does not induce SAR in the classical sense. However, the observation that the tissues were primed suggests that pyraclostrobin is an inducer of SAR or a SAR-like response. This study also illustrates the type of work that is needed to determine if and how materials reported to induce resistance actually function in disease suppression.

IV. SYSTEMIC BIOCHEMICAL CHANGES It is logical to assume that if plant tissues at some distance from an induction treatment are more effective in stopping pathogens than noninduced tissues, it follows that there are measurable biochemical changes in the induced tissues. This chapter will describe some of the biochemical changes that have been reported. A. PATHOGENESIS-RELATED PROTEINS

In the early 1970s, the systemic increase in novel proteins in N gene tobacco infected with TMV was reported (Van Loon, 1975; Van Loon and Van Kammen, 1970). These proteins were not found in uninfected tissues and were not peroxidases or other enzymes known to increase systemically after

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TMV infection (e.g., Simons and Ross, 1970). Van Loon and Van Kammen (1970) proposed that these proteins were involved in the SAR response. Following the work of White (1979) who found ASA and SA would induce resistance in tobacco, Van Loon and Antoniw (1982) reported that SA was also capable of inducing these SAR-associated proteins. Over a period of time, 17 families of proteins, now known as PR-proteins have been identified in a number of plant species and putative functions such as chitinase and -1,3-glucanase have been determined for some of these proteins (for reviews see Van Loon, 1997; Van Loon et al., 2006). B. OTHER PROTEINS

In addition to the classical PR-proteins, the accumulation of other putative defense-related proteins or expression of their genes has been associated with SAR. In some of A. Frank Ross’s early work on SAR induction in tobacco, he and colleagues reported on TMV-induced systemic increases in peroxidase, catalase and glucose-6-phosphate dehydrogenase activities (Simons and Ross, 1970, 1971a) but not polyphenoloxidase (Bozarth and Ross, 1964). A change in peroxidase activity was also observed by Van Loon and Geelen (1971). These results indicate an increase in oxidative metabolism as well as a possible increase in respiratory metabolism mediated by the increase in glucose-6-phosphate dehydrogenase. In cucumber and other cucurbits, a set of acidic, apoplastic peroxidases accumulates systemically upon infection of one leaf with necrotizing pathogens (Hammerschmidt et al., 1982; Smith and Hammerschmidt, 1988). These same peroxidases and their transcripts also accumulate in response to SA treatment (Rasmussen et al., 1991, 1994). Systemic increases in a microsomal callose synthase have been reported in cucumber plants expressing systemic resistance (Schmele and Kauss, 1990). As cell wall alteration involving callose deposition may be part of the defense of plants, activation of this enzyme may be part of the defense against cell wall penetrating pathogens. Hydroxyproline-rich glycoproteins (HRGP) are structural components of plant cell walls that have been implicated in host defense responses (e.g., Hammerschmidt et al., 1984; Stermer and Hammerschmidt, 1987). Accumulation of these proteins increases the resistance of cell walls to cell wall-degrading enzymes (Stermer and Hammerschmidt, 1987). Induction of resistance in tobacco by TMV results in systemic accumulation of HRGP in the host cell walls and resistance to Pe. tabacina (Ye et al., 1992) and Erysiphe cichoracearum (Raggi, 1998). Similarly, PVYN, which causes veinal necrosis in tobacco, also induced resistance to Ery. cichoracearum and caused a systemic increase in HRGP (Raggi, 2000). Treatment of tobacco leaves


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with SA or ASM resulted in local increases in HRGP content of cell walls. ASM, but not SA, also caused a systemic increase in HRGP (Raggi, 2007). This difference is likely the result of the different mobility of SA as compared to ASM after topical application. Since treatments with ASM and SA also result in increases in cell wall associated peroxidases, it is possible that one function of the induced peroxidase activity is to facilitate the incorporation of newly synthesized HRGP into cell walls (Sommer-Knudsen et al., 1998). In addition to proteins considered to be part of defense responses, induction of SAR also results in expression of genes encoding types of proteins used for pathogen detection. In 1999, Sakamoto et al. reported that induction of resistance in rice by probenazole was associated with the expression of a gene, RICE PROBENAZOLE-RESPONSIVE 1 (RPR1), which codes for a protein with a nucleotide binding site and leucine-rich repeats (NBS-LRR). SA treatment of rice resulted in induction of the NBS-LRR Pib rice-blastresistance gene family (Wang et al., 2001). Finally, a similar result was reported by Faize et al. (2007) who found that ASM treatment of Japanese pear resulted in expression of a gene for a leucine-rich repeat receptor-like protein kinase (LRR-RLK). Taken together, these reports suggest that one aspect of resistance induction may involve the expression of genes involved in pathogen detection and, therefore, may allow the induced plants the ability to more rapidly respond to attempted infections. C. SA ACCUMULATION

Induction of SAR in tobacco and cucumber results in the systemic accumulation of SA (Malamy et al., 1990; Me´traux et al., 1990). Because of the resistance-inducing properties of SA and the observation that this simple phenol was found in the phloem of cucumber during the induction process, it seemed plausible that SA was the mobile signal required for SAR development. In fact, there is evidence for the systemic transport of SA out of infected leaves of cucumber (Mo¨lders et al., 1996) and tobacco (Shulaev et al., 1995). Although some SA may be transported out of leaves undergoing necrotic lesion formation, work by Rasmussen et al. (1991) with cucumber and Vernooij et al. (1994) with tobacco cast doubt on the role of SA as the primary systemically transported signal, and suggested that it may be synthesized systemically in response to another, yet to be described, mobile signal. This concept was supported by observations that some SA was synthesized at a distance from inducing inoculations. Meuwly et al. (1995) found that both locally inoculated and systemically induced cucumber tissues produced more SA from phenylalanine than did control plants. The conversion of phenylalanine to SA was blocked by the

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phenylalanine ammonia-lyase (PAL) inhibitor 2-aminoindan-2-phosphonic acid. Also working with cucumber, Smith-Becker et al. (1998) found an increase in PAL in petioles of leaves inoculated with Ps. syringae pv. syringae and stem tissue above the inoculated leaf at 9 and 12 h after inoculation. Both SA and 4-hydroxybenzoic acid could be detected in petiole and stem phloem exudates at a time corresponding to increased PAL activity. More details on the role of SA and systemic signals can be found in the chapter by Champigny and Cameron (2009).

V. HOW SAR PROTECTS PLANTS AGAINST PATHOGENS How plants actually stop pathogen development and suppress disease symptoms in plants expressing SAR is slowly being revealed, and progress has been made since I asked this question a decade ago (Hammerschmidt, 1999a). The production of PR-proteins as a result of resistance induction is likely part of the defense process (Van Loon, 1997; Van Loon et al., 2006) and possibly serving as a ‘‘preformed’’ defense against infection. However, plants expressing high levels of these proteins may not be protected against disease. For example, the activity of chitinase, a major PR-protein in cucumber (Me´traux et al., 1988, 1989), remains high in leaves even after the acquired resistance levels decline (Dalisay and Kuc´, 1995). This work and other lines of evidence (see Van Loon, 1997, for review) suggest that the PR-proteins play some, but perhaps not a decisive defense role in SAR. A. PRIMING

Plants that are expressing SAR respond to infection by rapidly deploying defenses suggesting that the induced state also sensitizes the plant to detect new infections. This phenomenon, known as priming, has been demonstrated for both the SAR and other forms of induced resistance (Conrath et al., 2002, 2006). As the molecular regulation and other aspects of priming are discussed in detail by Conrath (2009), the remaining part of this section/chapter will examine which defenses are induced in SAR-expressing plants after infection with pathogens. B. PROTECTION AGAINST FUNGI AND OOMYCETES

1. Cucurbits The first report of SAR in cucumber described the ability of Co. orbiculare to induce resistance to itself (Kuc´ et al., 1975). Characteristics of the induced resistance to Co. orbiculare are the formation of both fewer and smaller


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lesions (Kuc´ and Richmond, 1977). These authors also reported that the SAR response was seen in cucumber cultivars that are susceptible as well as resistant to Co. orbiculare. Further work by Joe Kuc´ and others showed the induced resistance in cucumber was effective against other fungal pathogens (reviewed in Hammerschmidt and Yang-Cashman, 1995; Kuc´, 1982). The reason why SAR-expressing plants developed fewer lesions caused by Co. orbiculare was addressed by Richmond et al. (1979) using light microscopy. Prepenetration effects on expression of the resistance were ruled out by noting that the numbers of melanized appressoria produced by the pathogen were the same on both controls and SAR-expressing plants. Thus, the resistance could not be explained by the formation of a preformed antimicrobial compound on the surface of the plants. What Richmond et al. (1979) did find was a significant reduction in the number of successful penetrations from appressoria on the induced ones as compared to the control plants. At 60 h after inoculation, up to 40% of the appressoria on the control plants were associated with successful penetration into the host while only around a 5% success rate was found on the induced plants. This phenomenon was observed in several cucumber cultivars including some carrying genetic resistance to Co. orbiculare. Inoculation of resistant plants not expressing SAR resulted in the penetrated cells undergoing necrosis (possibly a HR). When the resistant plants were induced, there were far fewer penetrations, although those cells that were penetrated did become necrotic. Damage to SARexpressing leaves with Carborundum or needle punctures resulted in a loss of resistance to Co. orbiculare, suggesting that an intact epidermis was required for SAR expression. Removing the epidermis effectively eliminated the SAR-mediated resistance. Similar results were obtained by Jenns and Kuc´ (1980) using TNV as the inducing pathogen, thus demonstrating that the ability to reduce penetration efficiency in induced cucumber, like the reduction in disease severity, was independent of the inducing pathogen. A reduction in penetration is also a very likely explanation for why increasing inoculum concentration increased severity of disease development on both control and SAR-expressing cucumber (Dean and Kuc´, 1986b). Although the results discussed above show there was reduced penetration efficiency into SAR-expressing plants, they do not explain why. Histochemical staining of cucumber epidermal peels from induced and control plants inoculated with Co. orbiculare revealed the deposition of a lignin-like material under appressoria that had not penetrated into the induced plants (Hammerschmidt and Kuc´, 1982). The number of lignified sites was very similar to the numbers of successful penetrations into control tissues (as measured over time). Thus, it can be inferred that the pathogen was attempting to penetrate into the induced epidermal cells but was blocked

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by the formation of a structural barrier. Attempted infection of another cucumber pathogen, Cl. cucumerinum, into induced tissues also resulted in deposition of lignin at and near the site of penetration (Hammerschmidt and Kuc´, 1982). Papillae formation has also been associated with blocked penetration of Co. orbiculare into induced cucumber leaves. Stumm and Gessler (1986) reported that failure to penetrate was associated with fluorescent papillae directly under appressoria. Where penetration occurred, no papillae or very small, fluorescent papillae formed. Kovats et al. (1991a) confirmed the previous observations of Hammerschmidt and Kuc´ (1982) and Stumm and Gessler (1986) by showing the reduced penetration of Co. orbiculare was directly related to lignified papillae. Using Aniline Blue staining, they also found that the papillae contained callose. The latter is not surprising since callose is a common component of papillae (Aist, 1976). The deposition of callose also provides a role for the callose synthase that Schmele and Kauss (1990) reported to be more active in plants expressing SAR. An ultrastructural study of the response of induced cucumber to Co. orbiculare also supported the role of cell wall modifications and papillae in the resistance (Stein et al., 1993). Ultrastructural histochemistry coupled with energy dispersive X-ray analysis revealed that the cell wall alterations contained phenolic compounds. Silicon was also detected in the cell wall directly under appressoria that failed to penetrate into induced tissues (Kauss et al., 2003). Hyphae that successfully penetrated into the induced host were found to be encased in phenolic compounds (Stein et al., 1993). Diaminobenzidine staining provided evidence for peroxidase activity localized in the same regions where phenolic compounds were deposited (B. D. Stein and R. Hammerschmidt, unpublished data). However, it is not known if these peroxidases are the same as those induced systemically in cucumber after induction of SAR (Hammerschmidt et al., 1982; Smith and Hammerschmidt, 1988). SAR can also be induced in cucumber against powdery mildew caused by Sphaerotheca fuliginea using TNV as the inducing inoculum (Bashan and Cohen, 1983; Conti et al., 1990). In the former study, TNV induction reduced the numbers of powdery mildew colonies that formed on inoculated leaves as compared to the control. In addition, lignification of epidermal cells after Sp. fuliginea inoculation was observed much more frequently in the induced as compared to the control plants. In the latter study, histological observations revealed that papillae formed at infection sites on induced plants were very fluorescent and stained positively for phenolic compounds and lignin. In a subsequent study (Conti et al., 1994), Sp. fuliginea conidial germination, germ tube length and numbers of haustoria were reduced on TNV-induced plants as compared to controls. The decrease in germination and germ tube


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length on the surface of induced leaves suggested the presence of an inhibitor on the leaf surface. Increases in epidermal lignification as part of the response to Sp. fuliginea were also confirmed. Finally, treatment of cucumber plants with SA resulted in resistance against Sp. fuliginea that was very similar to the resistance induced by TMV in terms of disease suppression and cell wall modifications (Conti et al., 1996). SAR may not always protect plants against infection. Although it is now widely known that different forms of induced resistance ‘‘target’’ different pathogens types, one of the first ‘‘failures’’ was reported by Bashan and Cohen (1983). In the same paper in which successful resistance to powdery mildew of cucumber was reported, they also reported that TNV did not induce systemic resistance to the oomycete Pseudoperonospora cubensis (the cause of downy mildew). The reason for the failure to induce resistance was not elucidated, but the authors suggest that it may reside in the fact that this pathogen penetrates through stomata. This bypasses the epidermis which is where defense responses to true fungi, as discussed above, have been described. However, exogenous application of SA was later shown to induce resistance to Ps. cubensis (Okuno et al., 1991), thus contradicting the report of Bashan and Cohen (1983). The nature of the differences in these reports needs clarification since both TNV and SA should induce SAR in cucumber. However, Okuno et al. (1991) suggested that the concentration of inoculum used in the challenge may explain the differences between their results and those of Bashan and Cohen (1983). ASM also induces resistance in cucumber to Pythium (Benhamou and Be´langer, 1998a). In this interaction, roots from ASM-treated plants responded to infection by Pythium ultimum with the accumulation of phenolic materials in the cortex and vascular parenchyma cells. The phenolic materials appeared to block the progress of the pathogen toward the stele whereas the pathogen appeared to easily colonize root tissue in the noninduced plants. The observation that induced cucumber plants readily produce phenolic materials such as lignin suggests that these plants respond to infection by induction of enzymes involved in this process. ASM-treated cucumber plants respond to infection by Co. orbiculare by a rapid induction of genes encoding peroxidase and PAL (Cools and Ishii, 2002). As would be predicted by previous work on the cucumber SAR response (e.g., Hammerschmidt et al., 1982; Rasmussen et al., 1994), ASM treatment induced peroxidase gene expression. Cools and Ishii (2002) also reported the induction of PR-1 gene expression in response to ASM. PAL, however, was not induced by ASM, but its expression was enhanced in ASM-treated tissues after inoculation with Co. orbiculare. The effect of ASM as a priming agent for pathogen

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response in cucumber was later illustrated by the induction of a lignin peroxidase and a callose synthase gene after inoculation with Co. orbiculare (Deepak et al., 2006). Expression of these genes would be important for the production of papilla. Interestingly, genes for cinnamyl alcohol dehydrogenase and caffeoyl-CoA 3-o-methyl transferase were not induced after pathogen challenge (Deepak et al., 2006). Since the lignin-like material produced by cucurbits after infection is low in methoxylated components (Hammerschmidt et al., 1985), the lack of induction of the o-methyl transferase is perhaps not surprising. It would be interesting to determine the activity of the other enzymes that catalyze steps in lignin biosynthesis in induced plants before and after challenge. 2. Legumes Induced resistance (local, at a short distance and systemic from the point of induction) has been demonstrated in green bean (Pha. vulgaris) against the anthracnose fungus (Co. lindemuthianum). Early studies by Rahe et al. (1969) demonstrated that prior inoculation of bean hypocotyls with Helminthosporium carbonum, Alternaria sp. or incompatible races of Co. lindemuthianum induced resistance against compatible races of Co. lindemuthianum. Similarly, Skipp and Deverall (1973) reported that incompatible races of Co. lindemuthianum would induce resistance in green tissues of bean, including immature pods. However, as the inoculations in these studies were performed by inoculating entire plants or plant parts with inducer and challenge fungi, it is difficult to determine from these experiments if the induced resistance was due to the observed accumulation of phytoalexins elicited by the inducer inoculations (Rahe et al., 1969) or activation of the potential for resistance in tissues not directly affected by the inducing fungi. The question raised above was answered, in part, through a series of papers by Elliston et al. In 1971 they reported that inoculation of specific sites on bean hypocotyls with incompatible races of Co. lindemuthianum induced resistance in neighboring tissue to subsequent attack by compatible races of the fungus. Induced resistance was expressed if 18 or more hours elapsed between the time of the inducing inoculation and that of the challenge inoculation. Although the type of induced resistance described by Elliston et al. (1971) is not truly systemic in the sense in which the term is currently used (they challenged the tissue only 0.5 cm from the site of the inducer inoculation), this work did demonstrate that cells at a distance from the inducer site had become "conditioned" or ‘‘primed’’ to behave in a resistant manner. In a subsequent study, Elliston et al. (1976a) demonstrated that local and systemic induced resistance could be elicited by a number of different


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Colletotrichum species. Nonpathogenic races of Co. lindemuthianum as well as Co. orbiculare and Colletotrichum trifolii, induced a high level of local resistance to cultivar‐pathogenic races of Co. lindemuthianum. However, only Co. lindemuthianum and Co. orbiculare were able to elicit ‘‘systemic’’ resistance. This may be a reflection of the failure of Co. trifolii to cause a hypersensitive necrosis in bean tissue, whereas the other fungi elicited the hypersensitive response. Histological studies revealed that the development of compatible races of Co. lindemuthianum into control and induced tissues was initially the same (Elliston et al., 1976b). In induced tissue, however, the pathogen developed a limited amount of primary mycelium, but then was unable to develop any further. They found that the induced resistance was very similar to mature tissue resistance found in hypocotyls of all bean cultivars, and was distinctly different from the HR observed in tissue responding to an incompatible race of Co. lindemuthianum. Elliston et al. (1977) also reported that fungi capable of inducing systemic resistance elicited a localized accumulation of phytoalexins. However, there was no systemic change in phytoalexin content of the tissue. Induced tissue did, however, produce phytoalexins upon challenge with compatible races of Co. lindemuthianum. Thus, the induced resistance sensitizes tissues in a way that they will more rapidly produce phytoalexins in response to an attempted infection. Induction of phytoalexins by the inducer fungi may be involved in local induced resistance (Elliston et al., 1977). Local resistance was induced by Co. trifolii, even though it does not elicit phytoalexin accumulation. Although phytoalexin accumulation and an enhanced ability to produce phytoalexins may play a role in local and systemic induced resistance, respectively, it is obvious from the resistance induced by Co. trifolii that phytoalexin production was not the sole answer. True SAR also occurs in legumes. Sutton (1979) and Cloud and Deverall (1987) demonstrated that inoculation of primary leaves of Pha. vulgaris with Co. lindemuthianum induced resistance in upper trifoliate leaves against the same pathogen. In addition, inoculation of the hypocotyl also induced resistance in the leaves. This suggests that the induced resistance observed by Elliston et al. (1971) in hypocotyls may be a similar phenomenon. Fewer successful penetrations from appressoria occurred in leaves from plants with acquired resistance and cell wall appositions appeared to be part of this process (Cloud and Deverall, 1987). More rapid cell death of invaded cells occurred in the induced plants as compared to controls. SAR induced by ASM has been reported for the cowpea-Colletotrichum destructivum interaction (Latunde-Dada and Lucas, 2001). Penetration by Co. destructivum into ASM-induced tissues was greatly reduced as compared

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to controls, and, therefore, was similar to what is observed in both bean and cucumber. Where penetration into induced host tissues occurred, the invaded cells responded with a hypersensitive-like response and there was no transition by the invading pathogen to the more necrotrophic, secondary hyphal phase of pathogenesis. Challenge of ASM-induced tissues with Co. destructivum also resulted in a rapid, albeit transient, increase in activity of PAL and chalcone isomerase, both key enzymes in flavonoid biosynthesis. These two enzymes reached peak activity at 18 and 24 h postchallenge inoculation, respectively. Increases in the accumulation of the phytoalexins kievitone and phaseollidin also occurred more rapidly in the induced tissues. Results presented by Latunde-Dada and Lucas (2001) showed an increase in phaseollidin by 24 h and maximum accumulation at 96 h after inoculation. Phaseollidin was detected in inoculated control tissues at 48 h, and accumulated to a lower amount than observed in the induced tissues. Thus, it is likely that the expression of SAR in this host is associated with the ability to rapidly activate enzymes involved in phenolic compound biosynthesis and the accumulation of isoflavonoid phytoalexins. Other details on induced resistance in legumes can be found in Deverall and Dann (1995). 3. Solanaceous species In the early 1960s, systemic protection of tobacco foliage against Pe. tabacina was found to develop following stem infection by the same fungus (Cruickshank and Mandryk, 1960; Pont, 1959). In later studies, Cohen and Kuc´ (1981) further demonstrated that stem injection or inoculation of the stem–root interface of tobacco with conidia of Pe. tabacina induced resistance to foliar attack by the same pathogen. The induction of resistance was associated with necrosis of the external phloem and cambium of the stem. As in other induced resistance systems, a certain period of time was required between the inducing inoculation and expression of systemic resistance. Cohen and Kuc´ (1981) reported that 3 weeks were required to attain 95% protection. Inducing resistance in tobacco against Pe. tabacina by the methods of Cohen and Kuc´ (1981) and of Cruickshank and Mandryk (1960) also resulted in plant stunting and production of suckers. However, inoculating stem tissue exterior to the cambium resulted in an expression of induced resistance against Pe. tabacina and also induced increased growth of the plants (Tuzun and Kuc´, 1985). Thus the stunting, and therefore yield reduction, is not necessarily associated with the induction of resistance in tobacco to this disease. Although tobacco has been a model for many studies on SAR, most of the research has focused on the induction phase and the systemic regulation and


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accumulation of PR- and other proteins. There are a few studies that do provide some insight into how SAR-expressing tobacco and other solanaceous species respond to infection by fungi. A possible mechanism for the acquired resistance to Pe. tabacina was reported by Stolle et al. (1988) and Ye et al. (1992). In the former report, colonization of induced host tissues ceased after about 5 days and sporangia production was reduced by 70% as compared to noninduced controls. Sesquiterpenoid phytoalexin production was observed in both induced and control tissues, but higher concentrations of these compounds occurred in the Pe. tabacina-challenged controls. Further study is needed to determine if the phytoalexins are a component of resistance in SAR-expressing tissues. Using light microscopy, Ye et al. (1992) found that development of Pe. tabacina was restricted in the induced tissue and some of the hyphae exhibited cellular disorganization. Ultrastructural analysis indicated that host cell wall appositions and increased electron-opaqueness of the cell walls were also characteristic of the induced host response to the pathogen. Ribonuclease activity increased in induced plants more rapidly after infection than was observed in control plants (Lusso and Kuc, 1995). The relationship between an increase in this enzyme and defense was not determined. SAR in potato can be induced by a prior inoculation with Phy. infestans (Stromberg and Brishammar, 1991). Challenge inoculations of the induced plants with Phy. infestans revealed that the resistance was associated with a more rapid deposition of papillae and decreased efficiency of penetration (Stromberg and Brishammar, 1993). The development of hyphae that were able to enter the mesophyll tissues was much more restricted in the induced as compared to control plants (Stromberg and Brishammar, 1993). SAR can also be induced in tomato by prior inoculation with TNV (Anfoka and Buchenauer, 1997). In this case, the acquired resistance appeared to be associated with reduced penetration by Phy. infestans, but this apparently was not associated with callose deposition. In addition, a hypersensitive-like response occurred more frequently in the mesophyll of induced plants as compared to controls (Kovats et al., 1991b). Jeun et al. (2000) likewise induced resistance in tomato to Phy. infestans with TNV and also found that reduced penetration occurred into induced plants. However, these authors reported that the decrease in penetration was associated with callose deposition. Recently, the effect of NahG on basal resistance in potato to Phy. infestans has been reported (Halim et al., 2007). Although there was little difference in susceptibility observed at a macroscopic level, NahG expression in the plants did allow more growth of the pathogen in the tissue. Treatment of NahG plants with DCINA reversed the ability of the plants to support more pathogen growth. Callose deposition, a defense associated with SAR in

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potato (see preceding paragraph), was also decreased in NahG plants infected with Phy. infestans. Taken together, these results suggest that SA-mediated defenses may be needed for both basal and systemic resistance responses. ASM-induced resistance in tomato to Fusarium oxysporum f.sp. radicislycopersici was reported to be associated with rapid deposition of callose and phenolic materials in host cells walls (Benhamou and Be´langer, 1998b). This further demonstrated that SAR can be induced in roots and provided additional evidence for the role of cell wall modifications in the defense responses elicited by pathogens in induced tissue. 4. Arabidopsis Induction of SAR against the oomycete Hy. arabidopsidis was first reported by Uknes et al. (1992) who used DCINA as the inducing agent. Depending on the amount of DCINA applied to the plants, challenge of the plants with Hy. arabidopsidis resulted in single-cell host necrosis (HR) at the site of penetration to ‘‘delayed’’ necrosis of host cells along hyphae growing in the host. Arabidopsis SAR against Hy. arabidopsidis was further demonstrated by Mauch-Mani and Slusarenko (1994) who used F. oxysporum as the inducing agent. If the plants were challenged more than 4 days after the inducing inoculation, Hy. arabidopsidis induced single host cell hypersensitivity at the site of attempted penetration. If the plants were challenged before that time, the pathogen was able to infect, but there was the developing of host cell necrosis (‘‘trailing necrosis’’) along the pathogen hyphae. The deposition of lignin, based on histochemical staining, was reported to be associated with the trailing necrosis observed in Hy. arabidopsidis-infected plants (Mauch-Mani and Slusarenko, 1996). These authors concluded that lignification was part of the defense but not the deciding factor. These studies show that expression of SAR primes Arabidopsis to respond to Hy. arabidopsidis with a hypersensitive-like host response. Inoculation of Arabidopsis nim1 (non‐inducible immunity 1) mutants (allelic to npr1 in tobacco) or NahG plants resulted in increased susceptibility to Hy. arabidopsidis and greatly reduced expression of PR-1 (Donofrio and Delaney, 2001). In NahG plants, there was more pathogen biomass as well as less callose deposition around haustoria of Hy. arabidopsidis. Treatment of NahG plants with DCINA reversed the increased susceptibility and decreased ability to deposit callose. nim1 mutants were not as susceptible nor showed as much callose reduction when compared to NahG plants. These results support the role of SA in basal defenses and further indicate that SAR may be an enhancement of basal resistance that includes cell wall modifications.


