Recombinant Protease Inhibitors in Plants

BIOTECHNOLOGY INTELLIGENCE UNIT 3

Dominique Michaud

Recombinant Protease Inhibitors in Plants

BIOTECHNOLOGY INTELLIGENCE UNIT 3

Recombinant Protease Inhibitors in Plants Dominique Michaud, Agr., Ph.D. Département de Phytologie Centre de Recherche en Horticulture Université Laval Sainte-Foy, Québec, Canada

LANDES BIOSCIENCE GEORGETOWN, TEXAS U.S.A.

EUREKAH.COM AUSTIN, TEXAS U.S.A.

RECOMBINANT PROTEASE INHIBITORS IN PLANTS Biotechnology Intelligence Unit EUREKAH.COM Designed by Kimberly Mitchell Georgetown, Texas, U.S.A. Copyright ©2000 Eurekah.com All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the U.S.A. Please address all inquiries to the Publishers: Eurekah.com, 810 South Church Street, Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081 -ISBN: 1-58706-007-8

Library of Congress Cataloging-in-Publication Data Recombinant protease inhibitors in plants / [edited by] Dominique Michaud. p. cm. -- (Biotechnology intelligence unit) Includes bibliographical references and index ISBN 1-58706-007-8(alk. paper) 1. Plants--Disease and pest resistance. 2. Recombinant protease inhibitors. I. Michaud, Dominique. II. Series. [DNLM: 1. Protease Inhibitors--metabolism. 2. Plants--metabolism. 3. Recombinant Proteins--metabolism. QU 136 R311 1999] SB750.R43 1999 632' .96--dc21 DNLM/DLC for Library of Congress

CONTENTS 1. Protease/Inhibitor Interactions in Plant-Pest Systems: A Brief Overview .................................................................................... 1 Dominique Michaud 1.1. Introduction ................................................................................... 1 1.2. The Inhibition of Pest Extracellular Proteases ................................. 2 1.3. Plant Pest Control with PIs: After the Hits... the Misses ................. 3 1.4. Future Perspectives ......................................................................... 5 2. Control of Phytophagous Insect Pests Using Serine Proteinase Inhibitors ............................................................................................... 9 John A. Gatehouse, Angharad M.R. Gatehouse and David P. Bown 2.1. Serine PIs and Their Role in Plant Protection ................................ 9 2.2. Effects of Serine PIs on Insect Digestive Proteolysis ...................... 11 2.3. The Wounding Response in Plant Defsense ................................. 14 2.4. “First-Phase” Use of Foreign Serine PI Genes ............................... 16 2.5. Insect Responses to Dietary Serine PIs .......................................... 19 2.6. Conclusions .................................................................................. 23 3. Cystatin-Based Control of Insects, with Special Reference to Oryzacystatin ................................................................................... 27 Soichi Arai and Keiko Abe 3.1. Introduction ................................................................................. 27 3.2. Oryzacystatin and Other Plant Cystatins ...................................... 28 3.3. Cysteine Proteinases as Targets of Plant Cystatins ........................ 30 3.4. Control of Insects with Cystatins .................................................. 33 3.5. Regeneration of Cystatin-Expressing Transgenic Plants ................ 36 3.6. Conclusion ................................................................................... 38 4. Recombinant Protease Inhibitors as Management Tools to Suppress Parasitic Nematodes .......................................................... 43 Thierry C. Vrain 4.1. Introduction ................................................................................. 43 4.2. Proteases in Nematodes ................................................................ 44 4.3. Effects of Recombinant PIs .......................................................... 46 4.4. Outlook ....................................................................................... 48 5. The Control of Plant Pathogens with Protease Inhibitors: A Realistic Approach? ........................................................................... 53 Stephen Gleddie and Dominique Michaud 5.1. Introduction ................................................................................. 53 5.2. Extracellular Proteases in Plant Pathogens .................................... 54 5.3. Induction of PIs in Infected Plants ............................................... 57 5.4. Prospects for PI-Expressing Transgenic Plants .............................. 59 5.5. Conclusion ................................................................................... 62