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5. Japanese pear ASM also induces resistance in Japanese Pear to the scab fungus, V. nashicola (Ishii et al., 1999). Inoculation of ASM-treated pear leaves with V. nashicola resulted in the rapid, though transient, expression of transcripts encoding polygalacturonase inhibiting protein (PGIP) (Faize et al., 2004). Other defense-associated enzymes such as chitinase and peroxidase as well as certain PR-proteins were also expressed more rapidly after fungal infection in ASM-treated leaves as compared to controls. Ultrastructural analysis of V. nishicola-infected ASM-induced pear leaves (Jiang et al., 2008) revealed a higher incidence of fungal hyphae collapse and less pectin degradation in host cell walls suggesting that the induced chitinase and PGIP reported by Faize et al. (2004) may be components of the ASM-induced resistance expression. 6. Cereals Although there are numerous reports describing induced resistance in cereals, relatively few have documented the induced resistance as SAR and/or have examined the defenses activated after subsequent pathogen inoculations (Kogel and Langen, 2005). Using DCINA as the SAR activator, induced barley was shown to respond to infection with Bl. graminis f.sp. hordei in a way that resembled race-specific resistance conditioned by the Mlg gene (Kogel et al., 1994). In the induced plants, the numbers of fungal attacked host cells that produced papillae with strong autofluorescence increased over the noninduced controls. There was also a significant decrease in haustorium formation in the epidermal cells of induced plants, which was associated with a large increase in the number of attacked host cells responding hypersensitively. The induction treatment increased expression of peroxidase transcripts prior to inoculation, and the expression of this gene was increased even higher after challenge of the induced plants as compared to controls. In a subsequent study, DCINA‐induced barley seedlings were used to determine if the induced response was associated with superoxide radical anions (Kogel and Hu¨ckelhoven, 1999). As in the previous study (Kogel et al., 1994), much of the induced resistance response was associated with failure of Blumeria to invade the epidermal cells, an increase in hypersensitively responding host cells, and a decrease in successful haustorium formation. Although the formation of autofluorescent papillae and expression of hypersensitive cell death suggests that reactive oxygen species (ROS) were produced, no superoxide was detected in induced epidermal cells under attack by the pathogen. This was similar to what was observed in Mlg gene-mediated resistance to Bl. graminis in barley (Hu¨ckelhoven and Kogel, 1998).

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Saccharin-induced resistance of barley to Bl. graminis f.sp. hordei was associated with increases in peroxidase activity after challenge of induced plants (Boyle and Walters, 2006). These authors also reported an increase in cinnamyl-alcohol dehydrogenase after saccharin treatment that increased further upon challenge with Bl. graminis. Not surprisingly, the changes in activity were dependent on the amount of time between saccharin treatment and challenge.

C. PROTECTION AGAINST BACTERIA

1. Examples of induced resistance to bacterial pathogens Induction of resistance against bacterial pathogens is also well known. Although not truly systemic, Lovrekovich et al. (1968) found that inoculating the apical half of tobacco leaves with TMV induced resistance in the basal half of the leaf to Ps. syringae pv. tabaci. In this system, the line of tobacco used responded hypersensitively to TMV. In 1970, Lozano and Sequeira reported that the HR of tobacco leaves elicited by race 2 of Ralstonia solanacearum (formerly: Pseudomonas solanacearum) could be prevented if the area to be inoculated (by infiltration) with race 2 was previously infiltrated with heat killed cells of race 1 or race 2. Heat killed cells of other bacterial pathogens (e.g., Ps. syringae pv. lachrymans) also caused this effect. The induced resistance against the HR could be observed by 18 h after infiltration with the heat killed cells. From this time onward, the induced resistance was found to spread to neighboring cells and eventually (within 48 h) to become systemic. The induced resistance response was also effective against disease caused by a compatible isolate of race 1 of R. solanacearum (Lozano and Sequeira, 1970). Whether or not this resistance is SA-mediated has not been addressed in this system. In cucumber plants, inoculating one leaf with Ps. syringae pv. lachrymans, the angular leaf spot pathogen, induced resistance to Ps. syringae pv. lachrymans and to Co. orbiculare (Caruso and Kuc´, 1979). In this system, resistance was induced by a compatible interaction between the host and inducing pathogen and was thus similar to the induction of resistance in cucumber by Co. orbiculare against Co. orbiculare (Kuc´ et al., 1975). Systemic induction of resistance against Ps. syringae pv. lachrymans by Co. orbiculare was also reported by Doss and Hevisi (1981). SAR that is effective against bacterial pathogens has also been reported for Arabidopsis using both chemical (Uknes et al., 1992) and biological inducers (Cameron et al., 1994; Uknes et al., 1993).


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2. How SAR protects against bacterial pathogens How induced resistance reduces bacterial pathogen symptoms, whether the necrotic lesion associated with disease or with the HR, is not well understood (Lozano and Sequeira, 1970; Summermatter et al., 1995). Lovrekovich et al. (1968) and Caruso and Kuc´ (1979) found decreased populations of bacteria in the inoculated induced leaves as compared to controls. In the localized induced resistance described first by Lozano and Sequeira (1970), there was a much more rapid decrease in bacterial numbers in the induced plants (Sequeira and Hill, 1974). In Arabidopsis, SAR also resulted in a decrease in the growth of bacterial pathogens in induced tissues (Cameron et al., 1994; Uknes et al., 1993). These observations were further supported by Pieterse et al. (1998) who found that SAR induced in Arabidopsis by avirulent Ps. syringae pv. tomato decreased growth of virulent Ps. syringae pv. tomato in induced tissues. Suppression of symptoms, instead of reducing bacterial populations, has also been suggested as a factor in the induced resistance against these pathogens. Doss and Hevisi (1981) reported that systemic resistance in cucumber to Ps. syringae pv. lachrymans was not associated with an effect on bacterial growth in the induced tissues, thus contradicting the work of Caruso and Kuc´ (1979). In Arabidopsis, induction of SAR by the HR-inducing Ps. syringae pv. syringae resulted in a suppression of hypersensitive necrosis after challenge by the same bacterium in induced upper leaves (Summermatter et al., 1995). Similar to the observation of Doss and Hevisi (1981), there was little difference in bacterial growth in induced as compared to control leaves. These results suggest that the SAR phenomenon may be a focus of tolerance to disease rather than resistance. Suppression of disease symptoms was also reported in the systemic induction of resistance in tomato by both virulent and avirulent strains of Xanthomonas campestris pv. vesicatoria against the same pathogen (Block et al., 2005). PR-1a and PR-1b genes were expressed systemically after an inducing inoculation with either strain and both genes showed strong expression in systemically induced leaves at 24 h after challenge with a virulent strain of X. campestris pv. vesicatoria. Systemic resistance could also be induced by Ps. syringae pv. tomato against itself and X. campestris pv. vesicatoria. Xanthomonas also induced systemic resistance to Ps. syringae pv. tomato. These results are very much in line with one of the characteristics of SAR in which multiple pathogens can be used to induce resistance against different pathogens in the same host. What was interesting about these results is that the resistance induced by the bacteria did not result in the restriction of growth of either pathogen in the systemically induced tissues as compared to growth in the controls. Symptom suppression (i.e., reduced tissue damage)

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was observed, and the authors referred to this phenomenon as systemic acquired tolerance rather than SAR. Notably, treatment with ASM induced resistance that was characterized by a suppression of X. campestris pv. vesicatoria and Ps. syringae pv. tomato growth in induced tissues. This suggests that different mechanisms of disease and/or symptom suppression may be functioning depending on the nature of the inducer. An important question to address is how induced plants are able to actually resist or stop bacterial pathogens. In the case of reduced growth of bacteria in induced plants, it is not known which factor or factors may be involved. Rathmell and Sequeira (1975) reported that induced tobacco tissues produced an antimicrobial substance(s). The nature of this antimicrobial was not determined. Since this was also localized induced resistance, the nature of this defense may or may not be relevant to systemic responses. However, since this localized resistance is known to be induced by LPS (Graham et al., 1977), it is possible that the inhibitors reported by Rathmell and Sequeira (1975) are low molecular weight compounds such as feruloyl tyramines (Newman et al., 2001). It would be interesting to see if these types of inhibitors are induced systemically by inducing treatments or induced to a greater extent after challenge of SAR-expressing plants. As described above, an interesting systemic response is the lack of symptom/damage in induced plants after challenge that is not associated with a decrease in bacterial populations (e.g., Block et al., 2005; Doss and Hevisi, 1981; Summermatter et al., 1995). It is not at all clear why this may occur, although a suppression of necrosis as a result of increased antioxidant status of the induced plant has been suggested to reduce necrosis in SAR-expressing plants that have been challenged by necrotic-lesion inducing pathogens (e.g., Barna et al., 2003). Plant cells can respond to bacterial pathogens with cell wall modifications such as callose deposition (Kim et al., 2005), and an enhanced ability to structurally block the delivery of bacterial effector molecules to host cells may also be a cause of suppressed symptoms. As such, it would be interesting to determine if this interference in a pathogenicity mechanism, such as effector delivery, is part of the SAR defense response. The nonhost resistance response of Arabidopsis to Ps. syringae pv. phaseolicola is also associated with PR-1 protein accumulation and callose deposition. Inoculation of Arabidopsis mutants npr1 or sid2 (SA induction deficient 2) resulted in increased growth of Ps. syringae pv. phaseolicola suggesting a connection between SA signaling and basal defenses. There is no reason to believe that this would not also be the case for SAR responses to this bacterium. Another possibility is an ability of the induced host to interfere with the expression of genes controlling the bacterial type III secretion pathway.


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Although not dealing with a SAR type of response, Minardi (1995) reported that the resistance induced by protein-LPS in tobacco to the HR induced by Erw. amylovora was associated with altered expression of the bacterium’s hrp genes. Thus, interference with the basic mechanisms of pathogenicity may also be a part of the expression of resistance after challenge.

D. PROTECTION AGAINST VIRUSES

1. Decrease in lesion size and number SAR in N gene tobacco against TMV is expressed as a reduction in the number and size of local lesions caused by this virus (Ross, 1961b) and is, in essence, an increase in the expression of major gene resistance to this virus. In an attempt to explain the resistance, biochemical changes following challenge inoculations of SAR-expressing tobacco leaves with TMV were reported by Simons and Ross (1970, 1971a) and Van Loon and Geelen (1971). Challenge of the induced tissues resulted in faster increases and higher final peroxidase activity than in control leaves. Simons and Ross (1971b) also demonstrated that extractable PAL activity was greater after challenge of leaves with induced resistance than with control leaves. Challenge of both control and induced tissues resulted in peak PAL activity at about the same time after inoculation followed by a rapid decrease in extractable activity. The content of o-dihydroxyphenolic compounds decreased in the challenged tissue, with the greatest decrease in the induced leaves (Simons and Ross, 1971b). These results suggested that the resistance, as expressed by smaller and fewer necrotic lesions may be the result of viral localization by formation of phenolic structural barriers or perhaps highly reactive quinones formed from the phenolic compounds. This, of course, would be in line with what has been observed in defenses expressed against fungal pathogens in plants expressing SAR in which cell wall alterations appear to be a major factor. Another explanation for the decrease in disease severity after challenge is simply the suppression of necrosis formation by increases in antioxidant activity of SAR-expressing tissues. Activity of glutathione reductase and glutathione-S-transferase as well as glutathione content increased systemically in tobacco after inoculation of lower leaves with TMV or treatment with SA (Fodor et al., 1998). Further investigations on this system revealed that SAR-expressing leaves had increased superoxide dismutase activity. Challenge with TMV resulted in reduced hydrogen peroxide levels at infection sites (Barna et al., 2003). However, it is not clear from these studies if the reduced necrosis is, indeed, the cause or result of SAR. It is apparent from other studies that necrosis does not restrict the spread of virus in N gene

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tobacco (Wright et al., 2000). Thus, host cell death may be more of a result of resistance than an active cause. 2. Inhibition of virus replication One possible explanation for reduced lesion size and number is a decrease in viral replication in SAR-expressing tissues. Van Loon and Antoniw (1982) found that treatment of susceptible ‘‘Samsun’’ (nn genotype) tobacco with SA reduced TMV accumulation following inoculation. Similarly, White et al. (1983) reported that treatment of tobacco with ASA induced resistance to TMV in tobacco lines with and without the N gene for hypersensitive resistance. Associated with the induced resistance was a decrease in the accumulation of virus in both genotypes of tobacco. The results of Van Loon and Antoniw (1982) and White et al. (1983) suggested that SAR type resistance may be expressed by inhibiting the replication of the virus. Following up on this observation, Chivasa et al. (1997) found that SA pretreatment of an nn genotype of tobacco was able to greatly reduce the accumulation of TMV coat protein and RNAs after inoculation with the virus. Inhibition of virus replication has also been proposed as a mechanism of SA-induced resistance in tobacco to PVX (Naylor et al., 1998) and in Arabidopsis to turnip vein clearing virus (TVCV) (Wong et al., 2002). 3. Inhibition of cell-to-cell movement Murphy and Carr (2002) provided further evidence that SA can reduce TMV replication and that it can also reduce cell-to-cell movement of the virus in tobacco. What is interesting about this report is that the SA-induced resistance to TMV in tobacco is also tissue specific with cell-to-cell movement being affected in the epidermis while virus replication was reduced in mesophyll cells. The basis for inhibition of cell-to-cell movement was not elucidated, but it was shown not to be caused by changes in the size of the plasmodesmata (Murphy and Carr, 2002). Inhibition of cell-to-cell movement was also suggested to be the mechanism behind SA-induced resistance of squash to CMV (Mayers et al., 2005). 4. Inhibition of systemic movement Inoculation of lower leaves of cucumber plants with Co. orbiculare, Ps. syringae pv. lachrymans or TNV induced resistance to challenge inoculation with CMV (Bergstrom et al., 1982). Challenge with CMV by mechanical inoculation reduced the number of chlorotic primary CMV lesions on the challenged leaf and delayed appearance of systemic mosaic symptoms. Challenge of induced plants with CMV-infested melon aphids resulted in a delay in systemic spread of the virus but found no differences in symptoms on the


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aphid-infested leaves on control or induced plants. Using ASM as the inducer, Smith-Becker et al. (2003) found a reduction in systemic spread of CMV in cantaloupe following mechanical inoculation with the virus. In tobacco and Arabidopsis, SA treatment also induces resistance to CMV (Mayers et al., 2005; Naylor et al., 1998). These authors found that the induced resistance was based on inhibition of systemic movement of the virus and not due to inhibition of replication. In tobacco, SA did not affect normal translocation processes of photosynthetic assimilates as demonstrated by feeding plants 14CO2 and following the transport of the label in treated and control plants (Naylor et al., 1998). This demonstrated that SA-induced resistance to systemic movement was not due to changes in source–sink relationships or other normal functions of translocation. As noted above, the induction of resistance to CMV in squash was associated with reduced viral replication, and this indicates different mechanisms of resistance against the same virus are present in different plant species (Mayers et al., 2005).

5. Does SA induce a different form of resistance to viruses? SAR is typically associated with SA functioning upstream of NPR1 and the expression of PR genes. However, the role of SA in the SAR response to viruses may be different (Murphy et al., 1999). In the report by Chivasa et al. (1997), SA was found to inhibit TMV replication by a mechanism that is sensitive to salicylhydroxamic acid (SHAM), an inhibitor of mitochondrial alternative oxidase. This suggested that SA, a known inducer of the alternative oxidase pathway (Raskin, 1992), was inducing resistance via another route. Treatment of TMV susceptible (nn genotype) tobacco with SHAM had no effect on TMV replication as compared to SA which blocked replication (as based on TMV coat protein accumulation). However, co‐incubation of leaf tissue with SHAM and SA resulted in no inhibition of coat protein production. SHAM also delayed the SA-induced delay in systemic symptoms in n gene tobacco as well as resistance in N gene tobacco (Chivasa et al., 1997). In further support of the role of the alternative oxidase pathway in SA-mediated resistance, a respiratory inhibitor, cyanide, was able to restore the normal response of N gene tobacco to TMV in plants expressing NahG (Chivasa and Carr, 1998). Further support for the role of alternative oxidase in resistance to TMV was the ability of antimycin A, another inhibitor of respiration, to also induce resistance (Chivasa and Carr, 1998). These same authors also reported that SA and antimycin A induced transcripts of the alternative oxidase gene, thus further suggesting a role for the alternative oxidase pathway in induced resistance to TMV.

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To test the role of alternative respiration on SA-induced resistance, transgenic tobacco with increased or decreased alternative oxidase expression were generated (Gilliland et al., 2003). Altered alternative oxidase activity had no effect on SA-induced resistance to TMV in susceptible tobacco. Interestingly, antimycin A-induced resistance based on virus accumulation in inoculated leaves was inhibited in transgenic plants with enhanced alternative oxidase activity. Decreasing alternative oxidase, however, allowed short-term induction of resistance following SA or antimycin A treatment. Based on these results, Gilliland et al. (2003) suggested that SA-induced resistance to TMV is controlled by more than one mechanism, of which alternative oxidase plays one role. These authors further hypothesize that ROS in the mitochondria, the concentration of which would be affected by altered alternative oxidase expression as well as by treatment with SA or antimycin A, are likely important signals in the induced resistance response. SA-mediated resistance to viruses also differs from SAR described for bacteria and fungi in the expression of PR genes. Chivasa et al. (1997) found that SA induction of PR-1 protein accumulation was not inhibited by SHAM and was even induced by this inhibitor. The SA-induced resistance of Arabidopsis to TVCV was also shown to be separate from the induction of PR-proteins (Wong et al., 2002). These authors and Kachroo et al. (2000) also demonstrated that SA-induced resistance was also independent of NPR1 function as SA was able to induce resistance in npr1-1 mutants. Thus, SA-induced resistance to viruses appears to follow a signaling pathway that is different from that observed for fungi and bacteria (Murphy et al., 1999). This is of course an interesting revelation in light of the fact that classical SAR often uses the work of Ross (1961b) as a primary example. What these studies do demonstrate is that signal molecules that induce a similar ‘‘resistance phenotype’’ may do so by different pathways. Further details on the role of SA and related signaling molecules for induced resistance to viruses can be found in recent reviews by Singh et al. (2004) and Palukaitis and Carr (2008). E. MECHANISMS OF DEFENSE IN SUMMARY

1. Enhancing basal defense Having described a number of examples of how plants expressing SAR are thought to defend themselves against infection, it might be good to again consider the basic foundations and characteristics of this form of resistance. First of all, it should be remembered that SAR (and other forms of induced resistance) is functioning in a plant that is susceptible to multiple pathogens. Second, because of the diversity of pathogens that are resisted by a plant


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expressing SAR, it is logical that the resistance is associated with the expression of multiple defenses that are induced as a result of pathogen or chemical activator induction and those induced after a challenge. In other words, the nonspecificity of SAR is likely related to a diversity of defenses. Looking back over the defenses described in this review, it is clear that these can be broken into three stages representing SAR induction and expression. The first, or induction stage, requires inoculation of one leaf with a compatible pathogen that causes a necrotic lesion or an incompatible pathogen that elicits a HR. Both of these plant–pathogen interactions generate a systemically transported signal (or signals) that trigger(s) SA accumulation throughout the plant. Based on current information, the accumulated SA induces resistance to biotrophic or hemibiotrophic fungi and bacteria through an NPR1-mediated process that is accompanied by PR-protein accumulation. Although resistance induced by SA against viruses is also accompanied by PR-protein accumulation, the mechanism(s) involved appear to be different. At least in tobacco, SA induces resistance to virus through a SHAM-sensitive pathway. It is interesting to note that in the necrotic lesions that result from compatible interactions and lead to SAR, basal defenses such as lignification and phytoalexin accumulation occur (Hammerschmidt, 1999b; Hammerschmidt and Nicholson, 1999). In NahG plants, compatible interactions with necrotizing pathogens expand more rapidly than in untransformed controls (Delaney et al., 1994). It is important to note that SA-mediated defenses are not effective against necrotrophs which are typically resisted by the JA/ET pathway (Glazebrook, 2005; Thomma et al., 2001). This suggests that basal defenses are involved in limiting lesion spread. However, it seems unlikely that locally induced basal defenses are a major player in induction of the systemic resistance as inoculated NahG rootstocks were still capable of generating a systemic signal that induced SAR in wild-type scions (Vernooij et al., 1994). The second stage of SAR expression is the systemic accumulation of PR- and other proteins. Although the precise role of these proteins in defense is not clear (Van Loon et al., 2006), one possible function is to slow the development of the pathogen so that the SAR-expressing host has the needed time to induce additional defenses. However, there is little evidence to suggest this is happening. This is illustrated by the failure of plants constitutively expressing PR-proteins to be consistently more resistant (Van Loon et al., 2006). However, because it is known that slowing pathogen development by fungicides can result in the expression of defenses at the site of infection (Lazarovits and Ward, 1982), retarding pathogen growth (thus giving the plant more time to react) is still a possibility. Another alternative is that fungicide-sensitive pathogens release elicitors which trigger the

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localized defenses (Cahill and Ward, 1989). As some of the PR-proteins are chitinases or -1,3-glucanases that can act on fungal or oomycete cell walls, it would seem possible that the PR-proteins could also function in SAR by releasing cell wall elicitors that trigger localized defenses (Okinaka et al., 1995). However, there must be more to this than the presence of enzymes that can release elicitors from pathogens as extracellular chitinase activity remains high in cucumber even after SAR is no longer evident (Dalisay and Kuc´, 1995). Other systemic protein changes, such as the accumulation of HRGP in the cell wall, may function as structural barriers that slow the penetration of pathogens through cell walls and may provide an additional explanation for decreased fungal penetration into SAR expressing tissues. The last stage is the expression of defenses at the site of pathogen challenge. These types of responses clearly show that the SAR-expressing tissues are primed to respond to new infections (Conrath et al., 2000, 2002, 2006). As early as 1961, Ross (1961b) manipulated the size of TMV lesions produced in induced and noninduced leaves by varying environmental conditions. He found that the effects were similar in induced and noninduced leaves, and concluded that the mechanisms of lesion restriction were the same in noninduced and induced plants. With fungi, a general response observed in many hosts is modification of cell walls. These modifications (e.g., lignin and callose deposition) are not novel responses, but might, as suggested by Ton et al. (2006), be an enhancement of basal defenses primed by SAR. For example, one response of SAR expressing cucumber to Colletotrichum is the rapid deposition of lignin in outer epidermal cells walls (Hammerschmidt and Kuc´, 1982). Lignification also occurs, although more slowly and after penetration, in the necrotic lesions that result from the compatible interaction between Colletotrichum and cucumber. Other defenses, such as phytoalexin production, are enhanced in SAR-expressing tissue (Hammerschmidt, 1999a,b). Since these compounds are also produced in many compatible interactions (Hammerschmidt, 1999b), these observations further support the concept that SAR is an enhancement of basal defense responses. The idea that SAR and other forms of systemic induced resistance is an enhancement of basal defenses is supported in another, albeit a somewhat indirect, way: the failure of SAR to provide complete resistance. This should not be surprising since a SAR-expressing plant is really a susceptible plant with a layer of added defenses (i.e., the newly induced PR-proteins) and additional defenses elicited after challenge inoculation. Since basal defenses alone are not sufficient to stop compatible pathogens, there is no reason to expect these same defenses (even if enhanced) would be 100% effective. This is illustrated by the observation that SAR-expressing cucumber are only partially effective in blocking penetration by Co. orbiculare and infections


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that are successful result in typical, although smaller, anthracnose lesions (Jenns and Kuc´, 1980; Richmond et al., 1979). Furthermore, increasing the concentration of inoculum essentially eliminates the ability of SARexpressing cucumber to reduce anthracnose lesion development (Dean and Kuc´, 1986b). Thus it is not surprising that SAR is not always highly effective under field conditions (reviewed in Vallad and Goodman, 2004). B. WHAT DON’T WE KNOW?

There are many things that are not known about induced resistance and plant defense, in general. What follows are a few thoughts on this topic based on questions that arose while preparing this review. First, it is not known with absolute certainty which SAR-associated defenses are needed to stop pathogens from invading. There are obviously many good correlations, but it is important to further dissect and evaluate how each putative defense contributes to SAR. Studies using transgenic plants expressing individual PR-proteins have shown that one component is not sufficient to provide resistance, but this may not be surprising as SAR and other inducible defense responses are multicomponent. Obviously, a strategy using multiple defenses suggests there might be interactions between defenses that make the whole much more effective than individual responses. It may also be of use to look at defense expression as a process and not solely at how metabolic ‘‘end products’’ function in resistance. Ride (1978), in discussing the role of lignin in host defense, indicated that the process of lignification may be as important in defense as the static structural barrier of lignified walls. Of course, it is also possible that all of the ‘‘defenses’’ identified thus far are just host responses and have nothing to do with stopping the pathogen. This is illustrated by the work on SA-mediated resistance to viruses which appears to function differently from what was expected based on early studies. The similarities between the expression of defenses in SAR-expressing plants and those of basal resistance in noninduced plants suggest that comparative studies of these types of resistance might be helpful in evaluating which defenses are most important. The use of mutants in genes that are directly involved in the production of specific defenses is another approach that needs to be more thoroughly integrated into this line of research. Second, it is not known for certain why plants expressing SAR respond more quickly to pathogens. It is well known that SAR-expressing plants are primed to respond, but this does not explain how, or even whether the plant recognizes the presence of the pathogen more quickly. Perhaps the answer lies in the expression of genes encoding NBS-LRR proteins such as those

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induced by SAR activators (e.g., Faize et al., 2007; Sakamoto et al., 1999; Wang et al., 2001). Induction of these types of proteins might enable the induced plant to detect PAMPs more efficiently and thus more rapidly induce defenses at the infection site. This would also further support induced resistance as an enhancement of basal disease resistance. An equally likely explanation is that the primed, SAR-expressing plants respond faster to infection because of the enhancement of defense signaling pathways. Induced resistance also is not 100% effective. It is now clear that there are differences in systemic resistance expressed against biotrophs/hemibiotrophs as compared to necrotrophs that can be based on signaling pathways (Glazebrook, 2005), and this explains why SAR might not work against all pathogens. However, this does not explain why, for example, SAR does not fully protect plants such as cucumber against hemibiotrophs like Colletotrichum (e.g., Dean and Kuc´, 1986b). Histological studies have shown that a certain percentage of appressoria on SAR-expressing leaves produce penetrations that result in successful infections (e.g., Richmond et al., 1979). This suggests there is nonuniform expression of priming in the epidermis. Since SAR can be considered to be an enhancement of basal resistance to pathogens and some pathogens can suppress this type of resistance (e.g., Cooper et al., 2008), it is also possible that part of the ‘‘failure’’ of acquired resistance may be the result of suppression of defense responses by the ‘‘challenge’’ pathogen. Another item to consider is whether the apparent differences between SA-mediated (SAR) and JA/ET-mediated defense signaling that differentiates between biotrophs and necrotrophs (Glazebrook, 2005; Oliver and Ipcho, 2004) is a widespread phenomenon in the plant kingdom. This certainly appears to be the case with Arabidopsis, but whether it is universal needs to be determined. For example, the SAR activators ASM and DCINA induce resistance in soybean to Sc. sclerotiorum, a necrotroph (Dann et al., 1998). This is not only of intellectual interest, but this type of information would be of value in implementing induced resistance as part of practical disease management. Finally, it is important to determine how best to take advantage of SAR and other forms of induced resistance in disease management. As might be expected from the resources needed to synthesize PR-proteins, there are metabolic costs associated with SAR expression (Heil, 2007; Walters and Boyle, 2005; Walters and Heil, 2007). Thus, the use of SAR in practical management must take this into account. One way to possibly reduce the metabolic costs would be to combine SAR with other management approaches. It is interesting to note that fungicides are less effective in Arabidopsis plants impaired in SA signaling (Molina et al., 1998). This suggests that basal resistance may work in concert with fungicides in


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protecting plants against disease, and indicates that fungicides might act to enhance disease protection conferred by SAR. As reviewed by Reglinski et al. (2007), there are examples where activators such as ASM have been shown to improve the efficacy of standard fungicides. Understanding the biochemical bases for such interactions could shed further light on how to use induced resistance in the field.

VI. CONCLUDING COMMENTS Over the last 50 years, the study of SAR and other forms of induced resistance has evolved from a rather curious phenomenon studied by only a few researchers to a line of research that is providing new insight into signaling pathways that regulate disease resistance. In addition, the development of synthetic and natural plant resistance activators for use in production agriculture demonstrates that SAR may have a place in practical disease management. Based on a number of lines of evidence, it appears that SAR is an enhancement of the basal defenses that all plants have the capacity to express. Although this helps to explain why plants have the genetic capacity to resist disease, it does not help narrow down which defenses are most important. Thus, it is still important to determine the relative contribution of known defenses to disease resistance and to be open to other forms of defense that may not fit current thinking of what a defense looks like. Finally, one of the shortcomings of SAR research has been the limited number of plant species that have been studied. It is important to survey for the expression of SAR as defined by model systems in other species, to determine if the models are correct. This will also help determine the best strategies for implementing SAR and other induced resistance types in disease management.

ACKNOWLEDGMENT I wish to thank the Michigan Agricultural Experiment Station for its support.

REFERENCES Aist, J. R. (1976). Papillae and related wound plugs of plant cells. Annual Review of Phytopathology 14, 145–163. Anfoka, G. and Buchenauer, H. (1997). Systemic acquired resistance in tomato against Phytophthora infestans by pre-inoculation with tobacco necrosis virus. Physiological and Molecular Plant Pathology 50, 85–101.

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Rhizobacteria-Induced Systemic Resistance

¨ FTE1 DAVID DE VLEESSCHAUWER AND MONICA HO

Laboratory of Phytopathology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Gent, Belgium

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. PAMP- and Effector-Triggered Immunity ................................. B. Systemic Acquired Resistance or Salicylic Acid-Induced Systemic Resistance ............................................................ C. Rhizobacteria-Induced Systemic Resistance ............................... D. Rhizobacteria Known to Trigger ISR....................................... E. Scope of this Review ........................................................... II. Recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Flagella........................................................................... B. Lipopolysaccharides............................................................ C. Biosurfactants ................................................................... D. N-acyl-L-homoserine lactone.................................................. E. N-alkylated benzylamine ...................................................... F. Siderophores .................................................................... G. Antibiotics ....................................................................... H. Volatiles .......................................................................... I. Exopolysaccharides ............................................................ J. Other Bacterial Determinants ................................................ III. Signalling in Rhizobacteria-Induced Systemic Resistance . . . . . . . . . . . . . . . . . . A. The Arabidopsis–Pseudomonas fluorescens WCS417r System: A Paradigm for SA-Independent ISR Signalling .......................... B. SA-Dependent ISR Signalling ................................................ C. SA-Dependent and SA-Independent Signalling ...........................

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0065-2296/09 $35.00 DOI: 10.1016/S0065-2296(09)51006-3

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¨ FTE D. DE VLEESSCHAUWER AND M. HO

IV. Final Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 266

ABSTRACT Rhizobacteria-induced systemic resistance (ISR) is a type of systemically enhanced resistance against a broad spectrum of pathogens that is triggered upon root colonization by selected strains of non-pathogenic bacteria. Over the past decade, a myriad of bacterial traits operative in triggering ISR have been identified, including flagella, cell envelope components such as lipopolysaccharides, and secreted metabolites like siderophores, cyclic lipopeptides, volatiles, antibiotics, phenolic compounds, and quorum sensing molecules. This review provides an in-depth overview of these determinants, thereby focusing specifically on the molecular recognition processes in the plant. The putative mechanisms involved in microbial perception include high- and low-affinity membrane receptors, membrane bilayer perturbation, and siderophoremediated alterations in cellular iron homeostasis. In addition, details about the various defence signalling pathways reported to underpin rhizobacteria-mediated ISR are presented. Evidence is accumulating that there is not one definitive resistance pathway to ISR but that various hormone-dependent signalling conduits may govern the induced resistance phenotype depending on the rhizobacterium and the plant–pathogen system used.

I. INTRODUCTION A. PAMP- AND EFFECTOR-TRIGGERED IMMUNITY

Plants have evolved a powerful immune system to resist their potential colonization by microbial pathogens and parasites. Over the past decade, it has become increasingly clear that this innate immunity is, in essence, composed of two interconnected branches, termed PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI) (Eulgem and Somssich, 2007; Jones and Dangl, 2006). PTI is triggered by recognition of pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs), which are conserved molecular signatures decorating many classes of microbes, including non-pathogens. Perception of MAMPs by pattern recognition receptors (PRRs) at the cell surface activates a battery of host defence responses leading to a basal level of resistance (Chisholm et al., 2006). However, during the evolutionary arms-race between plants and their intruders, many microbial pathogens acquired the ability to dodge PTI-based host surveillance via secretion of effector molecules that intercept MAMP-triggered defence signals (Go¨hre and Robatzek, 2008). In turn, plants have adapted to produce cognate R (resistance) proteins by which they recognize, either directly or indirectly, these pathogen-specific effector proteins, resulting in a superimposed layer of defence variably termed ETI, gene-for-gene resistance, or R gene-dependent resistance (Jones and Dangl, 2006).

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In many cases, effector recognition culminates in the programmed suicide of a limited number of challenged host cells, clearly delimited from the surrounding healthy tissue. This hypersensitive response (HR) is thought to benefit the plant by restricting pathogen access to water and nutrients and is correlated with an integrated set of physiological and metabolic alterations that are instrumental in impeding further pathogen ingress, among which exists a burst of oxidative metabolism leading to the massive generation of reactive oxygen species (ROS) (Glazebrook, 2005; Greenberg and Yao, 2004). B. SYSTEMIC ACQUIRED RESISTANCE OR SALICYLIC ACID-INDUCED SYSTEMIC RESISTANCE

Systemic acquired resistance (SAR) or salicylic acid-induced systemic resistance (ISR), is a phenomenon whereby disease resistance to subsequent microbial infection is induced at the whole plant level by a localized pathogen inoculation (Durrant and Dong, 2004). Development of tissue necrosis used to be considered a common and necessary feature for SAR activation. This tissue necrosis can result from ETI-associated HR formation or originate from disease symptom development following infection by virulent pathogens. Mishina and Zeier (2007), however, have demonstrated that in Arabidopsis thaliana, SAR can also be triggered without tissue necrosis or HR by non-host or type III secretion-deficient Pseudomonas syringae strains or by local leaf infiltration of typical PAMPs such as bacterial lipopolysaccharides (LPS) and flagellin (Mishina and Zeier, 2007). These results indicate that both PTI and ETI can lead to SAR. PTI-induced SAR, however, is less pronounced than its ETI-induced counterpart (Mishina and Zeier, 2007). SAR requires endogenous accumulation of the signal molecule salicylic acid (SA) and is marked by the transcriptional reprogramming of a battery of SA-inducible genes encoding pathogenesis-related (PR) proteins. These PR-proteins, of which some possess antimicrobial activity, serve as hallmarks of SAR in several plant species and are thought to contribute to the state of resistance attained. The expression of a PR-1 gene or protein in particular is usually taken as a molecular marker to indicate that SAR was induced. All PR-1 genes in plants appear to be inducible by SA (Van Loon et al., 2006). Transduction of the SA signal requires the function of NPR1 (also known as NIM1), a master regulatory protein that was identified in Arabidopsis through genetic screens for SAR-compromised mutants (Cao et al., 1994; Shah et al., 1997). SAR is abolished in SA-non-accumulating NahG plants that express the bacterial SA-hydroxylase gene NahG, which converts SA to catechol (Lawton et al., 1996). NahG transgenes are available in various plants including Arabidopsis (Delaney et al., 1994), tobacco

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(Gaffney et al., 1993), tomato (Brading et al., 2000), and rice (Yang et al., 2004). Accumulation of SA is required for SAR, but only in the signalperceiving systemic tissue. Recently, it was demonstrated that the volatile molecule methyl salicylate (MeSA) can act as a long-distance mobile signal for SAR. MeSA itself appears to be biologically inactive, but in the systemic tissue MeSA is hydrolysed to SA by the MeSA-esterase activity of SA-binding protein 2 (Park et al., 2007; Vlot et al., 2008a,b). It is likely that MeSA can travel by both air and vascular transport to mediate long-distance induction of resistance in distal leaves that lack a direct vascular connection to the attacked leaf and in neighbouring plants (Heil and Ton, 2008). Besides MeSA, lipid-derived signals also play a role in triggering SAR. Vlot et al. (2008a) state that two lipid-associated signals may work in parallel with each other and with MeSA to regulate SAR. Possible candidates are galactolipids and jasmonic acid (JA). Recent findings show that auxin-related genes are repressed in the systemic tissue of SAR-induced Arabidopsis, and it was concluded that SA enhances resistance by inhibiting auxin signalling via SA-dependent stabilization of auxin repressor proteins (Wang et al., 2007). C. RHIZOBACTERIA-INDUCED SYSTEMIC RESISTANCE

Since the 1980s, numerous reports have ascribed a beneficial effect of root colonization by specific bacteria on plant development, and they were therefore, termed plant growth-promoting rhizobacteria or PGPR (Kloepper et al., 1980). Plant-growth promotion can result from a direct effect on plant growth, but is mostly related to a PGPR-mediated biological control of deleterious soil micro-organisms that can be on the basis of various mechanisms such as competition for nutrients, siderophore-mediated competition for iron, or antibiosis (Schippers et al., 1987). In 1991, three groups independently demonstrated that PGPR could also reduce pathogen infections on above-ground plant parts such as leaves and stems (Alstro¨m, 1991; Van Peer et al., 1991; Wei et al., 1991). Given the spatial separation of PGPR and pathogen, this effect had to be plant-mediated. Later on, similar results were obtained when root tips were treated with the PGPR, and the pathogen was inoculated on the root base (Leeman et al., 1995). Thus, colonization of plant roots by selected PGPR can lead to a type of systemic resistance that has been termed ‘induced systemic resistance’ (ISR) (Kloepper et al., 1992; Van Loon et al., 1998). To avoid a Babel confusion of terminology, we propose ISR to depict induced systemic resistance induced by non-pathogenic rhizobacteria or PGPR irrespective of the signalling pathway involved in this process, while the term SAR will be used to describe SA-dependent induced resistance triggered by a localized pathogen infection as explained above.


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Generally, the onset of ISR, unlike SAR, is not accompanied by the concomitant activation of PR genes. Even though colonization of the roots by ISR-triggering bacteria leads to a heightened level of resistance against a diverse set of intruders, often no defence mechanisms are activated in aboveground plant tissues upon perception of the resistance-inducing signal. Rather, these tissues are sensitized to express basal defence responses faster and/or more strongly in response to pathogen attack, a phenomenon known as priming (Conrath et al., 2002). As demonstrated recently, priming of the plant’s innate immune system confers broad-spectrum resistance with minimal impact on seed set and plant growth (Van Hulten et al., 2006). Hence, priming offers a cost-efficient resistance strategy, enabling the plant to react more effectively to any invader encountered by boosting infection-induced cellular defence responses (Beckers and Conrath, 2007; Conrath et al., 2006). In a series of influential studies using the reference rhizobacterial strain Pseudomonas fluorescens WCS417r, Pieterse et al. (1996, 1998, 2000) demonstrated that, at least in Arabidopsis, WCS417r-mediated ISR functions independently of SA, but requires components of the JA and ethylene (ET) response pathways. Like SAR however, WCS417r-mediated ISR is dependent on NPR1. In recent years, though, it is becoming increasingly clear that not all rhizobacteria trigger ISR by a JA-, ET-, and NPR1-dependent pathway. Table I shows that all kinds of variations can be found depending on the rhizobacterium and the plant–pathogen system used. The role of plant hormones in ISR signalling will be discussed further below. D. RHIZOBACTERIA KNOWN TO TRIGGER ISR

The first comprehensive review about rhizobacteria-mediated ISR was written by Van Loon et al. (1998). In this seminal paper ISR was reported in seven dicotyledonous plants (Arabidopsis, bean, carnation, cucumber, radish, tobacco, and tomato) and inducing bacteria were limited to 15 Pseudomonas strains and two Serratia strains. Since 1998, ISR has been reported in many different plant–pathogen systems. A comprehensive overview of PGPR able to trigger ISR is given by Bent (2006). A review by Van Loon and Bakker (2006) reports ISR in 19 different plant species including, beside the plant species listed above, banana, pea, chickpea, white clover, pepper, pine, Eucalyptus, potato, rice, tall fescue, and sugarcane. Besides Pseudomonas and Serratia, many Bacillus strains have the capacity to elicit ISR. The literature about ISR by Bacillus spp. up to 2004 has been reviewed by Kloepper et al. (2004). Numerous rhizobacteria able to trigger ISR appear to be endophytes that can be isolated from surface-sterilized plant tissue. Endophyte-mediated ISR has been summarized by Kloepper and Ryu

TABLE I Rhizobacteria that Induce Systemic Resistance in Plant–Pathosystems for Which Information is Available about Bacterial Determinants and/or Defence Pathways Involved Bacterial strain Arthrobacter oxidans BB1 Bacillus amyloliquefaciens IN937a Bacillus pumilus SE34

Plant species Arabidopsis Arabidopsis Arabidopsis Arabidopsis Arabidopsis

Bacillus pumilus T4

Tobacco Tomato Arabidopsis Arabidopsis Tobacco

Bacillus sp. L81

Arabidopsis

Bacillus sp. N11.37

Arabidopsis

Bacillus subtilis GB03

Arabidopsis

Bacillus subtilis S499 Burkholderia gladioli IN26

Bean Cucumber Tobacco

Pathogen Pseudomonas syringae pv. tomato Erwinia carotovora Cucumber mosaic virus Pseudomonas syringae pv. maculicola Pseudomonas syringae pv. tomato Peronospora tabacina Phytophthora infestans Pseudomonas syringae pv. maculicola Pseudomonas syringae pv. tomato Pseudomonas syringae pv. tabaci Pseudomonas syringae pv. tomato Xanthomonas campestris CECT95; 4480 Erwinia carotovora subsp. carotovora Botrytis cinerea Colletotrichum orbiculare Pseudomonas syringae pv. tabaci

Determinanta

Pathwayb

Plant lines tested/method usedc

Reference

ND

SA-dependent; JA-independent

NahG, jar1

Barriuso et al. (2008)

2R,3R-butanediol

ET/JA/NPR1/SA-independent

Ryu et al. (2004a)

ND ND

ND

SA-independent ET/JA/NPR1-dependent; SA-independent NPR1/SA-dependent; ET/JAindependent SA-independent ET/JA-dependent; SA-independent ET-dependent; SA/JA/NPR1independent ET-dependent; SA/JA/NPR1independent Induces PR-1a

ein2, coi1, npr1, NahG, cpr1 NahG ein2, fad3-2 fad7-2 fad8, npr1, NahG npr1, NahG, ein2, fad3-2 fad7-2 fad8 NahG Nr/Nr, def1, NahG ein2, NahG, fad3-2 fad7-2 fad8, npr1 ein2, NahG, fad3-2 fad7-2 fad8, npr1 GUS activity

ND

SA-dependent; JA-independent

NahG, jar1

Park and Kloepper (2000) Barriuso et al. (2008)

ND

SA/ET-dependent; JA-independent

NahG, etr1-1, jar1-1

Domenech et al. (2007)

2R,3R-butanediol

ein2, NahG, cpr1, coi1, npr1 ND ND

Ryu et al. (2004a)

Surfactin; fengycin EPS

ET-dependent; SA/JA/NPR1independent ND ND

ND

Induces PR-1a

GUS activity

ND ND ND ND ND

Ryu et al. (2004b) Ryu et al. (2003b) Ryu et al. (2003b) Zhang et al. (2002) Yan et al. (2002) Ryu et al. (2003b) Ryu et al. (2003b)

Ongena et al. (2007) Park et al. (2008a) Park and Kloepper (2000)

LPS

Induces PR-1

Quantitative PCR

Solano et al. (2008)

Arabidopsis

Pseudomonas syringae pv. tomato Verticillium dahliae

ND

Arabidopsis

Erwinia carotovora

ND

Pseudomonas aeruginosa 7NSK2

Arabidopsis

Unknown

Timmusk and Wagner (1999) Ran et al. (2005b)

Tobacco

Pseudomonas syringae pv. tomato Tobacco mosaic virus

sid2, eds5/sid1, npr1, jar1-1, etr1-1, pad3-1, pad4-1, NahG RNA differential display; RT-PCR NahG

Tjamos et al. (2005)

Paenibacillus polymyxa

SA/NPR1-dependent; JA/ET/ phytoalexin/NahGindependent Induces PR-1, HEL, VSP, ERD15, RAB18 SA-independent SA-dependent

NahG

De Meyer et al. (1999a)

Tomato

Botrytis cinerea

Tomato

Chryseobacterium balustinum AUR9 Paenibacillus alvei K165

Pseudomonas aeruginosa KMPCH

Pseudomonas chlororaphis O6

Arabidopsis

SA-dependent

NahG

Audenaert et al. (2002)

Meloidogyne javanica

Salicylic acid (role of pyocyanin not tested) Salicylic acid or pyochelin þ pyocyanin Unknown

SA-independent

NahG

Rice

Magnaporthe oryzae

Pyocyanin

SA-dependent

NahG

Bean

Botrytis cinerea

Salicylic acid þ pyocyanin

ND

ND

Bean

Salicylic acid (role of pyocyanin not tested) Salicylic acid þ pyocyanin

ND

ND

Tomato

Colletotrichum lindemuthianum Botrytis cinerea

SA-dependent

NahG

Cucumber

Corynespora cassiicola

ND

Northern hybridisation

Tobacco

Erwinia carotovora

Tetr18, NahG

Tobacco

Pseudomonas syringae pv. tabaci

2R,3R-butanediol; phenazines, other determinants 4-(aminocarbonyl) phenylacetate

Primed induction of galactinol synthase ET-dependent; SA-independent

Siddiqui and Shaukat (2004) D. De Vleesschauwer et al. (2006), D. De Vleesschauwer (unpublished data) De Meyer and Ho¨fte (1997) Bigirimana and Ho¨fte (2002) K. Audenaert et al. (2002), K. Audenaert et al. (unpublished data) Kim et al. (2008)

ET- and SA-independent

Tetr18, NahG

Spencer et al. (2003), Han et al. (2006b), Kang et al. (2007) Spencer et al. (2003), Park et al. (2008b)

(continues)

TABLE I Bacterial strain Pseudomonas fluorescens 89B-61 (¼ G8-4)

Plant species

Pseudomonas fluorescens Q2-87 Pseudomonas fluorescens SS101 Pseudomonas fluorescens WCS374

Determinanta

Pathwayb

Plant lines tested/method usedc

Reference

Arabidopsis

Pseudomonas syringae pv. maculicola

ND

NPR1-dependent; SA/JA/ETindependent

npr1, NahG, fad3-2 fad7-2 fad8, ein2

Ryu et al. (2003b)

Arabidopsis

Pseudomonas syringae pv. tomato Peronospora tabacina Pseudomonas syringae pv. tabaci Phytophthora infestans Hyaloperonospora arabidopsidis

ND

NPR1-dependent; SA/JA/ETindependent SA-independent Induces PR-1a

npr1, NahG, fad3-2 fad7-2 fad8, ein2 NahG GUS activity

Ryu et al. (2003b)

ND 2,4-diacetylphloroglucinol

ET/JA-dependent; SA-independent JA/NPR1/EIR1-dependent; SA/ET/ phytoalexin-independent

Tobacco

Tobacco necrosis virus

ND

Tomato

Meloidogyne javanica

Pyoverdine; unknown determinant 2,4-diacetylphloroglucinol

Nr/Nr, def1, NahG jar1, npr1, eir1, NahG, sid2-1, ein2-1, etr1-1, pad2-1 ND

SA-independent

NahG

Arabidopsis

Pseudomonas syringae pv. tomato Phytophthora infestans

2,4-diacetylphloroglucinol

ND

ND

Siddiqui and Shaukat (2003) Weller et al. (2004)

Massetolide A

SA-independent

NahG

Tran et al. (2007)

Flagella

ET/JA/NPR1-dependent; SA-independent SA/NPR1-dependent; ET/JA-independent ND

etr1, jar1, npr1, NahG, sid1 NahG, sid1, npr1, etr1, jar1 ND

Ran et al. (2005b), Djavaheri (2007) Djavaheri (2007)

ND

ND

ET/JA-dependent; SA-independent

471, hebiba, NahG

Leeman et al. (1995, 1996) De Vleesschauwer et al. (2008)

Tobacco Tobacco

Pseudomonas fluorescens CHA0

Pathogen

(continued)

Tomato Arabidopsis

Tomato Arabidopsis Arabidopsis

Pseudomonas syringae pv. tomato Turnip crinkle virus

Eucalyptus

Ralstonia solanacearum

Radish

Fusarium oxysporum f. sp. raphani Magnaporthe oryzae

Rice

ND ND

Pseudobactin þ salicylic acid Pseudobactin; unknown determinant Pseudobactin; LPS Pseudobactin