6. Regulation of Plant Defense Against Herbivorous Pests ....................... 65 Hisashi Koiwa, Ray A. Bressan and Paul M. Hasegawa 6.1. Introduction ................................................................................. 65 6.2. The Plant Response ...................................................................... 68 6.3. Future Perspectives ....................................................................... 73 7. The Response of Insects to Dietary Protease Inhibitors ........................ 80 Roxanne M. Broadway 7.1. Introduction ................................................................................. 80 7.2. Evaluation of PI Biological Activity .............................................. 80 7.3. Resistance of Insects to Dietary PIs ............................................... 81 7.4. Summary ...................................................................................... 84 8. Interference of Protease Inhibitors on Non-Target Organisms ............. 89 Louise A. Malone and Elisabeth P.J. Burgess 8.1. Introduction ................................................................................. 89 8.2. Pollinators .................................................................................... 89 8.3. Natural Enemies ........................................................................... 99 8.4. Soil Fauna and Microorganisms ................................................. 102 8.5. Summary .................................................................................... 103 9. Multiple Protease/Inhibitor Interactions in Plant-Pest Systems ......... 107 Savita Visal-Shah, France Brunelle and Dominique Michaud 9.1. Introduction ............................................................................... 107 9.2. First-Level Interactions: The Plant-Pest Continuum .................. 107 9.3. Multi-level Interactions: The Ecosystem ..................................... 108 9.4. Future Perspectives ..................................................................... 111 10. Using Natural and Modified Protease Inhibitors ................................ 114 Dominique Michaud and Binh Nguyen-Quoc 10.1. Introduction ............................................................................. 114 10.2.The Choice of Effective Inhibitors ............................................. 114 10.3. The Design of Hybrid Inhibitors .............................................. 119 10.3.1. Site-Directed Mutagenesis of Natural PIs .............................. 120 10.4. Conclusion ............................................................................... 124 11. Engineering Protease Inhibitors by Phage Display ................................................................................ 128 Jules Beekwilder and Maarten Jongsma 11.1. Introduction ............................................................................. 128 11.2. Phage Display ........................................................................... 128 11.3. Phage Display to Identify Natural Inhibitors ............................ 136 11.4. From Phage to Plant ................................................................. 137 11.5. Conclusion ............................................................................... 137

12. Using Protease Proregions as Regulators of Insect Digestive Proteinases ......................................................................................... 141 Mark A.J. Taylor 12.1. Introduction ............................................................................. 141 12.2. Proteinase Precursors ................................................................ 141 12.3. Precursor Processing ................................................................. 142 12.4. Protease Proregions as Selective Inhibitors ................................ 142 12.5. Discussion ................................................................................ 145 13. Expression of Protease Inhibitors in Potato ........................................ 147 Conrad Cloutier and Dominique Michaud 13.1. Introduction ............................................................................. 147 13.2. Rationale for a Low-Dose Approach with PI-Based Resistance ................................................................................. 150 13.3. The CPB Digestive Proteolytic System ..................................... 152 13.4. Fitness Consequences of PI Ingestion in CPB ........................... 156 13.5. OC-I-Expressing Transgenic Potato and CPB Natural Predators .................................................................................. 158 13.6. Conclusions and Future Perspectives ........................................ 159 14. Expression of Protease Inhibitors in Sweetpotato ............................... 166 Dapeng Zhang, Giselle Cipriani, Isabelle Rety, Ali Golmirzae, Nicole Smit and Dominique Michaud 14.1. Introduction ............................................................................. 166 14.2. The Insect Pests of Sweet Potato .............................................. 166 14.3. Integrated Pest Management for SPWs— The Success and the Lesson ...................................................... 167 14.4. Digestive Proteinases in SPW (Cylas) Species ............................ 168 14.5. Expression of Recombinant PIs in Sweet Potato ....................... 169 14.6. PIs in Sweet Potato, and Their Nutritional Impact ................... 172 14.7. Discussion ................................................................................ 174 15. Expression of Protease Inhibitors in Rapeseed .................................... 178 Lise Jouanin, Michel Bonadé Bottino, Cécile Girard, Jacques Lerin and Minh Hà Pham Delègue 15.1. Introduction ............................................................................. 178 15.2. Digestive Protease Types in Pests of B. napus ........................... 179 15.3. Expression of Recombinant PIs in B. napus .............................. 181 15.4. Discussion ................................................................................ 187