Zhang et al. (2002) Park and Kloepper (2000) Yan et al. (2002) Iavicoli et al. (2003)

Maurhofer et al. (1994)

Ran et al. (2005a)

Pseudomonas fluorescens WCS417

Pseudomonas syringae pv. tomato

LPS; other unknown metabolites

ET/JA/MYC2/NPR1/MYB72dependent; SA-independent

ein2, ein3 to ein7, eir1, etr1, eto1, jar1, myc2, npr1, myb72, NahG

Arabidopsis

Hyaloperonospora arabidopsidis Botrytis cinerea Alternaria brassicicola Fusarium oxysporum f. sp. dianthi Ralstonia solanacearum Fusarium oxysporum f. sp. raphani Botrytis cinerea

ND

MYC2/MYB72-dependent

myc2, myb72

ND ND LPS

MYB72-dependent MYB72-dependent ND

myb72 myb72 ND

LPS LPS; unknown ironregulated determinant N-alkylated benzylamine

ND ND

ND ND

SA-independent

Enzymatic assays

N-alkylated benzylamine ND ND

ND SA-independent ET/JA/NPR1-dependent; SA-independent SA-independent

ND Enzymatic assays etr1, jar1, npr1, NahG

Pseudobactin; LPS Pseudobactin; LPS

ND ND

ND ND

Meziane et al. (2005), Van Wees et al. (1997) Meziane et al. (2005) Meziane et al. (2005)

Pseudobactin Pseudobactin Pseudobactin; LPS LPS

ND ND ND ND

ND ND ND ND

Ran et al. (2005a) Van Loon et al. (2008) Meziane et al. (2005) Reitz et al. (2000, 2002)

Arabidopsis Arabidopsis Carnation Eucalyptus Radish Pseudomonas putida BTP1

Pseudomonas putida LSW17S Pseudomonas putida WCS358

Bean Cucumber Tomato Arabidopsis Arabidopsis

Bean Bean

Rhizobium etli G12

Van Wees et al. (1997), Pieterse et al. (1998), Knoester et al. (1999), Pozo et al. (2008), Van der Ent et al. (2008) Van der Ent et al. (2008)

Arabidopsis

Eucalyptus Tobacco Tomato Potato

Colletotrichum lagenarium Botrytis cinerea Pseudomonas syringae pv. tomato Pseudomonas syringae pv. tomato Botrytis cinerea Colletotrichum lindemuthianum Ralstonia solanacearum Erwinia carotovora Botrytis cinerea Globodera pallida

Pseudobactin; LPS; flagella

NahG

Van der Ent et al. (2008) Van der Ent et al. (2008) Van Peer and Schippers (1992) Ran et al. (2005a) Leeman et al. (1996) Ongena et al. (2004, 2005) Ongena et al. (2008) Akram et al. (2008) Ahn et al. (2007)

(continues)

TABLE I Bacterial strain

Plant species

Pathogen

(continued)

Determinanta

Pathwayb

Serratia liquefaciens MG1

Tomato

Alternaria alternata

N-acyl homoserine lactone

Probably SA- and ET-dependent

Serratia marcescens 90-166

Arabidopsis

Pseudomonas syringae pv. maculicola Pseudomonas syringae pv. tomato Cucumber mosaic virus

ND

Catechol-type siderophore ND ND

JA/NPR1-dependent; SA/ETindependent JA/NPR1-dependent; SA/ETindependent JA-dependent; SA/NPR1independent ND SA-independent SA-independent

EPS

Induces PR-1b

ND

SA-dependent; ET/JA-independent

ND

SA-dependent; ET/JA/NPR1independent SA/NPR1-dependent; ET/JAindependent

Arabidopsis Arabidopsis Cucumber Tobacco Tobacco Serratia sp. strain Gsm01 Stenotrophomonas sp. N6.8 Streptomyces sp. EN27

Tobacco Arabidopsis Arabidopsis Arabidopsis

Colletotrichum orbiculare Peronospora tabacina Pseudomonas syringae pv. tabaci Cucumber mosaic virus Xanthomonas campestris CECT95; 4480 Erwinia carotovora subsp. carotovora Fusarium oxysporum

ND ND

ND

Plant lines tested/method usedc Macroarray with pure N-acyl homoserine lactone fad3-2 fad7-2 fad8, npr1, NahG, ein2 fad3-2 fad7-2 fad8, npr1, NahG, ein2 fad3-2 fad7-2 fad8, NahG, npr1 ND NahG NahG

Reference Schuhegger et al. (2006a) Ryu et al. (2003b) Ryu et al. (2003b) Ryu et al. (2004b) Press et al. (2001) Zhang et al. (2002) Press et al. (1997)

Reverse transcriptasePCR NahG, etr1-1, jar1-1

Ipper et al. (2008)

NahG, npr1, etr1-3, jar1

Conn et al. (2008)

NahG, npr1, etr1-3, jar1

Conn et al. (2008)

Domenech et al. (2007)

ND, not determined; LPS, lipopolysaccharides; EPS, exopolysaccharides. ND, not determined; SA, salicylic acid; ET, ethylene; JA, jasmonate; PR-1, Pathogenesis-related protein 1, SA-dependent; VSP, Vegetative storage protein, JA-responsive; HEL, Hevein-like protein, ET-responsive; ERD15, drought stress-responsive; RAB18, ABA- and drought stress-responsive. c See Table II for explanation about plant lines. a b


233

(2006), who merely focus on Pseudomonas, Serratia, and Bacillus endophytes. A non-exhaustive list of other rhizobacteria with ISR-triggering capacities includes Lysobacter enzymogenes (Kilic-Ekici and Yuen, 2003), Paenibacillus alvei (Tjamos et al., 2005), Acinetobacter lwoffii (Trotel-Aziz et al., 2008), Chryseobacterium balustinum, Azospirillum brasilense (Ramos Solano et al., 2008), Curtobacterium sp., Arthrobacter oxidans (Barriuso et al., 2008), Stenotrophomonas (Domenech et al., 2007), and endophytic Actinobacteria (Conn et al., 2008). Although outside the scope of this review, it should be mentioned that various root-colonizing plant growth-promoting fungi also have ISR-eliciting capacities (see Bent, 2006, for an overview).

E. SCOPE OF THIS REVIEW

Rather than aiming at an exhaustive overview of different rhizobacteria able to elicit ISR, in this review we specifically focus on mechanisms of rhizobacteria-mediated ISR. We highlight recent progress in the identification of bacterial determinants, produced by rhizobacteria, that can trigger ISR, and pay special attention to how plants may recognize and respond to these resistance-inducing stimuli. In addition, details about the role of phytohormones in the various defence signalling pathways reported to underpin rhizobacteria–mediated ISR are presented. The plant defence responses that are being triggered during the onset and/or maintenance of ISR have been the subject of some excellent recent reviews (Van Loon, 2007; Van Loon and Bakker, 2005) and will therefore not be discussed in detail. A cursory overview of rhizobacteria for which information is available about bacterial determinants and/or signalling pathways involved in ISR, is listed in Table I.

II. RECOGNITION The only bacterial determinants mentioned in the review by Van Loon et al. (1998) are LPS and siderophores, including SA. Since that time a variety of additional resistance-inducing molecules have been described, including flagella, biosurfactants, N-acyl-homoserine lactones (AHL), N-alkylated benzylamines, antibiotics, and exopolysaccharides (EPS) (Table I). In the next paragraphs ISR-eliciting bacterial determinants and possible mechanisms by which plants can recognize or interfere with these compounds are discussed in detail.

234

¨ FTE D. DE VLEESSCHAUWER AND M. HO A. FLAGELLA

Bacterial motility is based on the flagellum and is important for the virulence of bacterial pathogens (Ramos et al., 2004) and for root colonization by rhizobacteria (De Weger et al., 1987b). The flagellum consists of a long helical filament and a rotary motor which is anchored in the cell surface. Flagellar filaments consist of thousands of flagellin protein subunits. The central part of flagellin, which forms the surface of the flagellum, is highly variable in sequence and length, while the N- and C-termini, which face the inside of the flagellar tube, are well conserved among bacteria. Flagellin is transported to the cell surface through the hollow core of the flagellum and assembled at the distal tip. Flagellin, however, can also accumulate in the environment as a result of leaks and spillover during the construction of flagellae, as well as by active shedding during biofilm formation and during cell death (Go´mez-Go´mez and Boller, 2002). In the case of Pseudomonas putida WCS358, the major secreted protein appears to be flagellin. Apparently, flagella from strain WCS358 are easily sheared from the cells and appear in the extracellular fraction (De Groot et al., 1996). In contrast, only a small amount of flagellin has been detected in the supernatant of Ps. fluorescens WCS374 (De Weger et al., 1987b). The conserved part of flagellin is recognized as a PAMP by the innate imune systems of plants and animals. In animal cells, a specific D1 domain of the conserved part of the flagellin polypeptide is recognized by the Toll-like receptor TLR5 (Ramos et al., 2004; Zipfel and Felix, 2005). Plant cells recognize a stretch of 15–22 amino acids close to the conserved N-terminal domain of flagellin. Flg22, a 22-amino acid peptide spanning the conserved domain is an extremely potent elicitor in cell cultures of different plant species such as Arabidopsis, tomato, potato, and tobacco. In tomato cells, flg22 was active at a threshold of about 1 pM, and the concentration required for half-maximal activity was 30 pM (Felix et al., 1999). In Arabidopsis, flagellin is perceived through its direct interaction with the transmembrane leucine-rich-repeat receptor kinase (LRR-RK) FLS2 (Chinchilla et al., 2006). FLS2 is normally present on the plasma membrane and was found to be internalized upon flg22 stimulation (Robatzek et al., 2006). Orthologues of FLS2 have been identified in Nicotiana benthamiana (Hann and Rathjen, 2007), tomato (Robatzek et al., 2007), Brassica spp. (Dunning et al., 2007), and rice (Takai et al., 2008). Flg22-type sequences can be found in flagellins from widely divergent bacteria, including Ps. putida and Pseudomonas aeruginosa. The flagellins of the plant-associated bacteria Agrobacterium and Rhizobium, however, have a highly divergent flagellin sequence, and their flagellins or the corresponding flg22-type sequences do not stimulate the


235

flagellin perception system (Felix et al., 1999). Infiltration of 200 nM flg22 into lower leaves of Arabidopsis accession Col-0 or Ler-0 resulted in induced resistance against Ps. syringae pv. maculicola in distal leaves. Induced resistance could not be triggered by flg22 in the Ws-0 ecotype, a natural fls2 mutant insensitive to flagellin as well as to ET (Ton et al., 2001). Flg22induced systemic resistance was lost in the SAR-deficient Arabidopsis mutants sid2, npr1, ndr1, fmo1, eds1, and pad4 (Mishina and Zeier, 2007; see Table II for further information about these mutants). In contrast, when whole plants were treated with 1 M flg22, mutants npr1, eds1, pad4, and other mutants impaired in ET- or JA-dependent defence signalling, still showed a significant flg22-induced reduction in bacterial growth upon leaf infection with the bacterial speck pathogen Ps. syringae pv. tomato (Zipfel et al., 2004). It was suggested that whole plant treatment with flg22 induces the activation of the SA, JA, and ET pathways in parallel and that knocking out a single pathway alone does not abolish the induction of resistance (Zipfel et al., 2004). Recently, it was demonstrated that the early response to flg22 involves activation of several components of the JA-, ET-, and SAdefence signalling pathways, while the late response involves activation of SA-regulated processes. Activation of the late response may be dependent on signal strength reaching some threshold, as SA-dependent PR-1 expression was observed when flg22 was applied to seedling medium at a final concentration of 1 M, but not when applied at a concentration of 10 nM (Denoux et al., 2008). Up to now, a role of flagellin in rhizobacteria-mediated ISR has only been reported in Arabidopsis. Soil inoculation with Ps. putida WCS358 or a root dip in a solution containing 8.35 g/ml isolated flagella (corresponding to 108 colony-forming units (cfu)/ml) from this strain triggered ISR to Ps. syringae pv. tomato in Arabidopsis. However, a non-motile Tn5 mutant of WCS358 lacking flagella (strain GMB6), was equally effective in triggering ISR as the wild-type strain (Meziane et al., 2005). This can be explained by the fact that WCS358 has additional determinants that are recognized by Arabidopsis and that can trigger ISR, such as LPS and the iron-chelating fluorescent siderophore PSB (or pyoverdine) (see further). It is likely that flagellin from WCS358 is recognized by the FLS2 receptor in Arabidopsis, because the amino acid sequence of the conserved part of the N-terminal domain of Ps. putida flagellin is identical to flg22 and, as stated above, the major extracellular protein of WCS358 corresponds to flagellin. It would be interesting to test the ability of Ps. putida WCS358 or its isolated flagella to trigger ISR on fls2 Arabidopsis mutants. Isolated flagella of Ps. putida WCS358, however, did not induce systemic resistance to Botrytis cinerea and/or Colletotrichum lindemuthianum in bean or tomato (Meziane et al., 2005) or to the rice-blast fungus Magnaporthe oryzae in rice (D. De Vleesschauwer, unpublished data).

TABLE II Description of Plant Mutants or Transgenic Lines Mentioned in Table I and/or in the Text of this Review Mutant/ transgenic line

Phenotypeb

Wild-type gene product/functionb

Reference

Arabidopsis coi1

Coronatine- and JA-insensitive

F-box protein, activation of JA-dependent responses

Feys et al. (1994), Xie et al. (1998)

coi1-16a

Coronatine- and JA-insensitive

Ellis and Turner (2002)

cpr1 eds1

SA overproducer, constitutive expressor of PR genes Impaired in SA signalling

F-box protein, activation of JA-dependent responses Negative regulator of SAR

Parker et al. (1996), Falk et al. (1999)

eds5/sid1

SA-induction deficient

eds8

Reduced sensitivity to MeJA

Lipase-like protein, SA and oxidative stress signalling MATE transporter, SA biosynthesis JA signalling

ein2

ET-insensitive

ein3 ein4 ein5, ein7

ET-insensitive ET-insensitive ET-insensitive

Positive regulator of ET responses Transcription factor ET receptor Exoribonuclease

ein6 eir1

ET-insensitive ET-insensitive in the roots

Involved in ET signalling Auxin transporter

eto1

ET overproducer

etr1

ET-insensitive

Negative regulator of ET biosynthesis ET receptor, ET sensitivity

Bowling et al. (1994)

Nawrath and Me´traux (1999), Nawrath et al. (2002) Glazebrook et al. (1996), Ton et al. (2002a) Guzmań and Ecker (1990), Alonso et al. (1999) Kieber et al. (1993), Solano et al. (1998) Roman et al. (1995), Hua et al. (1998) Roman et al. (1995), Potuschak et al. (2006) Roman et al. (1995) Roman et al. (1995), Luschnig et al. (1998) Guzmań and Ecker (1990), Wang et al. (2004) Bleecker et al. (1988), Chang et al. (1993)

jar1

Deficient in linolenic acid, no JA accumulation Flagellin-insensitive Impaired in SAR, no systemic SA accumulation JA-insensitive

jin1-1, jin1-2/myc2

JA-insensitive

myb72-1/myb72-2

Impaired in ISR signalling

NahG

No SA accumulation

ndr1

Impaired in SAR

npr1/nim1

SA-insensitive, non-expressor of PR genes SA induction-deficient

fad3-2 fad7-2 fad8 fls2 fmo1

sid2

pad3-1

Reduced camalexin levels, glutathione-deficient Camalexin-deficient

pad4-1

Impaired in SA signalling

pad2-1

Trienoic fatty acid biosynthesis

McConn and Browse (1996)

Flagellin receptor Flavin-dependent monooxygenase

Go´mez-Go´mez and Boller (2000) Mishina and Zeier (2006)

JA-amino synthetase, JA signalling MYC-type helix-loop-helix transcription factor MYC2 R2R3-MYB-like transcription factor Transformed with SA hydroxylase, breaks down SA to catechol Plasma membrane-localized protein, SA signalling Ankyrin-repeat protein, SA and JA/ET response regulator Isochorismate synthase 1, SA biosynthesis

-glutamyl cysteine synthetase, glutathione synthesis P450 monooxygenase, camalexin biosynthesis Lipase-like protein, SA and oxidative stress signalling

Staswick et al. (1992), Staswick and Tiryaki (2004) Lorenzo et al. (2004) Van der Ent et al. (2008) Delaney et al. (1994) Century et al. (1995, 1997) Cao et al. (1994) Nawrath and Me´traux (1999), Wildermuth et al. (2001) Glazebrook and Ausubel (1994), Parisy et al. (2007) Glazebrook and Ausubel (1994), Schuhegger et al. (2006b) Glazebrook et al. (1996), Jirage et al. (1999) (continues)

TABLE II Mutant/ transgenic line

Phenotypeb

Rice hebiba 417

JA-deficient ET-insensitive

NahG

Reduced SA accumulation

Tobacco NahG

No SA accumulation

Tetr18

ET-insensitive

Tomato def1 NahG

Impaired in JA biosynthesis No SA accumulation

never-ripe (nr)

ET-insensitive

a b

(continued)

Wild-type gene product/functionb

Reference

JA biosynthesis Transformed with OsEIN2 antisense construct Transformed with SA-hydroxylase gene, breaks down SA to catechol

Riemann et al. (2003) Jun et al. (2004)

Transformed with SA hydroxylase, breaks down SA to catechol Transformed with mutant ET-receptor gene etr1-1 from Arabidopsis

Gaffney et al. (1993)

Octadecanoid metabolism Transformed with SA-hydroxylase gene, breaks down SA to catechol ET receptor, ET sensitivity

Carries additional mutation in pen2, a gene involved in non-host resistance (Westphal et al., 2008). ET, ethylene; JA, jasmonate; MeJA, methyl jasmonate; SA, salicylic acid.

Yang et al. (2004)

Knoester et al. (1998)

Howe et al. (1996) Brading et al. (2000) Lanahan et al. (1994)


239

Although rice has a flagellin receptor, OsFLS2, flg22 induces only a very weak immune response without cell death in cultured rice cells (Takai et al., 2008), which may explain why flagella of WCS358 are not recognized by rice roots. It is presently unclear why purified flagella of WCS358 fail to induce resistance in tomato, as a high-affinity binding site for flagellin, flg22, and its 15-amino acid peptide flg15 has also been identified in tomato (Robatzek et al., 2007). One explanation is that the resistance pathway induced upon flagellin-receptor recognition in tomato is not effective against Bo. cinerea. In this context, it should be mentioned that purified flagella of Ps. fluorescens WCS374, Ps. fluorescens WCS417, and Ps. aeruginosa 7NSK2 did not trigger resistance to Bo. cinerea in tomato either (Meziane, 2005). It remains to be investigated whether purified flagella of WCS358 or other rhizobacteria can induce resistance to other pathogens in tomato. Van Wees et al. (1997) reported that unlike Ps. putida WCS358, Ps. fluorescens WCS374r did not induce resistance to Ps. syringae pv. tomato in Arabidopsis. But recently it was shown that Ps. fluorescens WCS374r does trigger ISR against Ps. syringae pv. tomato in Arabidopsis when the inoculum was grown at 33 8C rather than at 28 8C (Ran et al., 2005b), or when bacteria were applied at a low inoculum density (Djavaheri, 2007). When applied at an initial density of 103 cfu/g of soil, WCS374r reached a population density on Arabidopsis roots of around 107 cfu/g of root after 3 weeks. Apparently, active multiplication of WCS374r is required for bacterial elicitors of ISR to be produced or perceived by the plant. Two non-motile mutants of WCS374, L30 and L36 (De Weger et al., 1987b), lacking flagellin and defective in flagellin polymerization, respectively, failed to induce resistance to Ps. syringae pv. tomato. Isolated flagella from a culture of WCS374r significantly reduced disease incidence when applied to Arabidopsis roots 7 or 4 days prior to inoculation with the pathogen. Preparations of mutants L30 or L36, however, were not effective, but it should be noted that root colonization by these mutants was significantly impaired, which might be the reason for the lack of ISR-eliciting activity of these mutants. These results suggest that flagella of actively growing Ps. fluorescens WCS374r cells can induce systemic resistance to Ps. syringae pv. tomato in Arabidopsis, but it cannot be excluded that other determinants produced by actively growing cells are also involved in this process. It was hypothesized that WCS374r, when applied at high density, loses its flagella, whereas, if the number of cells is low, they would be incited to propel themselves towards the nutrient-rich zone of the rhizosphere through active flagellar movement. Perception of the flagella by the roots would then result in elicitation of the plant. Loss of flagella at high cell densities has been shown for Ps. syringae cells on moist bean leaves (Djavaheri, 2007). In this

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context, it is noteworthy that the defence pathway against Ps. syringae pv. tomato triggered by WCS374r applied at low densities in Arabidopsis was SA-independent (tested in genotypes NahG and sid1), while dependent on JA, ET, and NPR1 and probably similar to the signalling pathway described by Pieterse et al. (1998) for WCS417-mediated ISR (Djavaheri, 2007). These results may indicate that the flagella dose perceived by the plant is too low to trigger the late SA-mediated defences seen by Denoux et al. (2008) or the SA-dependent SAR described by Mishina and Zeier (2007). It is not known by which signalling pathway(s) isolated flagella or pure flg22 induce systemic resistance when applied to plant roots and this should be tested. As stated before, it cannot be excluded either that other determinants of WCS374r besides flagella are recognized by the plant. Ran et al. (2005b) showed that at cell densities of 5 107 cfu/g of soil Ps. fluorescens WCS374 triggered ISR to Ps. syringae pv. tomato in Arabidopsis when the inoculum was grown at 33 8C, but not when grown at 28 8C. It is not clear whether growth temperature may have an influence on flagella formation or on other bacterial determinants that may be implicated in WCS374-elicited ISR. Interestingly, mutants of Ps. aeruginosa 7NSK2 that failed to induce systemic resistance in tomato, tobacco, and bean, still induced systemic resistance to Ps. syringae pv. tomato in Arabidopsis by an SA-independent signalling pathway (Ran et al., 2005b). It is not unlikely that flagellin from Ps. aeruginosa 7NSK2 is involved in this elicitation, as flg22like sequences are found in the flagellin of Ps. aeruginosa. B. LIPOPOLYSACCHARIDES

LPS constitute the major structural component of the outer membrane of Gram-negative bacteria. LPS are complex molecules possessing both hydrophilic and lipophilic properties. LPS consist of three different components: a lipid, a core oligosaccharide, and an O-linked polysaccharide. The lipophilic part, called lipid A, is embedded in the outer membrane. Its structure is highly conserved among Gram-negative bacteria. The core oligosaccharide consists of a short chain of sugars, which connects the lipid A part to the hydrophilic O-antigen, which is composed of repeating oligosaccharide subunits made up of three to five sugar molecules. The individual chains can vary in length ranging up to 40 repeating units. The O-polysaccharide is much longer than the core oligosaccharide and extends out into the environment. The composition of the O-antigen part is highly variable between species and even between strains of Gram-negative bacteria (see Lerouge and Vanderleyden, 2002, for a comprehensive review). LPS has a structural function in stabilizing the outer membrane of the bacterium but also play a


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number of important roles in the interactions of bacteria with eukaryotic hosts. LPS are important virulence factors in animal pathogenesis. Lipid A is the toxic component of LPS and lipid A molecules are detected at picomolar concentrations by the innate immune system of animals. LPS molecules can dissociate from the surface of Gram-negative bacteria. The hydrophilic O-polysaccharide may allow diffusion or delivery of the toxic lipid in the hydrophilic environment. LPS first binds the serum LPS-binding protein. Then, the LPS, bound to the LPS-binding protein, is perceived by a complex that consists of the Toll-like receptor TLR4, the membrane receptor CD14, and an extracellular accessory protein MD-2 (Underhill and Ozinsky, 2002). LPS play an important role in bacterial interactions with plants also. The most studied effect of LPS on plant cells is their ability to prevent the HR induced in plants by either avirulent or nonhost bacteria. Following adhesion to cell wall components, the LPS bind to yet to be defined plasma membrane receptors. This response can be observed in various plants including tobacco, pepper, turnip, and Arabidopsis and is referred to as ‘localized induced resistance’ or ‘response’ (LIR) (see Dow et al., 2000, for a review). The minimal structure of LPS required for the prevention of the HR appears to be the lipid A attached to a truncated core oligosaccharide. LPS from plantpathogenic bacteria can also directly induce or activate defence-related responses in plants (Newman et al., 2007). In Arabidopsis, LPS from animal and plant pathogens at concentrations between 10 and 200 g/ml induced a rapid burst of nitric oxide (NO), a hallmark of innate immunity in animals. Lipid A was as effective as most LPS preparations. In addition, LPS of Burkholderia cepacia at 100 g/ml activated an array of defence genes in Arabidopsis plants and suspension cells, including glutathione S-transferases, cytochrome P450 and many genes encoding PR-proteins, such as PR-1, -2, -3, -4, and -5. Several of the LPS-induced genes were activated in systemic leaves too (Zeidler et al., 2004). Recently it was shown that the intact lipooligosaccharide (LOS) (LPS without the O-chain) of Xanthomonas campestris pv. campestris and the lipid A moiety, as well as the core oligosaccharide derived from it, could trigger the LIR response and the expression of PR-1 and -2 in Arabidopsis. In response to X. campestris pv. campestris LOS transcript levels showed an early but transient accumulation at 12 h and a later more substantial accumulation after 20 h. The core oligosaccharide only induced the early accumulation, while lipid A only induced a substantial response after 20 h. These data suggest that LOS is recognized through independent mechanisms involving the core oligosaccharide and lipid A moieties, respectively (Silipo et al., 2005). Infiltration of LPS preparations from Ps. aeruginosa or Escherichia coli at a concentration of 100 g/ml into primary leaves triggered resistance to