16. Production of Useful Protease Inhibitors in Plants ............................ 190 Dominique Michaud and Serge Yelle 16.1. Introduction ............................................................................. 190 16.2. High-level Accumulation of PIs in Plant Cells .......................... 191 16.3. Extraction of Recombinant PIs from Plant Tissues ................... 192 16.4. Purification of Recombinant PIs from Crude Extracts .............. 193 16.5. Conclusions .............................................................................. 197 17. Proteinase INhibitors in Health and Disease Control—Medical and Industrial Aspects ........................................................................ 202 Michiel F.J. Blankenvoorde, Henk S. Brand, Yvonne M.C. Henskens, Enno C.I. Veerman and Arie V. Nieuw Amerongen 17.1. Introduction ............................................................................. 202 17.2. Anti-microbial Properties of Cystatins ...................................... 204 17.3. Cystatins in the Control of Tumor Growth and Metastasis ............................................. 205 17.4. Protection Against Tissue Destruction ...................................... 206 17.5. Industrial Aspects ..................................................................... 208 18. Protease Inhibitors in Food Processing............................................... 214 Fernando L. García-Carreño, Haejung An and Norman F. Haard 18.1. Introduction ............................................................................. 214 18.2. Controlled Proteolysis in Food ................................................. 215 18.3. Safety of PIs in Food and Feed ................................................. 215 18.4. Plant PIs in Foodstuff ............................................................... 216 18.5. Conclusion ............................................................................... 219 Appendix I. Substrates and Inhibitors Useful in Protease Characterization ............................................................... 223 France Brunelle and Dominique Michaud Appendix II. Plant Protease Inhibitors: Available mRNA Sequences ......... 227 Binh Nguyen-Quoc Appendix III. Tertiary Structures of Proteases and Protease Inhibitors Available in the Brookhaven National Laboratory Protein Data Bank ................................................................................................... 230 France Brunelle and Dominique Michaud Index .................................................................................................. 238

EDITOR Dominique Michaud, Agr., Ph.D. Département de Phytologie, Centre de Recherche en Horticulture, Université Laval Sainte-Foy, Québec, Canada Chapters 1, 5, 9, 10, 13, 14 and 16 Appendices I and III

CONTRIBUTORS Keiko Abe Department of Applied Biological Chemistry Graduate School of Agricultural and Life Sciences The University of Tokyo Tokyo, Japan

Michel Bonadé Bottino Laboratoire de Biologie Cellulaire Institut National de la Recherche Agronomique Versailles, France

Chapter 3

David P. Bown Department of Biological Sciences University of Durham Durham, U.K

Haejung An Seafood Laboratory, Oregon State University Astoria, Oregon,U.S.A. Chapter 18

Soichi Arai Department of Nutritional Science, Faculty of Applied Biological Sciences Tokyo University of Agriculture Tokyo, Japan Chapter 3

Jules Beekwilder Centre for Plant Breeding and Reproduction Research (CPRO-DLO), Wageningen,The Netherlands Chapter 11

Michiel F.J. Blankenvoorde Department of Oral Biochemistry Academic Centre for Dentistry Amsterdam (ACTA) Amsterdam, The Netherlands Chapter 17

Chapter 15

Chapter 2

Henk S. Brand Department of Oral Biochemistry Academic Centre for Dentistry Amsterdam (ACTA) Amsterdam, The Netherlands Chapter 17

Ray A. Bressan Center for Plant Environmental Stress Physiology Purdue University West Lafayette, Indiana, U.S.A. Chapter 6