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Ps. syringae pv. maculicola in systemic leaves. This systemic resistance response was not observed when a de-esterified LPS lacking the lipid A part of the molecule was used. The LPS-elicited ISR response did not develop in the SAR-deficient Arabidopsis mutants sid2, npr1, ndr1, fmo1, eds1, and pad4 (Mishina and Zeier, 2007). To date, no LPS receptor has been identified in plants. However, fluorescently labelled LPS from X. campestris pv. campestris rapidly bound to tobacco cells and became internalized into endocytic vesicles, consistent with a receptor-mediated process and reminiscent of the mammalian system (Gross et al., 2005). In general, it can be stated that putative LPS receptors in plants are probably of a low-affinity type as micrograms-per-millilitre concentrations of LPS are needed to trigger plant defence responses (Zeidler et al., 2004). In the case of PGPR, the O-antigen of the LPS rather than the lipid A or the core oligosaccharide appears to be the moiety that triggers ISR in plants. In radish Ps. fluorescens WCS374 and Ps. fluorescens WCS417 triggered ISR against Fusarium oxysporum f. sp. raphani (Leeman et al., 1995). Under conditions of high iron availability, the O-antigen-minus mutants of these strains no longer reduced disease incidence, while their purified LPS were as effective as the wild-type strains. Ps. putida WCS358 or its isolated LPS were unable to trigger ISR against Fusarium wilt in radish. However, this strain triggered resistance to Bo. cinerea and C. lindemuthianum in bean and to Bo. cinerea in tomato. In these three cases, an O-antigen-minus mutant of WCS358 no longer triggered ISR, while purified LPS applied to plant roots was very active (Meziane et al., 2005). In Arabidopsis, LPS of Ps. fluorescens WCS374 is not effective, but LPS of both Ps. fluorescens WCS417 and Ps. putida WCS358 appear to be involved in ISR against Ps. syringae pv. tomato. Cell wall preparations of WCS417r triggered ISR, while cell walls of a mutant lacking the O-antigenic part of the LPS were less effective (Van Wees et al., 1997). Likewise, crude LPS of WCS358 triggered ISR (Meziane et al., 2005). The O-antigen-minus mutants of these strains, however, were equally effective as the wild-type strains (Meziane et al., 2005, Van Wees et al., 1997). These results indicate that other determinants of WCS417 and WCS358 that are still produced by the O-antigen-minus mutants are recognized in Arabidopsis. It cannot be excluded that the lipid A part or core oligosaccharides of the LPS molecules of WCS358 are recognized in Arabidopsis, as cell walls of its O-antigen-minus mutant were not tested. Alternatively, for Ps. putida WCS358 it is known that its flagella and PSB are also recognized in Arabidopsis. Ps. fluorescens WCS374 and Ps. putida WCS358 differ in their LPS composition; the LPS composition of Ps. fluorescens WCS417 is not known. The LPS of Ps. fluorescens WCS374 contains fucose and rhamnose (De Weger


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et al., 1987a), two sugars that are not found in the LPS of WCS358 but are typically present in O-chain structures from phytopathogenic bacteria (Newman et al., 2007). The O-antigenic side chain of WCS358, however, contains the aminosugar quinovosamine, which is absent in the LPS of WCS374 (De Weger et al., 1987a). In this context, it is interesting to note that quinovosamine is also present in the O-antigen of the LPS from Rhizobium etli. Mutants of R. etli and other Rhizobia that lack the O-antigen portion of the LPS are ineffective in infection and cause aberrant development of nodules on their host legumes. The LPS of R. etli mutant strain CE166 lacks quinovosamine and is as ineffective in symbiosis as mutants that lack the O-antigen completely. It was suggested that the symbiotic role of LPS requires a structural feature conferred by quinovosamine (Noel et al., 2000). Interestingly, the LPS of R. etli strain G12 acts as the inducing agent of systemic resistance against the potato cyst nematode Globodera pallida in potato roots (Reitz et al., 2000). In a follow-up study, however, it was shown that the oligosaccharides of the core region, rather than the O-antigen are the main trigger of systemic resistance in potato roots towards G. pallida infection (Reitz et al., 2002). C. BIOSURFACTANTS

Recently, it was shown that also biosurfactants, and more specifically cyclic lipopeptides, can act as bacterial determinants for ISR in plants. Cyclic lipopeptides are composed of a fatty acid tail linked to a short oligopeptide, which is cyclized to form a lactone ring between two amino acids in the peptide chain (Raaijmakers et al., 2006). Cyclic lipopeptides are produced by several plant-associated bacteria, including pathogenic and antagonistic Pseudomonas bacteria (reviewed by Raaijmakers et al., 2006) and antagonistic Bacillus strains (reviewed by Ongena and Jacques, 2008). Bacillus subtilis produces cyclic lipopeptides from the surfactin, iturin, and fengycin families. Surfactins are heptapeptides interlinked with a -hydroxy fatty acid with a length that may vary from 13 to 16 carbon units. Surfactins display haemolytic, anti-viral, anti-mycoplasma, and anti-bacterial activities but no marked fungitoxicity. Surfactins can readily associate with, and tightly anchor into lipid bilayers and thereby, interfere with biological membrane integrity in a dose-dependent manner. At low concentration, surfactins insert exclusively in the outer leaflet of the membrane, inducing only limited perturbation. At intermediate concentrations, surfactins cause a transient permeabilization, but membranes reanneal. At higher concentrations, irreversible pore formation and complete disruption and solubilization of the lipid bilayer can occur. Fengycins are lipodecapeptides with an internal

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lactone ring in the peptidic moiety and with a -hydroxy fatty acid chain ranging from 14 to 18 carbon units that can be saturated or unsaturated. Fengycins show strong fungitoxic activity, specifically against filamentous fungi. Mechanistically, the action of fengycins is less well known than that of surfactins, but they also readily interact with lipid bilayers and to some extent also possess the potential to alter cell membrane structure and permeability in a dose-dependent manner. As the presence of cyclic lipopeptides at concentrations of about 5 M was not associated with any phytotoxicity or adverse effect on bean or tomato plants (Ongena and Jacques, 2008), it is suggested that these molecules could interact with plant cells by inducing disturbance or transient channelling in the plasma membrane that can in turn activate a biochemical cascade of molecular events leading to defensive responses. Iturins are heptapeptides linked to a -amino fatty acid chain with a length of 14–17 carbons. They display a strong in vitro antifungal action against a wide variety of yeasts and fungi, but only limited antibacterial and no antiviral activities. Fungitoxicity is based on osmotic perturbation owing to the formation of ion-conducting pores in the fungal membranes and not on membrane disruption or solubilization. In addition, the presence of ergosterol in fungal membranes is important for iturin activity. The lack of effect of iturin on plants cells could be because of a different composition in phytosterols (Ongena and Jacques, 2008). Pure fengycins and surfactins provided a significant ISR-mediated protective effect on bean plants against Bo. cinerea, similar to the one induced by living cells of the strain Ba. subtilis S499 (Ongena et al., 2007). In addition, a significant protective effect against Bo. cinerea was gained by treating bean or tomato plants with surfactin and/or fengycin overproducing derivatives generated from wild-type Ba. subtilis Bs168, which is not able to synthesize cyclic lipopeptides and does not induce systemic resistance in plants. On tomato, surfactin appeared to be more effective than fengycin. However, fengycins, but not surfactins, could induce a defence response in potato tuber cells, while both surfactants elicited major defence-associated changes in tobacco cells. Iturin, however, does not show any ISR-eliciting activity in tomato plants, potato tuber slices, or tobacco cells. None of the surfactants had an effect on cucumber (Ongena and Jacques, 2008; Ongena et al., 2007). Raaijmakers et al. (2006) have classified the cyclic lipopeptides of Pseudomonas spp. into four major groups: the viscosin, amphisin, tolaasin, and syringomycin groups. Massitolide A is a member of the viscosin group which harbours cyclic lipopeptides with 9 amino acids linked with a 10-carbon hydroxy fatty acid chain. The massitolide-producing Ps. fluorescens strain SS101 was effective in preventing infection of tomato leaves by Phytophthora infestans and significantly reduced the expansion of existing


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late blight lesions. Ps. fluorescens SS101 or massitolide A could only prevent late blight infections through direct antagonism. Ps. fluorescens SS101 or massitolide A at a concentration of 50 g/ml (44 M) significantly reduced lesion area but not disease incidence when applied to the lower leaves of tomato plants, 24 h prior to challenge inoculation of the upper leaves with zoospores of Ph. infestans. A massitolide-negative mutant of Ps. fluorescens SS101 completely lost the ability to induce systemic resistance. Ps. fluorescens SS101 significantly reduced lesion area and sporangia formation per unit lesion area, but not disease incidence when applied to tomato seeds 5 weeks prior to inoculation with Ph. infestans zoospores on the tomato leaves. The massitolide-negative mutant showed effects intermediate between the control and SS101 treatments. These results show that massitolide A is a bacterial determinant of ISR in tomato (Tran et al., 2007). How massitolide A induces resistance in plants is not known, but it is likely that it can also be explained by effects on the plant plasma membrane. Cyclic lipopeptides produced by plant-pathogenic Pseudomonas spp. cause the formation of ion channels in the host plasma membrane. At high concentrations, cyclic lipopeptides can solubilize plasma membranes. Cell suspensions of Ps. fluorescens SS101 or massitolide A cause lysis of zoospores of oomycete pathogens within 60 s (De Souza et al., 2003). Cell suspensions of Ps. putida WCS358 also show a clear biosurfactant activity, such as lowering of the surface tension of water and drop collapse, but they do not have an adverse effect on zoospores (De Souza et al., 2003). There are no indications that biosurfactants of Ps. putida WCS358 play a role in ISR. It would be interesting to test whether there is a correlation between the ability of a biosurfactant to cause zoospore lysis and and its capacity to trigger ISR. D. N-ACYL-L-HOMOSERINE LACTONE

AHLs occur in various Gram-negative bacteria and act as signal molecules to control expression of various functions in a cell density-dependent manner. This phenomenon is known as quorum sensing (Miller and Bassler, 2001). Serratia liquefaciens MG1 produces two AHL molecules: N-butanoyl and N-hexanoyl homoserine lactones. S. liquefaciens MG1 can induce systemic resistance to Alternaria alternata when applied at a concentration of 1010 cfu/ ml around the shoots of tomato plants 3 days prior to challenge inoculation with the pathogen. An AHL-negative mutant of S. liquefaciens MG1 slowed down the development of A. alternata-induced cell death, but infected plants showed no significant alterations in response to the fungal pathogen when compared with the non-inoculated control. Inoculation with the AHL-producing Ps. putida strain IsoF also resulted in a marked reduction of leaf

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damage following challenge with A. alternata. This strain produces at least four different 3-oxo-AHL molecules with acyl side chains varying from 6 to 12 carbon units. An AHL-negative mutant, F117, was less effective than the wild-type strain IsoF, but still reduced necrotic cell death by about 50%, suggesting that additional factors which are not regulated by quorum-sensing compounds contribute to the biocontrol activity of Ps. putida IsoF. S. liquefaciens MG1 and pure N-hexanoyl homoserine lactone (10 M) significantly increased free and conjugated SA levels in tomato leaves, while this increase was not observed for the AHL-negative mutant. In addition, a macroarray analysis of defence gene expression in tomato leaves after application of 10 M N-hexanoyl or N-butanoyl homoserine lactone to the roots showed, among others, enhanced expression of PR-1a (P4), PR-1b (P6), and a 26 kDa acidic chitinase (Schuhegger et al., 2006a). In this study, however, the effect of application of pure AHL to plant roots on A. alternata leaf infection is not shown. It cannot be excluded that the bacterial determinant triggering resistance to A. alternata is not the homoserine lactone but a secondary metabolite regulated by quorum sensing. E. N-ALKYLATED BENZYLAMINE

Ps. putida BTP1 induces resistance to Bo. cinerea in bean and tomato and to Pythium aphanidermatum and Colletotrichum lagenarium in cucumber (Ongena et al., 1999, 2002, 2008). At least in bean and cucumber, N, N-dimethyl, N-tetradecyl-N-benzylammonium (NABD) appears to be the bacterial determinant responsible for ISR (Ongena et al., 2005, 2008). Pure benzylamine at a concentration of 1 M was also effective in triggering induced resistance in bean and cucumber, indicating that the aromatic amino part is important for the biological activity of the entire molecule. It was hypothesized that the aromatic phenol group, which is also present in thiamine (according to Ahn et al. (2005) another inducer of systemic resistance in plants), SA, and 2,4-diacetylphloroglucinol (DAPG), could constitute a general motif widely recognized by specific plant cell receptors (Ongena et al., 2008). F. SIDEROPHORES

Siderophores are low-molecular-weight molecules that are secreted by most aerobic and facultative anaerobic microorganisms to trap traces of ferric iron [Fe(III)] in the environment and deliver the iron to the cell. According to current concepts, ferric ion-loaded siderophores are selectively recognized and bound by high-affinity receptor proteins that are present on the outer


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bacterial membrane. Although competition for ferric iron between biocontrol bacteria and plant deleterious microorganisms is considered to be the main mode of action of siderophores, there is accumulating evidence that these compounds also function as important triggers of the plant immune response (Ho¨fte and Bakker, 2007).

1. Pseudobactins So far, research aimed at deciphering the role of siderophores in systemically induced resistance has tended to focus on the involvement of pseudobactin-type elicitors. Pseudobactin (PSB), also termed pyoverdin or fluorescein, designates an extensive group of diffusible green-fluorescent pigments that are historically recognized as the distinctive phenotypic trait of the rRNA homology group I species of the genus Pseudomonas (Visca et al., 2007). Following the seminal work of Meyer (Meyer and Abdallah, 1978; Meyer and Hornsperger, 1978), the structures of more than 50 PSBs from different strains have now been determined. All appear to comprise a conserved fluorescent dihydroxyquinoline chromophore joined to a highly variable peptide and acyl side chain (for review see Visca et al., 2007). Synthesis of PSBs and the corresponding membrane receptors occurs exclusively under conditions of iron starvation, and is repressed under iron-rich conditions (Meyer and Abdallah, 1978). In addition, some PSBs are known to trigger their own synthesis and uptake in a cell-density dependent manner, indicating a complex autoregulatory system that enables maximal expression of the cognate synthesis and receptor genes only when the siderophore is effective in Fe3þ delivery to the cell (Visca, 2004). A role for PSBs as determinants of rhizobacteria-triggered resistance has been reported in several systems. For instance, a clear-cut role for PSB in ISR was reported for Ps. putida WCS358 in the suppression of Ralstonia solanacearum in Eucalyptus urophylla (Ran et al., 2005a), Erwinia carotovora in tobacco (Van Loon et al., 2008), and Bo. cinerea in tomato (Meziane et al., 2005). In all three cases, the purified PSB358 was as effective as the wild-type strain, whereas a PSB-deficient mutant lost the ability to cause ISR. In bean, however, the situation appears to be more complex in that wild-type bacteria, the PSB-minus mutant, and isolated PSB358 all induced a significant level of ISR against Bo. cinerea and C. lindemuthianum, indicating redundancy of ISR-triggering traits in this system (Meziane et al., 2005). A similar phenomenon was observed for the ISR elicited by Ps. fluorescens WCS374r in Eu. urophylla. Although infiltration of the lower leaves with a siderophoreminus mutant of WCS374r 3–7 days before challenge inoculation with Ra. solanacearum, caused a similar reduction of bacterial wilt as the parental strain, purified PSB induced resistance as well (Ran et al., 2005b). Both the


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siderophore and another, yet unidentified, inducing determinant of WCS374r thus seem to be capable of causing ISR in Eucalyptus. In Arabidopsis, Ps. fluorescens WCS374r induced resistance to Turnip crinkle virus (TCV) irrespective of the bacterial density applied to the soil. Mutants impaired in PSB and/or SA production lost the ability to trigger ISR to TCV, suggesting that both compounds are required in this pathosystem (Djavaheri, 2007). In rice, however, PSB seems to be the only determinant responsible for elicitation of WCS374r-mediated ISR. Assessing the effect of several well-defined mutants and testing of the purified compound in the microgram range unambiguously demonstrated that WCS374r-afforded protection against the rice blast-causing ascomycete M. oryzae is based on PSBmediated priming for a multifaceted cellular defence response, comprising among others the concerted expression of a diverse set of physiological and biochemical defences, as well as a hyperinduction of H2O2 generation in the epidermis (De Vleesschauwer et al., 2008). Strikingly, the isolated PSBs of Ps. fluorescens WCS358 and Ps. aeruginosa 7NSK2 failed to induce ISR in rice against M. oryzae, whereas PSB of Ps. fluorescens C7R12 was as effective as that of WCS374 (our unpublished data; Fig. 1). Such variability between a

Ctrl BTH PSB374 PSBC7R12

b b b a

PSB358 PSB7NSK2

a 0 10 20 30 40 50 60 Sporulating blast lesions on leaf 5

Fig. 1. Effectiveness of benzothiadiazole (BTH; 0.05 mM)- and pseudobactin (PSB)-induced resistance in rice against the blast pathogen Magnaporthe oryzae. To trigger resistance, rice seedlings (5-leaf stage) were hydroponically fed with the various compounds by including the desired concentration in the half-strength Hoagland nutrient solution 3 days before challenge. Six days post inoculation, disease was rated by counting the number of susceptible-type lesions on leaf 5. All pseudobactins were isolated from the respective bacterial cultures and applied at a concentration of 100 g per root system, except for the purified PSB of Pseudomonas fluorescens WCS374, which was applied at a concentration of 70 g per plant. Different letters indicate statistically significant differences between treatments (Kruskal–Wallis and Mann–Whitney test, ¼ 0.05).


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PSBs has also been observed in radish, where the PSB siderophore of WCS374, but not those of Ps. fluorescens WCS417 and WCS358, was found to trigger ISR against Fusarium wilt (Leeman et al., 1996). In contrast, Van Loon et al. (2008) recently reported the capacity of all three of these siderophores to enhance defence against Er. carotovora in tobacco. Finally, PSB-conferred disease suppression was also found to be effective against Tobacco necrosis virus in tobacco, on the basis of the observation that CHA400, a PSB-deficient mutant of Ps. fluorescens CHA0, was less effective in reducing numbers and size of viral lesions than the parental strain (Maurhofer et al., 1994). Although the phenomenon of PSB-mediated resistance has received steadily increasing attention over the past decade, it is still unknown how PSB-type siderophores are perceived by the plant and ultimately give rise to ISR. An alternative to direct recognition of PSB elicitors by the plant is the perception of microbially induced alterations in the plant’s immediate environment, i.e. the rhizosphere. Given the scarcity of bioavailable iron [Fe(III)] in the rhizosphere, and the high affinity of PSBs for the ferric ion, PSBproducing rhizobacteria are thought to interfere with the iron acquisition of other soil organisms, including the host plant (Vansuyt et al., 2007). In this context, our recent observation that Ps. fluorescens WCS374r aggravates chlorosis symptoms of young rice plants grown under iron-limiting conditions is of particular interest (our unpublished data; Fig. 2). Strikingly, enhanced iron deficiency-induced chlorosis was not observed in response to root colonization by rhizobacteria producing non-ISR-eliciting PSBs, such as Ps. aeruginosa 7NSK2 or Ps. putida WCS358 (our unpublished data). Furthermore, purified PSB374, but not PSB358, was found to trigger intracellular iron depletion in systemic leaves as evidenced by the down-regulated expression of the iron homeostasis marker gene OsFer1 (our unpublished data). These findings suggest that the ability of a given PSB to increase blast resistance is related to its potential to deprive rice from iron. In a recent microarray study on iron-deficient rice, Kobayashi et al. (2005) found that iron deficiency in roots strongly elicits the transcriptional activation of nearly all genes involved in the methionine cycle, both in root and leaf tissue. Furthermore, several studies point to a role for the methionine cycle and its main intermediate, the universal substrate S-adenosyl-L-methionine (SAM), in rice defence to M. oryzae. Most tellingly in this regard, Seguchi et al. (1992) reported that the activity of the SAM-utilizing enzyme SAM decarboxylase was suppressed by as much as 50% in M. oryzae-inoculated rice plants, whereas such suppression was not observed in plants pretreated with the blast resistance-inducing chemical N-cyanomethyl-2-chloroisonicotinamide. Similarly, SAM-SYNTHETASE, a SAM-biosynthesis gene, was shown to be


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Distribution over classes

100%

80%

60%

Severe chlorosis Slight chlorosis No chlorosis

40%

20%

0% Ctrl

WCS374r

AT12

Fig. 2. Root colonization by the wild-type bacterium Pseudomonas fluorescens WCS374r aggravates iron-deficiency-induced chlorosis of young rice seedlings (5-leaf stage), while strain AT12, a salicylic acid-positive but pseudobactin- and pseudomonine-negative mutant derivative of WCS374r, does not induce chlorosis.

dramatically upregulated upon treatment of rice with probenazole, a blast fungicide and well-characterized inducer of resistance in several plant species (Shimono et al., 2003). The link between the methionine cycle, SAM metabolism, and resistance to M. oryzae is further strengthened by the rapid and specific expression of OsBISAMT1, encoding a putative SAM-methyltransferase, in incompatible rice–M. oryzae interactions (Xu et al., 2006), and the observation that topical application of methionine not only induces production of the rice phytoalexins sakuranetin and momilactone A, but also increases resistance to subsequent blast attack (Nakazato et al., 2000). Taking these facts into account, one may hypothesize that PSB-type siderophores enhance defence against M. oryzae by depriving rice roots from iron, leading to cytosolic iron depletion and resultant activation of the methionine cycle. This hypothesis has received further support recently from work by Liu et al. (2007). In line with disease-related alterations in iron homeostasis in animals (Hissen et al., 2005; Schaible and Kaufmann, 2004), these authors elegantly demonstrated that targeted redistribution of redox-active Fe inflicted by powdery mildew attack, acts as an underlying factor associated with the oxidative burst and regulation of disease resistance in cereals. A model implying PSB-inflicted iron stress on the roots as a primary event in the activation of rhizobacteria-mediated resistance may also


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hold for WCS417r-triggered ISR in Arabidopsis, as MYB72, a transcription factor gene required for the early onset of ISR (Van der Ent et al., 2008), was reported to be activated exclusively in response to low-iron conditions (Colangelo and Guerinot, 2004; Van de Mortel et al., 2006). Nevertheless, it should be noted that experiments conducted in radish indicate that iron deficiency alone is not sufficient to enhance defence against Fusarium wilt, implicating the necessity for at least one additional component to induce systemic resistance in this system (Leeman et al., 1996).

2. SA and SA-containing siderophores Under conditions of iron limitation, several Pseudomonas spp. not only produce PSBs but also the phenolic compound SA, which functions as a signalling hormone in the development of SAR (Durrant and Dong, 2004; Loake and Grant, 2007). Accordingly, the increased effectiveness of the pseudomonads WCS374r and WCS417r to suppress Fusarium wilt in an iron-deficient radish system was originally attributed to bacterial SA production (Leeman et al., 1996). Implicit here was the view that the ISR triggered by these strains would be reliant on SA signalling in the plant. However, Hoffland et al. (1995) failed to observe any activation of the SA-dependent signalling conduit in radish after treatment with WCS374. Moreover, root treatment with 5 107 cfu/g of soil of strain WCS374r did not induce systemic resistance in Arabidopsis against Ps. syringae pv. tomato, whereas exogenously administered SA did (Djavaheri, 2007; Van Wees et al., 1997). Additionally, in some elegant work on Arabidopsis, Ran et al. (2005b) demonstrated that many SA-producing rhizobacteria, among which are Ps. aeruginosa 7NKS2 and Ps. fluorescens WCS417, elicit systemic protection against Ps. syringae pv. tomato in an SA-independent manner (Ran et al., 2005b). In keeping with these results, several other studies describe that rhizobacterial SA production and triggering of ISR are not necessarily interrelated. For instance, elicitation of ISR in tobacco and cucumber by the versatile resistance-inducer Serratia marcescens 90-166 was shown to function independently of bacterially synthesized SA, while requiring production of a catechol-type siderophore (Press et al., 1997, 2001). Together, these reports constitute a large body of evidence suggesting that SA, despite being a potent elicitor of SAR, does not generally contribute to initiation of rhizobacteria-mediated ISR. This apparent discrepancy can be addressed by considering that bacterial SA often serves as a precursor in the biosynthesis of other siderophores

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containing a SA-moiety, such as pseudomonine in Ps. fluorescens WCS374r and pyochelin in Ps. aeruginosa 7NSK2. In this perspective, it is tempting to speculate that in the rhizosphere, where iron-limiting conditions tend to prevail, SA is not excreted by the bacteria but channelled into the production of SA-containing siderophores. Indeed, measuring SA levels on the roots of tomato colonized by either 7NSK2 or its chemical mutant KMPCH, which is unable to convert SA into pyochelin, demonstrated that only KMPCH produces nanogram amounts of SA in the rhizosphere, whereas 7NSK2 does not (Audenaert et al., 2002). In line with the latter observation, Audenaert et al. (2002) postulated that elicitation of ISR by 7NSK2 does not depend on SA per se, but rather is on the basis of a synergistic interaction between SA-derived pyochelin and the phenazine antibiotic pyocyanin. This notion was borne out by the observations that PHZ1, a mutant of 7NSK2 no longer able to produce pyocyanin because of an insertion in the phzM gene encoding an O-methyltransferase, failed to induce resistance in tomato against Bo. cinerea, whereas complementation for pyocyanin production or co-inoculation with the pyocyanin-overproducing but SA-, pyochelin-, and ISR-deficient mutant 7NSK2-562 restored induced resistance. Induction of resistance by mutant KMPCH, on the other hand, does appear to involve SA. Contrary to wild-type bacteria, treatment of bean and tomato roots with KMPCH resulted in elevated activity of phenylalanine ammonia-lyase (PAL), a key enzyme involved in the phenylpropanoid-driven SA biosynthetic pathway, and elicitation of ISR by KMPCH was blocked in SA-deficient NahG tomato plants (Audenaert et al., 2002; De Meyer and Ho¨fte, 1997; De Meyer et al., 1999b). Additional evidence supporting the involvement of bacterially produced SA in ISR comes from a 10-year-old report by Maurhofer et al. (1998), demonstrating that transfer of the SA-biosynthetic cluster from Ps. aeruginosa PAO1 to the non-SA-producing strain Ps. fluorescens P3 rendered this strain capable of SA production in vitro and significantly improved ISR in tobacco against Tobacco necrosis virus. Surprisingly, however, a pyocyanin-negative mutant of KMPCH lost the ability to trigger ISR against Bo. cinerea in bean and tomato (K. Audenaert and M. Ho¨fte, unpublished data), suggesting that also in strain KMPCH, SA and pyocyanin, rather than SA alone, are the determinants for induced resistance. SA has also been implicated in 7NSK2- and/or KMPCHmediated ISR against Tobacco mosaic virus (TMV) in tobacco (De Meyer et al., 1999a) and C. lindemuthianum in bean (Bigirimana and Ho¨fte, 2002). Nevertheless, pyocyanin-minus mutants were not tested and it cannot be excluded that also in these pathosystems a combined production of pyocyanin and SA (or pyochelin) is needed to trigger ISR.


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G. ANTIBIOTICS

1. 2,4-Diacetylphloroglucinol Like siderophores, antibiotics can play a dual role in rhizobacteria-mediated biocontrol by exerting a direct inhibitory effect on pathogens (Raaijmakers and Weller, 1998; Weller et al., 2002) as well as by triggering ISR. One of the most conclusive pieces of evidence for the involvement of antibiotics in rhizobacteria-triggered systemic resistance came from work carried out in the laboratory of Jean-Pierre Me´traux, where it was demonstrated that 2,4-diacetylphloroglucinol (DAPG) functions as a key determinant of ISR in Arabidopsis. In this plant system, DAPG produced by Ps. fluorescens CHA0 was shown to induce resistance against the oomycete Hyaloperonospora arabidopsidis (formerly known as (Hyalo)peronospora parasitica), whereas mutations interfering with DAPG production led to a significant decrease in ISR (Iavicoli et al., 2003). In trans complementation of the mutant restored the ability to induce resistance, further supporting the contribution of DAPG to CHA0-mediated ISR. In tomato, Ps. fluorescens CHA0 induces resistance against the root knot nematode Meloidogyne javanica. Also in this case, ISR seems to depend on the production of DAPG, as a DAPG-negative mutant was not effective in reducing the disease in split-root assays, and effectiveness could be restored by complementation of the mutant (Siddiqui and Shaukat, 2003). DAPG has likewise been implicated in ISR in Arabidopsis against Ps. syringae pv. tomato by Pseudomonas chlororaphis Q2-87, suggesting that DAPG is a major determinant of ISR in DAPG producers (Weller et al., 2004). To date, research aimed at elucidating the mode of action of DAPG has been confined to the study of Iavicoli et al. (2003). By screening an extensive set of Arabidopsis mutants and transgenic lines impaired in various structural components of known defence-signalling pathways, it was shown that, unlike ISR triggered by the wild-type bacteria, resistance induced by DAPG follows a signalling route that requires neither the master transcriptional regulator NPR1, nor a functional JAR1 protein. Particularly interesting, however, was the analysis of the eir1 (ethylene-insensitive root-1) mutant, which is ET insensitive in the roots only (Roman et al., 1995). This eir1 mutant was incapable of mounting ISR upon DAPG feeding, leading the authors to suggest that an intact ET signalling pathway is required for the establishment of DAPG-inducible resistance (Iavicoli et al., 2003). What should be noted, however, is that the EIR1 protein is not only obligatory for ET-signalling, but also is implicated in regulating the plant’s response to endogenously synthesized auxin (Luschnig et al., 1998; Sieberer et al., 2000). Especially noteworthy in this regard is a recent report by Brazelton et al. (2008)

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suggesting a connection between auxin signalling and DAPG-induced morphological alterations in tomato and tobacco. In these plant species, DAPG was shown to inhibit primary root growth and to stimulate lateral root formation. Indeed, roots of the auxin-resistant diageotropica mutant of tomato displayed reduced sensitivity to DAPG with regard to inhibition of primary root growth and induction of root branching, which is suggestive of a link between auxin signalling and DAPG sensitivity. Moreover, application of exogenous DAPG repressed the activation of an auxin-inducible GH3 promoter::luciferase reporter gene construct in transgenic tobacco hypocotyls in a manner similar to treatment with DAPG-producing Ps. fluorescens strains, whereas a DAPG-minus mutant displayed less inhibitory effects compared to the parental strain. These results clearly indicate that DAPG can alter root architecture by interacting with an auxin-dependent signal transduction pathway. Considering the well-documented role of auxin as a virulence factor for biotrophic microbes (Chen et al., 2007; Navarro et al., 2006; Spoel and Dong, 2008; Wang et al., 2007), it is not inconceivable that DAPG may induce systemic resistance through interference with pathogeninduced auxin signalling. 2. Pyocyanin Another antibiotic that has been identified as a crucial determinant of rhizobacteria-elicited ISR is the N-containing heterocyclic blue phenazine pigment pyocyanin. A well-characterized virulence factor in clinical isolates of Ps. aeruginosa (K. Britigan et al., 1992, 1997), pyocyanin produced by the rhizobacterium Ps. aeruginosa 7NSK2 is postulated to join forces with the SA-derivative pyochelin in triggering systemic resistance in bean and tomato against Bo. cinerea (Audenaert et al., 2002; unpublished data), as described in detail in the section on siderophores. In rice, however, no evidence could be found for the involvement of pyochelin in ISR against the rice blast pathogen, M. oryzae. This pyochelin-independency of 7NSK2-mediated ISR in rice was borne out by the observation that bacterial inoculum prepared from iron-rich medium was as effective as that prepared from iron-poor medium, and was further confirmed by the resistance-inducing potential of the pyochelin-negative mutant KMPCH (De Vleesschauwer et al., 2006). In contrast, treatment with the pyocyanin-deficient strains 7NSK2-phzM and KMPCH-phzM did not reduce the number of sporulating lesions after infection with M. oryzae. Because the inability of these strains to induce resistance did not result from insufficient rhizosphere populations, these results strongly suggested that pyocyanin production is an integral component of 7NSK2-mediated ISR against the pathogen. Additional support for the involvement of pyocyanin was


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provided by complementation experiments, as well as by the protective effect obtained upon hydroponic feeding of the purified compound at pico- and nanomolar concentrations. Strikingly, in bioassays with Rhizoctonia solani as challenging pathogen, a necrotrophic fungus which causes sheath blight, the situation appeared to be entirely different in that the wildtype bacteria were not effective, whereas the pyocyanin-negative mutants 7NSK2-phzM and KMPCH-phzM did induce resistance. In accordance with these findings, pyocyanin-treated rice seedlings exhibited a heightened level of susceptibility to Rh. solani. When considered together, these data clearly indicate that pyocyanin plays a dual role in 7NSK2-triggered ISR, acting as a positive regulator of resistance to M. oryzae while rendering plants hypersusceptible to attack by Rh. solani. Transient generation of low-level micro-oxidative bursts by redox-active pyocyanin in planta most likely accounts for the differential effectiveness of this phenazine antibiotic in 7NSK2-mediated ISR, as exogenous application of sodium ascorbate, a major anti-oxidant buffer and free radical scavenger (Foyer and Noctor, 2005), alleviated the contrasting effects of pure pyocyanin on M. oryzae and Rh. solani pathogenesis (De Vleesschauwer et al., 2006). In conclusion, it can be stated that while in bean, tomato, and tobacco, pyocyanin and SA/pyochelin act synergistically to induce resistance, in the monocot rice pyocyanin alone is sufficient to activate the host immune response. The outcome of pyocyanin-steered defence responses on disease resistance, however, seems highly dependent on the type of challenging pathogen. Numerous pharmacological studies have revealed that pyocyanin can undergo redox-cycling, with resultant formation of superoxide and H2O2 (Britigan et al., 1997; Hassan and Fridovich, 1979). These active oxygen species (AOS) apparently suffice to induce resistance against blast in rice. Yet, in dicot plants such as bean and tomato, establishment of ISR requires AOS to be converted into highly toxic OHradicals through the Haber–Weiss reaction in the presence of an iron-chelating catalyst such as ferrated pyochelin or Fe-SA (Audenaert et al., 2002). H. VOLATILES

A relatively new addition to the list of rhizobacterially produced compounds with a putative role in eliciting host defence is a class of volatile organic compounds (VOCs). Unlike airborne VOCs, such as C6 green-leaf volatiles that can be easily sampled by headspace collections of the living plant, rhizosphere emissions by root-colonizing bacteria present the complication of de-adsorbing low-molecular-weight compounds from the soil matrix (Pare´ et al., 2005). However, by physically separating plant seedlings

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from VOC-emitting rhizobacteria on divided Petri dishes, Ryu et al. (2004a) were able to unequivocally delineate the role of airborne bacterial metabolites in eliciting ISR. Exposure of Arabidopsis seedlings to volatile blends from Ba. subtilis GB03 and Bacillus amyloliquefaciens IN937a for 4 days was sufficient to activate ISR, as reflected by a marked reduction in the number of symptomatic leaves 24 h after inoculation with the soft-rot pathogen Er. carotovora. Extensive gas-chromatographic analysis of the complex bacterial bouquet emitted by the Bacillus strains revealed the release of a series of low-molecular-weight hydrocarbons, the most abundant component being the growth-promoting volatile 2R,3R-butanediol. The importance of this 2R,3R-butanediol in ISR was surmised in Arabidopsis when pre-exposure of plants to low doses (pg to ng range) of the pure compound activated resistance, while this ability was alleviated in bacterial mutant derivatives emitting reduced levels of 2,3-butanediol and acetoin (Ryu et al., 2004a). Additional support for a role of VOCs in activating ISR came from Han et al. (2006b). In this study, activation of systemically induced resistance against Er. carotovora in tobacco by Ps. chlororaphis O6 was found to be inherently linked to the production of 2R,3R-butanediol, the latter being subject to positive regulation by the global sensor kinase GacS (Han et al., 2006b). It remains to be investigated whether the site of plant VOC perception is above or below ground for soil-grown plants and how plants perceive VOC signals. It is not clear either whether an endogenous signal is involved in VOC-mediated ISR (Pare´ et al., 2005). Heil and Ton (2008) hypothesized that green-leaf volatiles may provoke changes in transmembrane potentials and thereby induce gene activity. Possibly, rhizobacterially produced VOCs may be perceived by a similar mechanism. Although our understanding of the molecular mechanisms underpinning volatile-mediated ISR is still rudimentary, it is evident from the limited data available that, at least in some plant–bacterium interactions, responsiveness to ET is key to optimal manifestation of the VOC-triggered immune response (Ryu et al., 2004a; Spencer et al., 2003). Considering the role of microbial volatiles in regulating an array of cellular processes, including plant growth and development, pathogen defence, and abiotic stress adaptation (Cho et al., 2008; Han et al., 2006b; Ryu et al., 2003a, 2004a), there may be yet other signalling pathways extant. For instance, VOCs might also operate through an auxin-dependent mechanism, as was suggested for the antibiotic DAPG. In support of this assumption, Zhang et al. (2007) recently demonstrated that volatile blends from the Ba. subtilis strain GB03 orchestrate cell expansion in Arabidopsis by modulating the auxin-signalling infrastructure.


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I. EXOPOLYSACCHARIDES

EPS are a group of high-molecular-weight carbohydrates secreted by both pathogenic and beneficial bacteria. In pathogenic bacteria, EPS are necessary to cause disease symptoms such as wilting and water-soaking. EPS also promote survival and colonization within host tissues (Denny, 1995). In beneficial bacteria, EPS can function as signalling molecules that trigger a developmental response in the plant or suppress host defence responses (see, for instance, Jones et al., 2008). In Arabidopsis, leaf infiltration with bacterial EPS from plant, insect, and human pathogens and symbionts (10 mg/ml) suppressed calcium influx and defences induced by MAMPs such as flg22 (Aslam et al., 2008). In contrast, EPS from Pantoea agglomerans activated the oxidative burst response of tobacco and parsley cell cultures directly and primed rice and wheat cells for augmentation of H2O2 accumulation after subsequent induction by a chitin hexamer elicitor (Ortmann et al., 2006). Foliar-applied EPS from two pathovars of X. campestris induced local and systemic protection against coffee rust caused by Hemileia vastatrix. Protection was also observed when coffee plants were treated with different concentrations of commercially available xanthan gum (Guzzo et al., 1993). Recently it was shown that EPS from the PGPR Burkholderia gladioli IN26 can induce systemic resistance to Colletotrichum orbiculare on cucumber when infiltrated in leaves or applied via seed soaking. The most effective concentration appeared to be 200 ppm. In addition, EPS induced the PR-1a promoter in transgenic tobacco plants in a concentration-dependent manner (Park et al., 2008b). EPS from Serratia sp. strain Gsm01 sprayed at a concentration of 200 ppm on tobacco leaves affected Cucumber mosaic virus (CMV) accumulation. EPS-treated plants showed an enhanced accumulation of PAL, peroxidase, and phenols, and PR-1b expression was increased (Ipper et al., 2008). The culture filtrate of Serratia Gsm01 also controlled CMV infection in tobacco. In these experiments, no bacterial mutants impaired in EPS production were used, so it is not clear to what extent EPS production is important in rhizobacteriamediated ISR. From the examples cited above, it is clear that depending on the conditions, EPS can either trigger or suppress resistance responses in plants. Aslam et al. (2008) found that while pure xanthan suppressed defence responses, calcium-saturated xanthan elicited PR-1, PDF1.2, and PAL genes in Arabidopsis and also xanthan oligomers could function as MAMPs. Structural differences in EPS may explain their differential effects on plants.

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¨ FTE D. DE VLEESSCHAUWER AND M. HO J. OTHER BACTERIAL DETERMINANTS

Park et al. (2008a) identified 4-(aminocarbonyl) phenylacetate as an ISReliciting metabolite produced by Ps. chlororaphis O6 against the wildfire pathogen Ps. syringae pv. tabaci in tobacco by ISR bioassay-guided fractionation of O6 culture supernatant. Application of 68 mM 4-(aminocarbonyl) phenylacetate on in vitro grown tobacco plants yielded protection against Ps. syringae pv. tabaci. This concentration appears to be very high; the effect of lower concentrations was not reported. It remains to be investigated whether this compound can trigger ISR at physiologically realistic concentrations and what role it plays in Ps. chlororaphis O6-mediated ISR. Han et al. (2006a) isolated a variety of mutants of Ps. chlororaphis O6 impaired in ISR against Er. carotovora in tobacco. Genes disrupted in these mutants were involved in various functions including biosynthesis of purines, phospholipase C, transport of branched-chain amino acids, chemotaxis, and ABC transporters. Most of these mutants, except the ABC-transporter mutants, were seriously impaired in root colonization, indicative of the importance of this trait in ISR.

III. SIGNALLING IN RHIZOBACTERIA-INDUCED SYSTEMIC RESISTANCE A. THE ARABIDOPSIS–PSEUDOMONAS FLUORESCENS WCS417R SYSTEM: A PARADIGM FOR SA-INDEPENDENT ISR SIGNALLING

Following the perception of resistance-inducing stimuli, plants activate an elaborate matrix of signal-transduction pathways in which phytohormones such as SA, JA, ET, and abscisic acid (ABA) act as key signalling molecules. A mechanistic understanding of ISR in terms of signalling has derived largely from studies in Arabidopsis where, as with pathogen-induced SAR, mutant and transgenic lines have identified the key roles of phytohormonal signals, effectors, and potential points of cross-talk among the signals. Driving this research initially was the observed disconnection between resistance induced by the Dutch reference strain Ps. fluorescens WCS417r and the accumulation of transcripts for certain PR genes that are hallmarks of SAR and SA action (Hoffland et al., 1995; Pieterse et al., 1996; Van Wees et al., 1997). Measurements of SA levels in ISR-expressing Arabidopsis plants revealed that ISR, unlike SAR, is not associated with alterations in endogenous SA content, neither before nor after challenge inoculation (Pieterse et al., 2000). Moreover, WCS417r-mediated ISR was maintained in SA non-accumulating Arabidopsis NahG transformants (Pieterse et al., 1996; Van Wees et al., 1997), leading the


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authors to suggest that WCS417r-mediated ISR is a SA-independent resistance mechanism and that WCS417r-mediated ISR and pathogen-induced SAR are regulated by largely distinct signalling conduits. The non-involvement of SA in WCS417r-mediated ISR prompted Corne´ Pieterse and colleagues to investigate the possible requirement for other regulatory factors implicated in plant defence. To this end, the Arabidopsis JA-response mutant jar1 and the ET-response mutant etr1 were tested for their ability to mount ISR. Intriguingly, both mutants were unable to develop ISR against Ps. syringae pv. tomato upon colonization of the roots by WCS417r bacteria (Pieterse et al., 1998), illustrating the dependency of ISR signalling on these phytohormones. Bioassays with other mutants in JA and ET signalling yielded similar results. For instance, Arabidopsis mutant eds8, which was previously shown to display increased susceptibility to Ps. syringae pv. maculicola (Glazebrook et al., 1996), was impaired in both WCS417r-mediated ISR (Ton et al., 2002a) and JA signalling (Glazebrook et al., 2003; Ton et al., 2002a). Furthermore, of a large set of well-characterized ET-signalling mutants, none displayed enhanced resistance against Ps. syringae pv. tomato upon root treatment with WCS417r (Knoester et al., 1999), indicating that an intact ET signalling pathway is a prerequisite for the establishment of ISR. Bioassays with the eir1-1 mutant, which is insensitive to ET in the roots only, shed additional light on the pathway by which ISR is induced. In this mutant, application of WCS417r to the roots did not result in an ISR response, whereas infiltration of the inducing bacteria into the leaves did. The eir1-1 leaves are normally sensitive to ET, in contrast to the etr1-1 and several ein mutants, which display complete ET insensitivity. These results demonstrated that for induction of ISR in Arabidopsis by WCS417r, responsiveness to ET is required at the site of application of inducing bacteria, be it roots or leaves (Knoester et al., 1999). Further evidence for the involvement of the ET-response system came from the identification of the Arabidopsis ISR1 locus (Ton et al., 1999, 2002b). Genetic analysis of the progeny from crosses between WCS417rresponsive and non-responsive accessions demonstrated a single dominant locus, designated ISR1, to be important for ISR signalling against different pathogens (Ton et al., 2002b). Interestingly, accessions carrying the recessive isr1 allele display reduced sensitivity to ET (Ton et al., 2001), as well as enhanced susceptibility to Ps. syringae pv. tomato (Ton et al., 1999). These findings suggest that ISR1 encodes a novel component in the ET signalling pathway that is integral to both basal and rhizobacteria-induced resistance in Arabidopsis. Treatment of wild-type Arabidopsis plants with JA, ET, or the ET precursor 1-aminocyclopropane-1-carboxylic acid (ACC) induced systemic

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protection identical to that elicited by WCS417r rhizobacteria, and protected plants against Ps. syringae pv. tomato (Pieterse et al., 1998). However, etr1-1 plants failed to respond to either ACC or JA, whereas the jar1-1 mutation abrogated the response to JA but not to ACC, indicating that JA operates upstream of ET in the ISR signalling cascade (Pieterse et al., 1998). Surprisingly, the ISR response does not appear to depend on changes in the endogenous levels of JA or ET, as significant changes in the levels of these phytohormones did not occur during induction of resistance by WCS417r (Pieterse et al., 2000), and transcripts of classic JA- and ET-regulated genes (e.g., LOX1, LOX2, PAL1, HEL, CHI-B, PDF1.2) were not upregulated, neither before nor after pathogen challenge (Van Wees et al., 1999). However, it is not inconceivable that activation of ISR by WCS417r requires responsiveness to JA and ET, rather than increased levels of these defence regulators. In this scenario, the sensitivity to JA and ET is likely to be boosted as a result of ISR elicitation. Two lines of evidence support this hypothesis. First is the observation that in ISRexpressing plants the capacity to convert ACC to ET is significantly enhanced, providing a greater potential to produce ET upon pathogen attack (Hase et al., 2003; Pieterse et al., 2000). Secondly, in induced plants, after pathogen challenge JA- and ET-responsive gene expression is enhanced relative to non-induced plants (Pozo et al., 2008; Verhagen et al., 2004). Thus, it seems that induced plants are sensitized for perception of pathogen-induced JA and ET, a notion which is consistent with priming or potentiation of the plant to respond more rapidly and with greater intensity to subsequent pathogen attack (Conrath et al., 2006). Recent findings pinpoint the helix-loop-helix transcription factor MYC2 as a potential regulator in this rhizobacteria-induced priming for enhanced defence (Pozo et al., 2008). Promoter analysis of ISR-primed and methyl jasmonate-responsive genes revealed over-representation of a G-box-like motif known to serve as a docking-site for MYC2. In addition, the MYC2impaired mutants jin1-1 and jin1-2 were unable to express ISR against Ps. syringae pv. tomato and the downy mildew pathogen H. arabidopsidis upon elicitation by WCS417r, further highlighting the important regulatory function of this transcription factor in priming for enhanced JA-responsive gene transcription during WCS417r-mediated ISR. Intriguingly, mutant jin11 plants were recently shown to be compromised also in the ability to express pathogen-induced SAR, a finding which is consistent with the view of JA as an important early signal establishing broad-spectrum systemic immunity (Truman et al., 2007). To delineate the possible role of the SAR master regulatory protein NPR1 in ISR response triggered by WCS417r, bioassays were performed with the


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Arabidopsis npr1 mutant. In contrast to other mutations in the SA-signalling pathway, npr1 failed to express ISR upon elicitation by WCS417r (Pieterse et al., 1998). Hence, NPR1 seems to play a pivotal role in reaching the induced state, whether triggered by avirulent pathogens or benign rhizobacteria. Further analysis placed NPR1 downstream of the requirements for both JA and ET in the ISR-signalling cascade. Considering that SAR is associated with NPR1-controlled PR-transcript accumulation, and ISR is not, one would expect the action of NPR1 in ISR to be substantially different from that in SAR. Especially noteworthy in this regard is that these supposedly distinct activities are not mutually exclusive, as simultaneous activation of pathogen-induced SAR and WCS417r-governed ISR was found to act independently and additively to increase resistance of Arabidopsis against Ps. syringae pv. tomato (Van Wees et al., 2000). To date, the function of NPR1 in ISR is still unresolved, although some studies portend a role for NPR1 in the stress-induced augmentation of the protein-secretory pathway (Van der Ent et al., 2009; Wang et al., 2005). Recently, the R2R3-MYB-like transcription factor gene MYB72 was identified as a novel player in WCS417r-mediated ISR (Van der Ent et al., 2008). T-DNA knockout mutants myb72-1 and myb72-2 were incapable of mounting ISR against a fairly broad range of pathogens with different lifestyles (i.e. biotrophs, hemibiotrophs, necrotrophs), indicating that MYB72 is essential to establish broad-spectrum ISR. However, overexpression of MYB72 did not result in enhanced resistance against any of the pathogens tested, leading the authors to suggest that MYB72 acts in concert with another signalling component. A clue for the identity of this accomplice was provided when yeast-two hybrid analysis revealed MYB72 to physically interact in vitro with the EIN3-like transcription factor EIL3. This interaction with EIL3 links MYB72 to the ET response pathway, further establishing this transcription factor as a crucial signalling intermediate required in early signalling steps of WCS417r-mediated ISR (Van der Ent et al., 2008). Emerging from this extensive series of studies on WCS417r-induced Arabidopsis is a model providing fascinating insights into the various aspects of ISR signalling, ranging from the early ET-regulated and MYB72-dependent onset of ISR following perception of resistance-inducing stimuli in the roots, to long-distance signalling and ET-, JA-, and NPR1-dependent priming of effector responses in systemic leaves. Priming by several other rhizobacteria, such as Ps. putida LSW17S, also is reliant on ET, JA, and NPR1, and not on SA. However, augmented expression of PR genes was observed in Arabidopsis treated with LSW17S and challenged with Ps. syringae pv. tomato. Pathogen challenge likewise resulted

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in higher H2O2 accumulation and enhanced callose deposition in bacterized plants compared to control ones (Ahn et al., 2007). These reactions have not been observed in WCS417r-primed plants, suggesting that the molecular and biochemical events associated with ISR elicited by LSW17S overlap only partly with those defined for ISR against Ps. syringae pv. tomato, as induced by WCS417r. Ryu et al. (2003b) found that Bacillus pumilus SE34 triggered ET/ JA- and NPR1-dependent ISR against Ps. syringae pv. maculicola in Arabidopsis. Surprisingly though, ISR against Ps. syringae pv. tomato triggered by the same bacterium appeared to be JA/ET-independent. Ps. fluorescens WCS374r-mediated ISR against Ps. syringae pv. tomato in Arabidopsis is also mediated by a signalling pathway that depends on ET, JA, and NPR1. In rice, WCS374r-elicited ISR against M. oryzae is maintained in SA-non-accumulating NahG transformants, but completely abolished in the ET-insensitive antisense line 471 and the JA-deficient mutant hebiba, suggesting that in the latter plant–pathogen system also, WCS374r-mediated ISR derives primarily from JA/ET-driven effects (De Vleesschauwer et al., 2008). Consistently, root treatment with the purified PSB siderophore of WCS374r, which faithfully mimicks living bacteria in activating ISR, does not lead to direct transcriptional activation or priming of SA-responsive PR-like genes, such as OsPR-1b and OsPBZ1. Plant growth-promoting fungi such as Penicillium sp. GP162 (Hossain et al., 2008) and Trichoderma asperellum T203 (Shoresh et al., 2005) have been shown to induce systemic resistance in an ET- and JAdependent manner as well. Moreover, T. asperellum T34-mediated ISR required, besides NPR1, the MYB72 transcription factor, suggesting that MYB72 functions as a node of convergence in induced defence triggered by soilborne beneficial microorganisms (Segarra et al., 2009; Van Wees et al., 2008). Whereas JA- and ET-dependent signalling mechanisms are co-required for the elicitation of, among others, Ps. fluorescens WCS417r- and Ps. putida LSW17S-mediated ISR in Arabidopsis (Ahn et al., 2007; Pieterse et al., 1998), as well as Ba. pumilus SE34-mediated ISR in tomato (Yan et al., 2002), alternative signalling pathways have been demonstrated in other plant–bacterium interactions. For instance, in Arabidopsis resistance against H. arabidopsidis triggered by DAPG-producing Ps. fluorescens CHA0 follows a pathway that is JA- and NPR1-dependent and needs a functional EIR1 protein in the roots (Iavicoli et al., 2003). Pure DAPGtriggered resistance, however, only depends on a functional EIR1 protein and is NPR1-independent. As discussed above, it is possible that the mutation in EIR1 acts independently of ET signalling and is rather implicated in auxin signalling. ISR induced by strain CHA0 was ineffective against Ps. syringae and Bo. cinerea (Iavicoli et al., 2003), a further


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indication that the induced signal-transduction pathway is different from that underpinning WCS417r-mediated ISR. S. marcescens 90-166 likewise seems to elicit a defence pathway that is mediated by NPR1 and JA, but is SA- and ET-independent (Ryu et al., 2003b; Table I). This strain was not tested on an eir1 mutant of Arabidopsis. However, unlike Ps. fluorescens CHA0, S. marcescens 90-166 is effective against Ps. syringae in Arabidopsis, indicating that these two strains do not trigger the same defence pathway. Still other variations are indicated by the capacity of rhizobacteria such as Ba. pumilus T4, Ba. subtilis GB03, and S. marcescens 90-166 to induce disease resistance signalling that depends only on either ET or JA (Ryu et al., 2003b, 2004a,b).