Roxanne M. Broadway Department of Entomology, New York State Agricultural Experiment Station, Cornell University Geneva, New York, U.S.A. Chapter 7

France Brunelle Département de Phytologie, Centre de Recherche en Horticulture, Université Laval Québec, Canada Chapter 9, Appendices I and III

Stephen Gleddie Eastern Cereal and Oilseed Research Centre, Central Experimental Farm Agriculture and Agri-Food Canada Ottawa, Canada Chapter 5

Elizabeth P.J. Burgess The Horticulture and Food Research Institute of New Zealand Ltd Auckland, New Zealand

Ali Golmirzae Centro Internacional de la Papa Lima, Peru

Chapter 8

Chapter 14

Giselle Cipriani Centro Internacional de la Papa Lima, Peru

Norman F. Haard Department of Food Science and Technology University of Califormia Davis, California, U.S.A.

Chapter 14

Conrad Cloutier Département de Biologie Centre de Recherche en Horticulture Université Laval Québec, Canada Chapter 13

Fernando L. García-Carreño Centro de Investigaciones Biologicas del Noroeste, S.C., La Paz, Mexico Chapter 18

Angharad M. R. Gatehouse Department of Biological Sciences University of Durham Durham, U.K. Chapter 2

John A. Gatehouse Department of Biological Sciences University of Durham Durham, U.K. Chapter 2

Cécile Girard Laboratoire de Biologie Cellulaire Institut National de la Recherche Scientifique Versailles, France Chapter 15

Chapter 18

Paul M. Hasegawa Center for Plant Environmental Stress Physiology, Purdue University West Lafayette, Indiana, U.S.A. Chapter 6

Yvonne M.C. Henskens Laboratory for Clinical Chemistry and Hematology Diagnostic Center SSDZ Delft, The Netherlands Chapter 17

Maarten A. Jongsma Centre for Plant Breeding and Reproduction Research (CPRO-DLO) Wageningen, The Netherlands Chapter 11

Lise Jouanin Laboratoire de Biologie Cellulaire Institut National de la Recherche Agronomique Versailles, France Chapter 15

Hisashi Koiwa Center for Plant Environmental Stress Physiology, Purdue University West Lafayette, Indiana, U.S.A. Chapter 6 Jacques Lerin Laboratoire de Zoologie Institut National de la Recherche Agronomique Luzignan, France Chapter 15

Louise A. Malone The Horticulture and Food Research Institute of New Zealand Ltd Auckland, New Zealand Chapter 8 Binh Nguyen-Quoc Département de Phytologie Centre de Recherche en Horticulture Université Laval Québec, Canada Chapter 10, Appendix II

Arie V. Nieuw Amerongen Department of Oral Biochemistry Academic Centre for Dentistry Amsterdam (ACTA) Amsterdam, The Netherlands Chapter 17

Minh-Ha Pham Delegue Laboratoire de Neurobiologie Comparée des Invertébrés Institut National de la Recherche Agronomique Bures sur Yvette, France Chapter 15

Isabelle Rety Département de Phytologie Centre de Recherche en Horticulture Université Laval Québec, Canada Chapter 14

Nicole Smit Centro Internacional de la Papa Kampala, Uganda Chapter 14

Mark A.J. Taylor Institute of Food Research Reading, U.K. Chapter 12

Enno C.I. Veerman Department of Oral Biochemistry Academic Centre for Dentistry Amsterdam (ACTA) Amsterdam, The Netherlands Chapter 17

Savita Visal-Shah Département de Phytologie Centre de Recherche en Horticulture Université Laval Québec, Canada Chapter 9 Thierry C. Vrain Pacific Agri-Food Research Centre Agriculture and Agri-Food Canada Summerland, Canada Chapter 4

Serge Yelle Département de Phytologie Centre de Recherche en Horticulture Université Laval Québec, Canada Chapter 16 Dapeng Zhang Centro Internacional de la Papa Lima, Peru Chapter 14