B. SA-DEPENDENT ISR SIGNALLING

Although rhizobacteria-mediated ISR is perhaps the best-studied example of induced resistance that is not regulated by SA, it is becoming increasingly clear that not all rhizobacteria-triggered ISR is mediated by JA/ET (Table I). The role of SA in ISR was first studied for Ps. aeruginosa 7NSK2 and its SA-producing mutant Ps. aeruginosa KMPCH. Both strains clearly induce resistance in a SA-dependent manner as the resistance response to TMV is no longer expressed in NahG tobacco (De Meyer et al., 1999a), while resistance to Bo. cinerea can no longer be triggered in NahG tomato (Audenaert et al., 2002). Although some strains of Bacillus spp. operate through a JA/ET-dependent mechanism and require NPR1 similar to Ps. fluorescens WCS417r, ISR induced by other Bacillus strains requires SA but not JA and NPR1 (Barriuso et al., 2008; Ryu et al., 2003b). Also ISR against Verticillium dahliae in Arabidopsis in response to root inoculation with the rhizobacterial strain Pa. alvei K165 requires a SA-dependent resistance mechanism, as evidenced by the ISR-minus phenotype of the SA-signalling mutants eds5 and sid2 (Tjamos et al., 2005). However, Pa. alvei K165 was still fully active in NahG Arabidopsis plants. Although difficult to explain, this result indicates that other plant lines besides NahG should be tested in order to unambiguously exclude a role for SA in ISR. There are other examples in Arabidopsis where diseases are diminished by a SA-dependent rhizobacteria-induced resistance. For instance, Bacillus strain N11.37 induces ISR to X. campestris in Arabidopsis by a SA- and ETdependent pathway, while only SA-signalling seems to be operative in the case of resistance induced by the Stenotrophomonas strain N6.8 (Domenech et al., 2007). Soil inoculation with Bacillus sp. strain L81 or Art. oxidans

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strain BB1 protected Arabidopsis plants against Ps. syringae pv. tomato and increased plant tolerance to salt stress. The SA-dependent pathway was involved in the defence response against biotic and abiotic stress induced by both strains. Inoculation with Art. oxidans strain BB1 increased PR-1 gene transcription, while this was not observed for Bacillus sp. strain L81. The authors, however, did not analyze gene expression after stress challenge (Barriuso et al., 2008). Interestingly, also Ps. fluorescens WCS374r-mediated ISR against TCV in Arabidopsis is SA- and NPR1-dependent (Djavaheri, 2007).

C. SA-DEPENDENT AND SA-INDEPENDENT SIGNALLING

From Table I and the examples cited above, it is evident that both SA-dependent and SA-independent signalling conduits can contribute to rhizobacteria-mediated induced resistance, making it difficult to assign a definitive signalling pathway to ISR. Rather, it appears that plants display a remarkable flexibility in the molecular processes governing the perception and subsequent transduction of resistance-inducing stimuli produced by root-colonizing bacteria. Another challenge in finding coherence among models for ISR signalling is added by the fact that even within the same plant–rhizobacterium interaction, different signal transduction pathways can be activated depending on the type of intruder encountered. For instance, Conn et al. (2008) recently demonstrated that inoculation of Arabidopsis with selected endophytic actinobacteria results in a low-level induction of both SA and JA/ET marker genes in systemic healthy leaves. However, whereas resistance to the bacterial pathogen Er. carotovora subsp. carotovora required the JA/ET pathway, resistance to the fungal pathogen F. oxysporum proved to involve primarily the SA-regulated SAR pathway. The endophytic actinobacteria thus appear to be able to prime both the SA and JA/ET pathways, upregulating genes in either pathway depending on the infecting pathogen. Induction of resistance in Arabidopsis by Ps. fluorescens WCS374r is another case in point. In the case of TCV, to which defence responses in Arabidopsis are SA-dependent, ISR is abolished in NahG and sid1 plants, as well as in npr1, but not in jar1 and etr1 mutant plants. In contrast, WCS374r-mediated ISR against Ps. syringae pv. tomato is abrogated in npr1, jar1, and etr1 plants, but still functional in NahG and sid1 plants (Djavaheri, 2007). Hence, similar to actinobacteria, root colonization by WCS374r appears to sensitize Arabidopsis for augmented infection-induced activation of multiple defence signalling pathways that all add to establish broad-spectrum WCS374r-triggered ISR.


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IV. FINAL REMARKS A detailed understanding of the complex, yet fascinating mechanisms by which resistance-inducing bacterial stimuli elicit host immune responses may open new doors to design innovative strategies for biologically based, environmentally friendly, and durable disease control. Emerging from the numerous studies dealing with ISR elicitation is a greater awareness that plants are able to perceive a plethora of resistance-inducing bacterial components reminiscent of the large variety of chemical or pathogen-derived elicitors that stimulate the plant’s immune response (Schreiber and Desveaux, 2008). Some bacterial determinants such as flagellin, SA, pyocyanin, DAPG, N-alkylated benzylamine, and VOCs are recognized at concentrations in the pico- to nanomolar range, whereas other compounds only trigger protective effects in the g/ml range, with examples of the latter including LPS, EPS, PSB, and biosurfactants. Moreover, a clear dose-response effect is typically observed for the determinants that need higher concentrations to be effective, whereas for determinants recognized at very low doses, no such relationship seems to hold. A lack of dose-response is characteristic of recognition by high-affinity receptors such as the LRR-RK FLS2 (Jones and Dangl, 2006). This would imply that plants possess high-affinity receptor kinases able to recognize and respond to compounds such as SA, pyocyanin, DAPG, and N-alkylated benzylamine. Considering that all of these metabolites contain an aromatic phenol group, it is tempting to speculate that the latter moiety may constitute a general motif widely recognized by specific plant receptors (Ongena et al., 2008). On the other hand, compounds such as LPS that require higher doses to be effective may possibly be perceived by low-affinity receptors (Gross et al., 2005; Newman et al., 2007). These resistance determinants may, however, also enhance the plant’s defensive capacity by inducing disturbance or transient channelling in the plasma membrane, thereby activating a cascade of molecular events culminating in the primed or direct activation of immune responses, as was suggested for biosurfactants (Ongena and Jacques, 2008). Alterations in intracellular iron homeostasis caused by siderophore-mediated iron deprivation in the rhizosphere can be put forward as an alternative mechanism by which rhizobacteria activate the plant’s immune response. Interestingly, these proposed types of non-self recognition all have equivalents in the mammalian immune system (Nu¨rnberger et al., 2004; Zipfel and Felix, 2005), suggesting that fundamental modes of microbe sensing and subsequent resistance elicitation have been evolutionarily conserved across biological kingdoms. In general, ISR leads to a level of resistance that is less pronounced compared to ETI or ETI-triggered SAR. In this perspective, it can be stated that ISR resembles, to some extent, PTI-triggered SAR (Mishina and Zeier, 2007).

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Considering that PGPRs do not produce virulence factors such as toxins and pectinolytic enzymes that inflict tissue necrosis, and probably do not inject effector proteins into host cells to circumvent, or attenuate MAMP-triggered defences, rhizobacterial ISR is not expected to be associated with a full-blown ETI or SAR response in the absence of a challenging pathogen. However, in order to establish a compatible interaction with their hosts, PGPRs are likely equipped with diverse mechanisms to alleviate the local plant defence response triggered upon recognition of the aforementioned bacterial traits. Plant hormones such as auxin and JA are possibly involved in this process. Auxins are commonly produced by PGPRs (Spaepen et al., 2007) and besides their growth-promoting effects, they are known to suppress SA-dependent defence responses (Robert-Seilaniantz et al., 2007; Wang et al., 2007). In a similar vein, local triggering of the JA response may be a strategy to suppress SA-regulated defences in the root, while leading to the enhancement of the defensive capacity in naive systemic leaves similar to what has been reported in the context of plant–mycorrhiza interactions (Pozo and Azcoń-Aguilar, 2007). Alternatively or in addition, LPS or EPS production may also fulfil such a dual role in the ISR response. In this respect, the observation that a mutant of Ps. fluorescens WCS417r lacking the O-antigenic side chain of its outer membrane LPS, displays a reduced capacity to colonize the interior but not the exterior root tissues of tomato, is of particular interest (Duijff et al., 1997). Moreover, suppression of local defences by bacteria-produced LPS or EPS may also explain why purified compounds are often more effective in triggering ISR and reducing disease severity relative to treatment with living bacteria. It is also apparent that rhizobacteria-mediated ISR is not conditioned by one definitive signalling pathway. Rather, the pathway triggered is depending on the PGPR, the plant, and the challenging pathogen. In many cases, and especially in Arabidopsis, multiple bacterial determinants tend to be involved in the elicitation of the ISR response, resulting in the sensitization of various signalling conduits. The enhanced signalling capacity in these primed plants not only facilitates a faster and stronger activation of basal defences upon microbial infection, but also confers flexibility to fine-tune the plant’s immune response to the type of invading pathogen.

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Plant Growth-Promoting Actions of Rhizobacteria

STIJN SPAEPEN,*,1 JOS VANDERLEYDEN* AND YAACOV OKON{

*Department of Microbial and Molecular Systems, Centre of Microbial and Plant Genetics, K.U.Leuven, Kasteelpark Arenberg 20—Box 2460, 3001 Heverlee, Belgium { Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Modes of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Plant Growth-Promoting Substances........................................ B. Nitrogen Transformations .................................................... C. Phosphate and Micronutrient Availability ................................. D. Emerging Signals ............................................................... E. Biocontrol in the Rhizosphere................................................ III. Agricultural Aspects and Relevance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. PGPR and Endophytes—Role of Bacterial Numbers .................... B. PGPR and Other Symbiotic Systems such as Rhizobium-Legumes..... C. Vegetative Growth and Grain Filling ....................................... D. Inoculant Technology.......................................................... E. Probiotics in Agriculture ...................................................... IV. Perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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0065-2296/09 $35.00 DOI: 10.1016/S0065-2296(09)51007-5

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ABSTRACT The rhizosphere as compared to bulk soil is rich in nutrients because of root exudation and deposits. As a consequence, the number of bacteria surrounding plant roots is 10–100 times higher than in bulk soil. These rhizobacteria, based on their effects on plants, can be largely divided into beneficial, deleterious, or neutral bacteria. The beneficial bacteria, also called plant growth-promoting rhizobacteria (PGPR), exert their beneficial effect through either direct or indirect mechanisms or both. In this chapter, the different mechanisms of plant-growth promotion and their impact are discussed. The mechanisms comprise the production of plant growth-promoting substances, nitrogen transformations, increasing bioavailability of phosphate and micronutrients, and biological control, as the best documented cases. In addition, the bacterial production of some molecules with recently described plant growthpromoting effects is discussed. To indicate the impact of PGPR, the applications and relevance of these bacteria in agricultural practices are highlighted. The importance of the plant genotype, inoculum density and technology, and co‐inoculation practices, in terms of plant responsiveness are discussed. It is concluded that basic research should remain a priority in order to be able to develop performing and reliable bacterial inocula as a means to support sustainable agriculture.

I. INTRODUCTION The term ‘‘rhizosphere’’ was for the first time defined by Lorenz Hiltner in 1904 as ‘‘the soil compartment influenced by the root’’ (Hiltner, 1904). Rhizodeposition describes the total carbon transfer from the plant roots to the soil and comprises exudates (small molecules), secretions (macromolecules such as enzymes), lysates from dead cells, and mucilage. Rhizodeposition promotes microbial abundance and activities in the rhizosphere, which can therefore be described as the most active microbial habitat of the soil (Burdman et al., 2000; Dobbelaere and Okon, 2007; Hartmann et al., 2008; Lucy et al., 2004; Smalla et al., 2006; Van Loon, 2007). The rhizosphere is a highly dynamic open system with temporal and spatial changes of biotic (such as resulting from physiological and morphological changes of the growing root system) and abiotic (such as rain, irrigation, drought) factors. Consequently, it is difficult to understand the microbial adaptations to each particular situation. The rhizosphere is often depicted as a soil cylinder of given radius around the root, drawing a boundary between the rhizosphere and the bulk soil. In the last decade, there have been many developments and new theories trying to understand the architectural features and gradients of the rhizosphere (Hinsinger et al., 2005). Novel in situ techniques and modeling will help in providing a holistic view of the rhizosphere function. However, the new knowledge about root architecture, root growth, function, and rhizosphere is not yet ready for application in the management and manipulation of the rhizosphere.

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Through the dynamic rhizodeposition, it is suggested that roots can regulate the soil microbial community in their immediate vicinity. Some bacterial species living in the rhizosphere and inside the roots can affect growth in either a positive or a negative way. Bacteria that favorably affect plant growth and yield of commercially important crops are denominated as plant growthpromoting rhizobacteria (PGPR) (Burdman et al., 2000; Dobbelaere and Okon, 2007; Lucy et al., 2004; Steenhoudt and Vanderleyden, 2000). The well-known PGPR include members of the genera Azospirillum, Bacillus, Paenibacillus, Pseudomonas, Enterobacter, Klebsiella, Burkholderia, Serratia, Gluconacetobacter, Herbaspirillum, Azoarcus, Arthrobacter, among others. Beneficial bacteria that are able to establish a nitrogen-fixing symbiotic relationship with leguminous plants (and collectively called Rhizobia) are usually not considered within this group. However, recently some of these bacteria have been shown to be plant-growth promoting on non‐legumes, through mechanisms different from nitrogen fixation. Nevertheless, these will not be considered further, as the mechanisms invoked are not different from those of the well-known and better documented PGPR. Within the PGPR group, it is also common to discriminate between bacteria that are usually found at the plant root surface and those that are found most often or exclusively within the plant root tissue, the so-called endophytes. For the latter, we refer to recent reviews (Hardoim et al., 2008; Rosenblueth and MartinezRomero, 2006; Ryan et al., 2008). Direct plant-growth promotion can be derived from phosphorus solubilization, production of plant growth regulators such as auxins, gibberellins (GAs), and cytokinins, by eliciting root metabolic activities and/or by supplying biologically fixed nitrogen. Indirect plant growth-promoting mechanisms used by PGPR include induced systemic resistance (ISR), antibiotic protection against pathogens, reduction of iron availability in the rhizosphere by sequestration with siderophores, synthesis of fungal cell wall-lysing or lytic enzymes, and competition for nutrients and colonization sites with pathogens (Burdman et al., 2000; Dobbelaere and Okon, 2007; Lucy et al., 2004). These direct and indirect mechanisms will be discussed in detail in this chapter and in other chapters in this volume.

II. MODES OF ACTION A. PLANT GROWTH-PROMOTING SUBSTANCES

Plants synthesize several hormones, which act as chemical messengers to regulate plant growth and development. Phytohormones or plant growthpromoting substances are chemical compounds that in small amounts

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promote and influence the growth, development, and differentiation of cells and tissues. In view of this, changes in hormone concentrations alter plant growth and development in a drastic way (possibly with a positive or negative outcome, as in the case of auxins for which an optimal dose-response curve exists). Although phytohormones have been intensively studied over many years, the exact mode of action of some molecules in plants is still not very clear. It is generally accepted that there are five major classes of plant hormones: auxins, cytokinins, GAs, abscisic acid (ABA), and ethylene (ET) (Table I). However, more phytohormones are presently known, such as strigolactone, a carotenoid-derived molecule that inhibits shoot branching and acts as signal molecule in symbiotic interactions with arbuscular mycorrhizal fungi; new ones are still be discovered (Gomez-Roldan et al., 2008; Umehara et al., 2008). Other identified plant growth regulators include: brassinolides, salicylic acid (SA), jasmonates (JA), polyamines, plant peptide hormones, and nitric oxide (for a recent review: Santner et al., 2009). The production of plant growth-promoting substances by bacteria has been reported for many bacterial species, and ideas that this production contributes to the growth-promoting effects of some bacteria have been launched more than 50 years ago (Barea et al., 1976). Although the list of soil and plant-associated bacteria, capable of producing phytohormones, is extensive, the direct evidence for the role of phytohormones in the plant growth-promoting capacities of these bacteria is scarce. The lipochitooligosaccharides (LCOs) produced by rhizobia that evoke nodule formation in legumes, however, are by now very well documented in terms of receptor and signal transduction pathway (D’Haeze and Holsters, 2002; Geurts et al., 2005; Oldroyd and Downie, 2008) and will not be discussed here. This part of the chapter aims to discuss the bacterial production of several phytohormones and highlights the evidence for their role in plant growth promotion. For background information on the biosynthesis, signal transduction, and action of plant hormones, we refer to the excellent book edited by Davies (2004). 1. Auxins Auxins are an important class of phytohormones, driving different plant processes, such as embryogenesis, organ differentiation, root and shoot architecture, apical dominance, and tropistic responses (Teale et al., 2006). The best characterized and most abundant member in the auxin family is indole-3-acetic acid (IAA). The biosynthesis of IAA by plants has been an intensive research topic over many years, but till now some pathways are still poorly characterized or under debate. The aromatic amino acid tryptophan is the main precursor for IAA synthesis, but also a tryptophan-independent


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TABLE I Plant Hormones, Produced by Plant Growth-Promoting Rhizobacteria Class

Example

Structure

Effect on plant

N H

Root and shoot architecture Apical dominance Tropistic responses

HO

Auxins

IAA

O

HO

H3C

Cytokinins

NH

Zeatin

Inhibition of root elongation Leaf expansion by cell enlargement Delay of senescence

N

N

N H

N

H

Gibberellins

Ethylene

GA3

O

OH

HO O

H CH3 O

H2C

CH2

H3C CH3 OH

Abscisic acid

O

CH2

OH

CH3

CH3

O

OH

Seed germination Stem and leaf growth Floral induction and fruit growth Stress and ripening hormone Flower and leaf senescence and abscission Adaptation to abiotic and biotic stresses Stomatal closure Bud dormancy Adaptation to abiotic and biotic stresses

Abbreviations: IAA, indole-3-acetic acid; GA3, gibberellic acid.

pathway has been described (Woodward and Bartel, 2005). Based on feeding studies and mutant analyses, multiple pathways have been suggested, although for most pathways identification of single steps is still lacking, as exemplified by the recent identification of the aromatic amino acid

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transferase 1 in Arabidopsis thaliana, involved in the IAA biosynthetic pathway via indole-3-pyruvate (Stepanova et al., 2008; Tao et al., 2008). For diverse bacteria, production of IAA has been reported. Around 80% of the bacteria isolated from the rhizosphere are estimated to be capable of producing IAA (Khalid et al., 2004; Patten and Glick, 1996), indicating a potential role in the interaction with the plant. As in plants, different biosynthetic pathways have been described in bacteria, and these are mostly similar to those in plants (Fig. 1). The best characterized pathways are those via indole-3-acetamide (IAM) and indole-3-pyruvate (IPyA) intermediates. The former pathway consists of two distinct steps. In a first step, tryptophan is metabolized to IAM by a tryptophan monooxygenase (encoded by iaaM). IAM is further converted to IAA by an IAM hydrolase (encoded by iaaH). This pathway has been characterized in various plant pathogens and some rhizobial strains (Clark et al., 1993; Glickmann et al., 1998; Manulis et al., 1998; Morris, 1995; Sekine et al., 1989; Theunis et al., 2004). There is no evidence for the presence of this pathway in plants, although IAM could be detected in Arabidopsis (Pollmann et al., 2002). In the IPyA pathway, tryptophan is firstly transaminated to IPyA by an (aromatic) aminotransferase. In the next step, IPyA is decarboxylated by an indole-3-pyruvate or phenylpyruvate decarboxylase to

Nitrilase

Indole-3-acetonitrile

Trp mono-oxygenase

OH O

Indole-3-acetamide

IAM-hydrolase

HO O

NH2

N H

N H

Tryptophan side-chain oxidase

Indole-3-acetic acid

Tryptophan

Amino transferase

Trp decarboxylase

Indole-3-pyruvate

Tryptamine

IPDC/PPDC

Amine-oxidase

IAAId dehydrogenase

Indole-3-acetic acid IAA conjugates

Fig. 1. Indole-3-acetic acid biosynthetic pathways in bacteria. Most biosynthetic pathways for IAA start from the main precursor tryptophan (Trp). The pathways are mostly named by the molecule that is used as an intermediate. It should be noted that not all pathways are characterized to the same extent and that multiple pathways can exist in a single organism. The pathways via indole-3-acetamide and indole-3-pyruvate are well known, as they are the predominant pathways in bacterial pathogens and PGPR. IAA can be conjugated to sugars (ester linked) or amino acids (amide linked). The functions for these conjugates are storage, transport, compartmentalization, and protection against degradation (Cohen and Bandurski, 1982), although the process of conjugation is not well characterized in bacteria. Abbreviations: IAAld, indole-3acetaldehyde; IAM, indole-3-acetamide; IPDC, indole-3-pyruvate decarboxylase; PPDC, phenylpyruvate decarboxylase.