PREFACE

A

survey of the current scientific literature clearly shows the implication of proteolytic enzymes and their inhibitors in various biological systems, in volving processes as diverse and important as the degradation of dietary proteins, the regulation of cellular protein catabolism and the inhibition of pathogen extracellular proteases during infection. Together, the rapidly growing body of literature in the broad research field of proteolysis —about 15 papers are published every day, and the many patents issued each year regarding the control of specific protease-related events in biological systems demonstrate the importance of proteolysis in biological processes and the increasing interest for this research field in the scientific community. While gene regulation is important to control the synthesis of proteins in living cells, proteolysis is important to mediate both their processing and their degradation. From a practical point of view, proteases may be seen as interesting targets for the control proteins after they are synthesized, as genes represent interesting targets to control proteins before they accumulate in the cell. In this context protease inhibitors (PIs), the ‘natural regulators’ of proteases, represent a useful tool to regulate these enzymes in biological systems. The inactivation of proteases with specific inhibitors may prove useful, for instance to control several protease-related physiological disorders in humans, to stop pathogen invasion by hindering the hydrolysis of the host tissue proteins, to protect defenserelated proteins produced by the host to counteract predation or infection, or to interfere with the digestion of dietary proteins in plant pests and pathogens. The use of PIs in plant protection, in particular, represents an attractive way to protect economically-important plants from predation or infection. With the growing concerns worldwide about the negative effects of chemical pesticides in the environment, several alternative approaches for pest control are now currently devised. The design of genetically-modified plants expressing recombinant PIs, notably, has been proposed as a way to protect plants from various organisms. It is now increasingly evident that most herbivorous insects and pathogens secrete extracellular proteases to mediate important processes like the digestion of plant proteins or the invasion of host tissues. In this perspective, altering proteolytic processes in target pests by allowing the plant to produce appropriate PIs certainly represents a potentially useful way to reduce the fitness of several target pest organisms, and thus help minimize the use of potentially harmful synthetic pesticides in the field. In the last ten years PI-encoding cDNA sequences have been integrated into the genome of plants as important as cereals, rapeseed, potato and sweetpotato, and convincing protective effects have been noted in some cases, especially against lepidopteran insects and parasitic nematodes. The general usefulness of protein PIs in plant protection, however still remains to be clearly established. During the course of their evolution, pests and pathogens

have been in contact with a variety of PIs produced naturally by their host, and they developed efficient strategies to elude their inhibitory effects, including the use of complex proteolytic systems composed of proteases differentially inhibited by PIs, compensation of inhibited proteolytic functions by the production of ‘insensitive’ proteases, and degradation of the inhibitors by nontarget proteases. As a result of these coevolutive processes protease/inhibitor interactions in plantpest systems became remarkably complex, and anyone wishing to alter specific proteolytic functions in a given target organism has to take into account not only the inhibitory effect of the PI against the target proteases, but also several additional factors associated with the release of such a novel biologically active compound in the whole system. The capacity of the target pest to elude the effect of the introduced inhibitor, or the possible negative effects of this inhibitor against nontarget organisms at the ecosystem level are just a few issues that should be considered to ensure the successful implementation of recombinant PIs in the field. With the aim of summarizing our current knowledge on the use of recombinant PIs in plant protection, this book presents a collection of articles written by experts actively involved in the field worldwide. After a brief introduction on some basic concepts related to protease/inhibitor interactions in plant-pest systems (Chapter 1), the first part of the book summarizes recent advances on the use of recombinant PIs to protect plants from predation by herbivorous insects (Chapters 2 and 3) and from infection by parasitic nematodes (Chapter 4) and pathogens (Chapter 5). The second part of the book deals with some basic aspects of the biological systems assessed, including the response of plants to herbivorous pests (Chapter 6), the response of herbivorous insects to dietary PIs (Chapter 7), the risks associated with the release of recombinant PIs in the environment (Chapter 8), and how all these elements interact with each other in complex biological systems (Chapter 9). Based on these considerations, strategies are then proposed for the choice and the development of effective PIs: Chapter 10 discusses the main biochemical criteria to consider for the choice and the design of appropriate inhibitors; Chapter 11 describes the potential of molecular phage display as a mean to improve the binding characteristics of PIs; and Chapter 12 discusses the potential of protease proregions as regulators of exogenous proteases in biological systems. The third section of the book, finally, deals with the practical and industrial aspects of recombinant PI expression in plants. The first three chapters assess the usefulness of PIs in the protection of three model plants of major economic importance: potato (Chapter 13), sweetpotato (Chapter 14) and rapeseed (Chapter 15). The following three chapters deal with alternative uses of recombinant PIs expressed in plants: Chapter 16 addresses the basic principles to consider when planning the use of plants as bioreactors to produce useful PIs, while Chapter 17 and Chapter 18 discuss the potential of these recombinant inhibitors in two fields of industrial importance, medicine and food science. Dominique Michaud Puétec, January 1999

CHAPTER 1

Protease/Inhibitor Interactions in Plant-Pest Systems A Brief Overview Dominique Michaud

1.1. Introduction

T

he metabolism of any living cell depends on proteolysis. Proteases, which form a diverse group of enzymes capable of cleaving peptide bonds, are implicated in various essential processes ranging from the fine control of protein catabolism and the selective degradation of damaged proteins to the bulk hydrolysis of dietary proteins. Considering the recent developments in the broad research field of proteolysis, it now appears obvious that proteases are essential not only in providing cells with simple metabolites essential for growth and development, but also in mediating a variety of key processes like the cleavage of specific peptide bonds in immature proteins or the removal of targeting signals in preproteins after their translocation to the appropriate cell compartment. Whereas proteolytic enzymes were previously seen as ‘destroying catalysts’ mostly involved in the bulk hydrolysis of dietary proteins, they are now considered as central control elements in the development of most living organisms.1 During the ‘life cycle’ of a protein, proteolysis is essential in regulating maturation and catabolism, as gene control is essential to regulate anabolism. Simply put, proteolytic enzymes mediate two types of hydrolytic processes:

1. limited proteolysis, in which only one or a few peptide bonds are cleaved to release a biologically active protein, and 2. extensive proteolysis, in which the protein is completely degraded through the hydrolysis of most of its peptide bonds.

Limited proteolysis is generally associated with the control of specific metabolic events; extensive proteolysis usually results in complete elimination of the protein, either to regulate its intracellular level or to recycle the resulting amino acids (Fig.1.1). Together, these processes contribute to the overall metabolism of living organisms by mediating important cellular functions like the final processing of pre- or proproteins before they play their role in the cell, the selective removal of these proteins when they are no longer useful, or the recycling of amino acids needed to synthesize novel proteins. Besides these endogenous processes, proteases mediate several molecular interactions taking place between the different organisms of a given environment. Intracellular parasites, for instance secrete proteases implicated in both their interactions with the host, their survival into the host cell environment and the processing of host

Recombinant Protease Inhibitors in Plants, edited by Dominique Michaud. ©2000 Eurekah.com

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Recombinant Protease Inhibitors in Plants

Fig. 1.1. The ‘life cycle’ of a protein. Protein metabolism in living cells may be seen as a two-step process in which the amino acids used as building blocks for protein synthesis are recycled as a result of protein turnover. While gene control appears of crucial importance to regulate and direct the synthesis of proteins (grey area), proteolysis is essential in mediating their posttranslational cleavage, either to remove targeting signals after the translocation of preproteins in the cell, to activate immature proproteins by removal of their regulatory propeptide, to hydrolyze a protein no longer useful in the cell, to remove ‘abnormal’—including foreign—proteins from the cell, or to digest dietary proteins obtained from food to provide the organism with the amino acids needed for growth and development (white area). Proteolysis is generally restricted to one or a few peptide bonds during preprotein or proprotein processing, while extensive hydrolysis of the polypeptide chain takes place during protein turnover. proteins for nutritional purposes.2,3 Likewise, processes like the hydrolysis of plant dietary proteins in the midgut of herbivorous insects, the digestion of these same proteins by our own digestive proteases, or the degradation of host tissue proteins during microbial infection all represent extracellular proteolytic processes implicating the different living entities of a biological system. In the course of their evolution, living organisms have acquired complex protease systems efficient

in hydrolyzing exogenous proteins, a process essential both to obtain the nutrients needed for growth, and to compete with the other organisms of the surrounding environment.