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indole-3-acetaldehyde, which is subsequently oxidized to IAA by non‐ enzymatic oxidation or by an aldehyde oxidase/dehydrogenase (Costacurta and Vanderleyden, 1995; Patten and Glick, 1996). The IPyA pathway has been identified in several bacteria, such as Pantoea agglomerans, Bradyrhizobium, Azospirillum, Rhizobium, Enterobacter cloacae, and Pseudomonas putida, mostly associated with the plant growth-promoting capacities of these bacteria. The key enzyme in the IPyA pathway is the indole-3-pyruvate decarboxylase or phenylpyruvate decarboxylase, and these enzymes and genes have been characterized from Azospirillum brasilense, E. cloacae, Pa. agglomerans, and Ps. putida (Brandl and Lindow, 1996; Costacurta et al., 1994; Koga et al., 1991; Patten and Glick, 2002; Schu¨tz et al., 2003). Based on biochemical studies and a phylogenetic analysis, the group of indole-3-pyruvate decarboxylases and phenylpyruvate decarboxylases can be divided into two subgroups, with distinct activities (Spaepen et al., 2007b). Besides the two major pathways via IAM and IPyA, other pathways have been described (Fig. 1). In the pathway via tryptamine (TAM), tryptophan is decarboxylated to TAM and subsequently converted to indole-3-acetaldehyde by an amine oxidase. By the conversion of exogenous TAM, this pathway could be demonstrated in Azospirillum; however, further biochemical and genetic data are missing (Hartmann et al., 1983). In Pseudomonas fluorescens strain CHA0, the tryptophan side-chain oxidase (TSO) pathway was identified (Oberhansli et al., 1991) and some nitrilases, catalyzing a key step in the pathway via indole-3-acetonitrile (IAN), were characterized (Kobayashi et al., 1993, 1995; Nagasawa et al., 1990). For many years, it has been accepted that the production of auxins by PGPR is the main factor in the growth-promoting capacities of these bacteria. In Az. brasilense, the mechanism of plant-growth promotion by the production of IAA has been intensively studied. In early reports, the importance of bacterial IAA production in plant stimulation could be shown by inoculation studies with mutant (mostly mutants in IAA biosynthetic genes) or overexpressing strains (chemical mutants selected for resistance to the toxic compound 5-fluorotryptophan) (Barbieri and Galli, 1993; Barbieri et al., 1986; Harari et al., 1988). A direct link between IAA production and altered root morphology was demonstrated by Dobbelaere et al. (1999). Inoculation with the wild-type strain results in a shortening of the root length and enhanced root hair formation, and this effect could be mimicked by the addition of pure IAA. A mutant strain, strongly reduced in IAA production by mutation in the ipdC gene, failed to induce these changes. Mutant strains in which the native ipdC promoter was exchanged for a constitutive or plantinducible promoter, showed the same effects as the wild-type strain at lower inoculum concentrations (Spaepen et al., 2008).

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It is of interest to mention the environmental regulation of the ipdC gene of Az. brasilense in relation to plant root colonization. In Az. brasilense IAA production and expression of the key gene ipdC have been shown to be increased under carbon limitation, during reduction in growth rate, and at acidic pH values (Ona et al., 2003, 2005; Vande Broek et al., 2005). As Az. brasilense cells are colonizing plant roots, the bacterial cells are using root exudates for proliferation. When root exudates are becoming limited for bacterial growth, Az. brasilense increases IAA production, thereby triggering lateral root and root hair formation resulting in more root exudation. In this way a regulatory loop connecting plant root proliferation and bacterial growth is created. All mutants in specific genes involved in IAA biosynthesis, still show residual IAA production, even though levels are reduced by 90–99%. Attempts to isolate null mutants failed, indicating the redundancy of IAA biosynthetic pathways in bacteria. The presence of multiple pathways in a single organism suggests a distinct contribution of pathways to the IAA pool, for example, by differential regulation (Barbieri et al., 1986; Carreno-Lopez et al., 2000; Hartmann et al., 1983; Prinsen et al., 1993; Spaepen et al., 2007a). The exact contribution and regulation of different IAA biosynthetic pathways has clearly been demonstrated in the pathogen Pa. agglomerans. Mutants in genes of the IAM pathway cause a large reduction in gall size, while mutants in ipdC genes show no decreased pathogenicity. The latter mutants have reverse effects on the epiphytic fitness of the bacterium. The difference in both pathways is also reflected in the gene regulation: the ipdC gene is enhanced during plant colonization, whereas the iaaM gene is upregulated at later phases of the interaction (e.g., during gall formation) (Manulis et al., 1998). Although it is quite clear that rhizobacterial auxin production drastically affects plant root morphology, information on other plant processes affected by bacterial IAA synthesis is still fragmentary, or even speculative. Nevertheless, recent publications on the role of auxin in defense of Arabidopsis against the leaf pathogenic bacterium Pseudomonas syringae pv. tomato (Chen et al., 2007; Navarro et al., 2006) could open new research perspectives to better understand the role of IAA produced by PGPR. Both studies illustrate very well a prominent role for host auxin signaling in a particular aspect, that is, effector-triggered susceptibility and pathogen-associated molecular pattern (PAMP)-triggered immunity, respectively, of the ZigZag model of the plant immune system proposed by Jones and Dangl (2006). Both studies demonstrate that exogenous application of auxin to the host promotes susceptibility to the pathogen and disease development. The link to the ability of some bacterial pathogens to produce free IAA in culture was


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made in both publications. It is tempting to postulate that auxin production by PGPR could promote the colonization of plant roots by these PGPR as bacterial IAA production might contribute to circumvent the host defense system by derepressing auxin signaling (Remans et al., 2006). However, there is no direct evidence so far, and the dynamic differential distribution of auxin within plant tissues should be taken into account when comparing events in the phyllosphere versus the rhizosphere. Several research groups are currently studying the transcriptome of Arabidopsis roots colonized by PGPR. This together with quantitative imaging of roots colonized by IAA-producing PGPR and IAA-minus mutants, respectively, will allow testing of this hypothesis. Biotrophic pathogens and plant-beneficial bacteria are possibly coming closer to each other when taking an auxin perspective, recently referred to as an ‘‘information-processing system’’ (Vogel, 2006). Clearly, answering questions in one direction (phytopathology) raises new fascinating questions in another direction (phytostimulation). 2. Cytokinins The balance between auxins and cytokinins regulates the outcome on cellular differentiation in plants: if the balance is shifted toward auxin, root development is favored, while in the case of more cytokinin shoot growth is induced. When equimolar amounts of cytokinin and auxin are added in in vitro plant tissue cultures, undifferentiated callus cells proliferate. In contrast to the group of auxins, cytokinins are a broad group, mainly identified in bioassays as inducers of cell division, and are derived from 6N-substituted aminopurines. After biosynthesis in root tips and developing seeds, cytokinins are transported to the shoot via the xylem, where they regulate several processes, such as cell division, leaf expansion, and delay of senescence. The main representatives are zeatin and kinetin (Table I). As for auxins in bacteria, cytokinins were first discovered in pathogens, before cytokinin production was identified in PGPR. Exogenous application of this phytohormone can cause different responses in plants, including abnormal differentiation, promotion of tillering, and activation of seed germination. For several pathogens, the massive production of both auxins and cytokinins is an important virulence factor as they induce gall formation, as exemplified for the Agrobacterium tumefaciens oncogenes that are transferred into the plant DNA, Pa. agglomerans and Ps. syringae (Barash and Manulis-Sasson, 2007; Costacurta and Vanderleyden, 1995; Jameson, 2000). In parallel to auxin production, the capacity to produce cytokinins is widespread among rhizosphere bacteria and the spectrum of cytokinins does not differ from that in plants (Barea et al., 1976; De Salamone et al., 2001; Frankenberger and Arshad, 1995; Tien et al., 1979). Cytokinins are synthesized from isopentenyl

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pyrophosphate and 50 -AMP and several genes involved in this catalysis have been characterized, mainly from pathogenic bacteria such as Pa. agglomerans, Rhodococcus fascians and several Agrobacterium strains (Akiyoshi et al., 1984; Crespi et al., 1992; Lichter et al., 1995a,b). In a next step, isopentenyladenosine-50 -monophosphate is converted to isopentenyladenosine, which can be further modified (Kakimoto, 2003; Prinsen et al., 1997). It was even hypothesized at some time that all cytokinins present in plants are produced by microbial symbionts and are not plant-derived (Holland, 1997). Proof for the mode of action or mechanistic insights into the role of cytokinins in microbe–plant interactions is very scarce due to the lack of mutants that would allow measuring the contribution of bacterial cytokinin production to the plant growth-promoting effect. It is thought that bacterial cytokinin production contributes to the plant cytokinin pool, thereby influencing its growth and development. Recently, it has been shown that bacterial cytokinins are perceived by plant cytokinin receptors. In combination with the inadequate elimination of bacterial cytokinins by the plant, this ultimately leads to developmental changes and tissue proliferation (Pertry et al., 2009). Many PGPR produce both auxins and cytokinins. Consequently, the effect of these PGPR on plant growth and development depends on the balance between these two. Van Laer (2003) has made a quantitative survey of the auxin and cytokinin production by different PGPR.

3. Gibberellins The phytohormone group of the GAs consists of more than 100 members. These compounds are mainly involved in division and elongation of plant cells and influence almost all stages of plant growth, including seed germination, stem and leaf growth, floral induction, and fruit growth. As with auxins and cytokinins, they mainly act in combination with other phytohormones. Due to the use of diverse species and experimental models and the existence of over 120 different gibberellinic compounds, determination of the exact role of GAs is challenging, as it is difficult to discriminate between biologically active and precursor compounds (Yamaguchi, 2008). GAs can be classified as tetracyclid diterpenoic acids, with the typical ent-gibberellane basis synthesized from mevalonic acid. In bacteria, not much is known about GA biosynthesis. Therefore, most suggested pathways are based on those typical for plants and fungi. For Bradyrhizobium japonicum, a biosynthetic pathway different from plants and fungi was demonstrated by biochemical characterization and sequence comparison with known players in early steps of fungal and plant GA biosynthesis, such as diterpene cyclases, cytochromes


293

P450 and dioxygenases, which are involved in the formation of the GA precursor ent-kaurenoic acid (Morrone et al., 2009). In the culture medium of some (plant-associated) bacteria, low concentrations of GAs could be measured, and it was demonstrated that GAs are released into the rhizosphere (Frankenberger and Arshad, 1995; Rademacher, 1994). For rhizobial strains, it was observed that root nodules contain higher amounts of GAs in comparison with noninfected roots. Since the GA spectrum in the nodules was identical to that of the bacteria, the nodule-derived GAs appeared to originate from the rhizobia. However, it cannot be ruled out that the rhizobial strains also induce GA biosynthesis in the nodules (Dobert et al., 1992; Ferguson and Mathesius, 2003). Several Azospirillum species are capable of producing different GAs and in addition metabolize exogenously applied GAs. Based on the observation that Azospirillum can hydrolyze GA conjugates, it has been suggested that the plant growth-promoting effect of rhizobacteria is a combination of both bacterial production of GAs and release of GAs from stored plant conjugates (Piccoli et al., 1997). The strongest evidence for the effect of bacterial GAs was demonstrated in an Azospirillum–maize system. The dwarf phenotype of the dwarf-1 line of maize or dwarfism induced by inhibitors of GA biosynthesis could be reversed by inoculation with Az. brasilense or Azospirillum lipoferum. However, whether bacterial GA biosynthesis or bacterial production of enzymes that release GA from conjugates is involved in this process is not clear (Lucangeli and Bottini, 1996, 1997). As GAs can act at several stages of plant growth and development, the bacterial production of GAs can interfere in very different ways. The exact mechanism of plant growth promotion by GAs is not known, although root-colonizing PGPR probably act by promoting root growth, more particularly by increasing root hair density in root zones involved in nutrient and water uptake, as shown for Az. lipoferum inoculation of maize seedlings (Fulchieri et al., 1993). 4. Ethylene The phytohormone ET was first named the ripening hormone, as it induces fruit ripening. Later, it was shown that the molecule also has a role in other processes, such as seed germination, cell expansion, leaf and flower senescence and abscission, and plant–pathogen interactions. In addition, ET is produced under both abiotic and biotic stress conditions and is therefore known as the stress hormone. High ET concentrations have an inhibitory effect on root growth, resulting in reduced plant growth (Abeles et al., 1992; Mattoo and Suttle, 1991). ET is produced by shuttling methionine out of the methionine cycle yielding S-adenosylmethionine (SAM) by SAM synthetase. SAM is converted in the rate-limiting step to 1-aminocyclopropane-1-carboxylate

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(ACC) and 50 -deoxy-50 methylthioadenosine (MTA) by the enzyme ACC synthase. MTA can be recycled to methionine. Finally, ACC is converted to ET, CO2 and cyanide by ACC oxidase (Chae and Kieber, 2005; Glick et al., 2007). ET biosynthesis is highly regulated mostly at the key step catalyzed by the enzyme ACC synthase at different levels, such as transcriptional regulation, enzyme degradation by the 26S proteasome, and phosphorylation. Factors altering ET biosynthesis are abiotic and biotic stresses (e.g., drought, wounding, and pathogen infection), hormones (cytokinins, auxins, and brassinosteroids), and developmental cues. Their action is exerted mainly through regulation of ACC synthase (Argueso et al., 2007; Chae and Kieber, 2005). Some PGPR express the enzyme 1-aminocyclopropane-1-carboxylate deaminase (AcdS), which can degrade ACC to -ketobutyrate and ammonia. The acdS gene is probably more widespread in bacteria than originally thought, and has been found in both pathogenic and plant-beneficial bacteria (Blaha et al., 2006). Inoculation of diverse plant species with bacteria expressing ACC deaminase activity stimulates plant growth, probably by lowering the level of ET inside the plant (Glick, 2005; Glick et al., 2007; Saleem et al., 2007). A model was proposed by Glick et al. (1998) to explain the role of bacterial ACC deaminase in the plant growth-promoting effect of these bacteria. As ACC may be exuded by plant roots (Bayliss et al., 1997), it can be taken up and hydrolyzed by bacteria with AcdS activity to -ketobutyrate and ammonia, which are then metabolized by the bacteria. The ACC concentration outside the roots decreases and more ACC is exuded by the plant. As a result, ACC levels in the plant are lowered and the ET content is reduced as biosynthesis of ET is inhibited by the lack of precursor (Glick et al., 1998). This results in an increased plant root and shoot length, increased biomass, and reduction of inhibitory effects of ET as a consequence of diverse stresses (Contesto et al., 2008; Glick et al., 2007; Saleem et al., 2007). In addition to expressing AcdS activity, most PGPR are capable of producing IAA. IAA excreted by these bacteria can contribute to the plant IAA pool, thereby inducing the effects described in Section II.A.1, as well as synthesis of plant ACC synthase, leading to more root-excreted ACC (Argueso et al., 2007). Bacteria with ACC deaminase activity have been extensively used for alleviating diverse stresses in plants. By reducing the stress hormone ET, these bacteria are able to protect plants from the growth inhibition caused by ET under several stress conditions, such as flooding, toxic compounds (both organic compounds and heavy metals), high salt concentrations, drought, and pathogenic attack. Transgenic expression of bacterial ACC deaminase genes in plants results in tolerance toward those stresses, although to a lesser


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extent compared to bacterial inoculation, as the bacteria possess also other mechanisms for plant-growth promotion (for reviews: Glick et al., 2007; Saleem et al., 2007). Production of ET has also been reported for microorganisms, including bacterial pathogens, as exemplified by Ps. syringae. In different pathovars ET production is mostly contributing to bacterial virulence, probably by creating a hormonal imbalance in the infected plant. Higher levels of ET found in diseased plant tissues could be correlated to ET production by inoculated bacteria. (Weingart and Volksch, 1997; Weingart et al., 2001). Bacterial ET biosynthesis differs from plant biosynthesis. Two distinct pathways have been described. In the first, the transaminated derivative of methionine, 2-keto-4-methyl-thiobutyric acid, is formed, which is further converted to ET by hydroxyl radicals formed by an NADH:Fe(III)EDTA oxidoreductase. In the second pathway, 2-oxoglutarate is the precursor for ET production by the ET-forming enzyme. The latter pathway is mainly used by ET-producing pathogens and some fungi (Tsavkelova et al., 2006; Weingart et al., 2001). ET also activates several defense responses in plants and is a necessary component in the induction of systemic resistance by rhizobacteria. The dual role of ET as a virulence factor of fungal and bacterial pathogens and as a signaling compound in disease resistance makes it difficult to define the exact role of ET produced by different strains of PGPR (Van Loon et al., 2006).

5. Abscisic acid Also ABA is involved in plant responses to biotic and abiotic stresses. As a hormone, it induces stomatal closure, inhibits seed germination and fruit ripening, and is involved in bud dormancy. Moreover, it mediates protective responses against adverse growth conditions such as drought, salt stress, and metal toxicity. ABA is synthesized in all plant parts and can be readily translocated through the plant. In plants, ABA is synthesized from the isoprenoid molecules isopentenyl diphosphate and dimethylallyl diphosphate via terpenoid intermediates (Taylor et al., 2005). Production of ABA in the culture medium of bacteria has been shown for Az. brasilense and some Br. japonicum strains, but a mechanistic proof or a biosynthetic pathway has not yet been provided (Boiero et al., 2007; Cohen et al., 2008). It can be hypothesized that ABA-producing bacteria can increase plant growth by interfering with the plant cytokinin pool, as ABA inhibits the synthesis of cytokinins (Miernyk, 1979). In addition, under stress conditions bacterially produced ABA can alleviate plant stress as suggested by Boiero et al. (2007).

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S. SPAEPEN ET AL. B. NITROGEN TRANSFORMATIONS

1. Biological nitrogen fixation Following reports of the isolation of rhizosphere and endophytic diazotrophs and their potential biological nitrogen fixation (BNF) activities (Dobereiner and Campelo, 1971; Dobereiner et al., 1972), there has been a large effort to demonstrate substantial BNF in different associative (nonsymbiotic) systems and their contribution to the nitrogen needs of plants. All the bacterial species studied were found to be vigorous nitrogen fixers in pure culture when an appropriate energy source, optimal temperatures, pH, and O2 concentration in the growth medium were available (Elmerich, 2007). There are probably many more N-fixing, nonculturable species, as revealed by the presence of the nifH and other marker genes in DNA extracted from soil (Schmid and Hartmann, 2007). BNF has been measured variously by the acetylene-reduction assay, 15N dilution technique, 15N fixation, and Kjeldahl N-content measurements (Okon, 1985). In most systems and conditions, the nitrogen fixation values for many grain and forage grass crops, when extrapolated to fixed nitrogen, generally did not amount to more than 10 kg N ha1 year1. Under some growth conditions, in few cultivars of sugar cane and rice (colonized by rhizosphere bacteria and endophytes representing several bacterial species) and in Kallar grass in Pakistan, BNF values indicated potentially significant BNF of up to 50% of the N content of the plant (Elmerich, 2007). From 15N dilution studies it was concluded that in annual crops, the fixed N probably remained in the bacterial cells, and that the bacterial cells could not be readily mineralized, preventing the uptake of combined nitrogen by the plant (Okon, 1985). In most cases, the contribution of biologically fixed N is far too low to contribute significant amounts of N to field crops. For example, a hybrid maize cultivar is commonly fertilized with 250 kg N ha1 per growth season. 2. Nitrate uptake by roots as affected by bacteria Enhanced mineral uptake in plants inoculated with PGPR has been reported repeatedly, both in greenhouse experiments (Lin et al., 1983) and in the field (Dobbelaere and Okon, 2007; Dobbelaere et al., 2003). The major nutrient involved was nitrogen in the form of nitrate (Dobbelaere and Okon, 2007). Using a hydroponic system containing NO3, during plant growth both the surface area of wheat roots and the uptake of NO3 from the mineral nutrient solution were found to increase upon inoculation with Az. brasilense. However, no significant changes were obtained in the NO3 uptake/root surface area ratio, indicating that the increased NO3 ion uptake by wheat was due to a general increase in root surface area, and not because of


297

an increase in specific uptake rate (Kapulnik et al., 1985). On the other hand, direct effects on specific uptake mechanisms cannot be excluded, as it was observed that Azospirillum–inoculated corn and sorghum plants take up NO3 from solutions at faster rates than non‐inoculated plants (Lin et al., 1983). Section II.B.3. 3. Denitrification A large number of PGPR species that are used for inoculation in greenhouse and field experiments and commercially are also capable of dissimilatory nitrate reduction (denitrification). Under soil conditions of high NO3 content and low O2 availability all bacterial denitrifiers, and not just PGPR denitrifiers, will be active in soil denitrification (Steenhoudt and Vanderleyden, 2000). Denitrification is the respiratory process whereby NO3 is successively reduced to NO2, N-oxides (N and N2O), and dinitrogen (N2) (Fig. 2). (Steenhoudt and Vanderleyden, 2000). The first step, the reduction of nitrate to nitrite, can be catalyzed by a membrane-bound respiratory, or a periplasmic dissimilatory nitrate reductase. It has been proposed that bacterial nitrate reductase activity plays a significant role in PGPR–plant associations. It was also proposed that nitrite could mimic the effects

NO3 − (1) (2)

(3)

(6)

NO2 − (6) NO, N2O, N2 (4)

NO2 −

(2) (3)

NH4 + (5) Organic N

Fig. 2. The biological nitrogen cycle. Nitrogen can be transformed into different chemical forms by microbes, ranging from oxidation state þ5 (NO3) to 3 (NH4þ). The different reactions, carried out by different microbial groups under different ecological conditions, are indicated by numbers: (1) denitrification, (2) nitrification, (3) assimilatory and dissimilatory nitrate reduction, (4) biological nitrogen fixation, (5) ammonium assimilation, and (6) anaerobic ammonium oxidation (anammox). Processes (1), (3), and (4) require an electron donor, whereas process (2) requires an electron acceptor. NO3 and NH4þ are the main forms of nitrogen that can be readily used by plants.

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of IAA in several plants tested (Zimmer et al., 1988). However, all these observations have not been further substantiated (Steenhoudt and Vanderleyden, 2000). C. PHOSPHATE AND MICRONUTRIENT AVAILABILITY

1. Phosphate Phosphorus is an important nutritional element for growth and development of plants. The concentration of bioavailable phosphorus is rather low in soil (GIDVvT00015510001_ PF000697 P450, 1 PF00314 thaumatin, signal sequence A5AEX9

Oryza sativa

PF00069: protein kinase : transmembrane

Fig. 4.

PF00067: cytochrome P450 enzymes are heme containing mono-oxygenases that are important in plants for the biosynthesis of hormones, defensive compounds and fatty acids

(continued )

Species

Accession numbers

Domains with Pfam accession numbers (in order from N-terminus)

Protein structure

Domain function

PF01151: ELO is a family of membrane proteins involved in the synthesis of ceramides and sphingolipids. They may have roles in controlling glucose signaling and plasma membrane H+-ATPase.

Vitis vinifera

>GSVIVT00026571001 A7PYT6

PF01151 ELO, PF00314 thaumatin, signal sequence

Zea mays

AC187883.4_FGT009

PF00314 thaumatin, PF00069 protein kinase, signal sequence

PF00069: protein kinase

PF00314 thaumatin, PF03129 HGTP anticodon, signal sequence

PF03129: this is believed to be the anticodon binding domain of His-, Gly-, Thr- and Pro-tRNA synthases

: transmembrane

AC212473.1_FGT011 Zea mays

AC193988.3_FGT028 Q8SA98

Fig. 4. Thaumatin domain fusion proteins in plants. Domain searches were performed with THAUMATIN or PF00314 as query terms at Pfam 23.0 database (http://pfam.sanger.ac.uk/) (Finn et al., 2008), Superfamily 1.69 database (http://supfam.mrc-lmb.cam.ac.uk/) (Gough et al., 2001), and manually at Grape genome browser (http://www.genoscope.cns.fr/). Domains predictions were verified manually by querying the predicted proteins against InterPro at http://www.ebi.ac.uk/ (Hunter et al., 2009; Quevillon et al., 2005). Domain descriptions can be found at the Conserved Domain Database (http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) (Marchler-Bauer et al., 2009). Accession numbers are from UniProtKB (http://www.uniprot.org/uniprot/; The UniProt Consortium, 2009), MaizeSequence.org Release 3b.50 (http://maizesequence.org/), the Grape Genome Browser (http://www.genoscope.cns.fr/), J. Craig Venter database (http://www.tigr. org/), and BGI Rise Rice Genome Database (http://rice.genomics.org.cn/). Some of the indicated signal sequences have transmembrane domains and others do not. Domains and their descriptions are in identical colors. Truncated domains are indicated with jagged edges. These data were verified on March 24, 2009 and may change as annotations in the databases are revised.

PR-PROTEINS CONNECT ENERGY METABOLISM AND IMMUNITY

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interacts with G-protein-coupled taste receptors to induce the sensation of sweet taste, lending further support to the notion that the THN domain has broad, yet poorly understood, target recognition functions (Temussi, 2006).

D. COMPARISON OF THN DOMAIN WITH C1q-TNF DOMAINS

Several intriguing concepts regarding the role of PR-5 proteins can be established by comparing similarities in the properties, location, and functions of adiponectin and adiponectin-like mammalian proteins with those of PR-5 proteins. Adiponectin is a protein hormone secreted by adipocytes, that is, mammalian fat cells (Diez and Iglesias, 2003; Fang and Sweeney, 2006). It is an abundant plasma protein. Many PR-5 proteins share a similar extracellular location (Bayer et al., 2006; Charmont et al., 2005; Melchers et al., 1993; Rep et al., 2002; Uknes et al., 1992) (Fig. 1), as the mammalian plasma is equivalent to apoplastic fluid, xylem, and phloem sap of plants. Adiponectin is composed of two domains: an N-terminal collagen domain and a C-terminal globular domain that has a clearly recognizable fold, annotated in databases as the C1q domain (Pfam23.0 database, accession PF00386; http://pfam.sanger.dc.uk; Finn et al., 2008). The adiponectin C1q domain is necessary and sufficient for interaction with adiponectin receptors (Tomas et al., 2002; Yamauchi et al., 2003a,b). Although the sequence identity between osmotin and adiponectin is insignificant (

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