1.2. The Inhibition of Pest Extracellular Proteases In reaction to these hydrolytic processes, several organisms also developed strategies to counteract the adverse effects of exogenous

Protease/Inhibitor Interactions in Plant-Pest Systems

proteases. Plants challenged by a pathogen or a predator, for instance actively produce proteinaceous protease inhibitors (PIs), which react with proteases as pseudo-substrates to hinder their activity. It is now well established that PIs are used by a wide variety of organisms not only to direct the control of endogenous proteolytic functions, but also to ensure their protection against predation or infection.4-9 Like extracellular proteases, PIs actively contribute to the complex array of molecular interactions taking place between the different organisms of an ecosystem, by acting as regulators of extracellular proteolytic events. In this context, it is not surprising that PIs were proposed as a tool for the control of various pests and pathogens. Assuming that every protease or group of proteases in given target organisms mediates at least one useful process, one can speculate that altering this process by inactivating the protease(s) involved may represent a way to affect the development of these organisms, and thus eventually protect their host from predation or infection. Until now pest control strategies based on the inhibition of proteolytic enzymes with selective inhibitors have been devised to control pests as diverse as viruses, herbivorous insects, parasitic nematodes and microbial pathogens.6,10,11 Although the exact metabolic functions altered by the inhibitors remain to be elucidated in most cases, the importance of extracellular proteolysis in the target organisms appears obvious. Repressive effects of dietary PIs on growth and fecundity of herbivorous insects, for instance have been described for several species (see Chapters 2 and 3, this volume), and the implication of extracellular proteases in pathogenic processes has been documented in several instances (see Chapters 4, 5 and 17). Based on these findings, the use of protein PIs expressed in transgenic systems or delivered by any other appropriate mean has been proposed as a way to protect a wide variety of hosts from their natural enemies. The genetic transformation of plants with PI-encoding cDNA sequences, in particular, represents an attractive way to control a variety of herbivorous pests and pathogens attacking

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plants (see Chapters 2-5).10-15 Many studies have demonstrated the ability of protein PIs to efficiently inhibit the extracellular proteases of various herbivorous insects, root-parasitic nematodes and fungal pathogens, and deleterious effects of several proteinaceous inhibitors included in artificial diets or culture media were reported for several insects16-20 and pathogens.21-23 Based on these observations, the use of recombinant PIs was proposed as an effective means of protecting plants from their natural enemies, and several plants of economic importance were genetically modified with PI-encoding cDNA sequences. In the last ten years the genes of several different PIs were integrated into the genome of almost fifteen different plants,12 clearly showing the growing interest of scientists for this control approach.

1.3. Plant Pest Control with PIs: After the Hits... the Misses Despite these promising developments, the general usefulness of recombinant PIs in plant protection still remains to demonstrate. The inhibitory spectrum of protein PIs is usually limited to proteases in one of several mechanistic classes, leaving free proteases in the surrounding medium after inhibition (see Figure 1.2 for a classification of proteases and PIs).24 Probably due to a progressive adaptation of plant pests to the continuous occurrence of PIs in the diet, the inhibitory spectrum of protein inhibitors against the extracellular proteases of several pests is even more limited, being often restricted to the family level.19,25-34 Nontarget proteases, that may allow metabolic compensation of inhibited proteolytic functions,35 may also challenge the structural integrity of several PIs and thus potentially affect their effectiveness in vivo.36 At this point, understanding the dynamic interactions implicating protein PIs and pest extracellular proteases appears important to correctly assess the actual usefulness of extracellular protease inhibition in plant protection (see Chapters 6, 7 and 9). Until now the adverse effects observed with PIs against several insects and pathogens were

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Recombinant Protease Inhibitors in Plants

Fig. 1.2. Classification of proteases and PIs—a simple overview. According to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, proteases belong to the fourth subgroup of Group 3 enzymes, which includes hydrolytic enzymes. Given their huge structural and functional diversity, proteases may be classified based on three major criteria: the type of reaction catalyzed, the chemical nature of the catalytic site, and the structural evolutionary relationship between the different proteases.24 For practical reasons the classification of proteases based on the catalytic site appears quite useful since protein PIs, which act in several cases as pseudo-substrates on their target proteases, may be classified according to the same scheme. Based on this approach proteases are categorized into four main groups: serine proteases, which possess a catalytic triad composed an aspartate, a serine and a histidine; cysteine proteases, which possess a catalytic triad composed of a cysteine, a histidine and an aspartate; aspartate proteases, which rely on two aspartate residues for activity; and metalloproteases, which rely on the presence of a metallic ion, usually Zn2+. Protein PIs, which as a general rule show affinity for proteases from one of these groups, may be classified into four groups: serine PIs, cysteine PIs, aspartate PIs and metallo-PIs, thereby providing an easy way to rapidly determine candidate PIs potentially useful in the control of specific groups of proteases. presumably due to a drastic alteration of their protein digestive functions, but alternative or parallel effects cannot be excluded. Proteases excreted by fungi or parasitic nematodes, for instance may be associated not only with the uptake of dietary amino acids, but also with specific pathogenic processes like the activation of zymogens, the penetration of host tissues or the irreversible inactivation of certain defense-related proteins in the host cells. Similarly, inhibiting the midgut proteases of herbivorous insects or the

proteases released by microbial pathogens in the surrounding environment may not only alter dietary protein hydrolysis, but also protect the integrity of host defense proteins by preventing their degradation.36 While major progress has been made in the last several years regarding our knowledge on the roles of protein PIs in plant defense, their induction in plant tissues following predation or infection, and the complex physiological processes triggered in target pests by the inhibition of their extracellular proteases,4,5,35,37 our current

Protease/Inhibitor Interactions in Plant-Pest Systems

understanding of protease/inhibitor interactions in plant-pest systems still appears limited. Extensive studies remain necessary, for instance to understand basic processes like the sequential degradation of dietary proteins in the midgut of herbivorous insects, or the degradation of cell wall proteins by pathogenic fungi or bacteria when they infect plant tissues. While many important questions remain for a correct assessment of protease/PI interactions, however recent progress in the field suggests that several successful control strategies based on recombinant PIs should be available in the near future. The processes involved in the hydrolysis of plant proteins by pest extracellular proteases have not yet been elucidated in detail, but one can already assume that a PI-based control strategy will be effective if compensatory and PI- degradation processes in the target organism are avoided.35,36,38 From a practical point of view, recombinant PIs could be effective, for instance if they strongly inhibit the target proteases,31,36,39-42 and if the number of insensitive proteases is kept to a minimum.36,38 Several successful strategies have been devised recently to improve the efficiency of protein PIs (see Chapters 10-12), including the improvement of their binding capacity by site-directssis,40,43 the design of hybrid PIs with an extended inhibitory spectrum,44,45 the isolation of effective inhibitor variants by phage display,46,47 the identification from plant tissues of stress-induced PIs with a broad inhibitory spectrum,48,49 the use of insect PIs exhibiting high affinity for their cognate protease,50 and the use of protease regulatory propeptides as a complement to the currently used protein PIs.34,51 When expressed in transgenic plants these PIs, tailored for the inhibition of specific protease systems, should contribute significantly to the development of effective PI-based control strategies, especially in those systems where complex protease systems are found.

1.4. Future Perspectives With the recent developments in plant molecular biotechnology, DNA sequences encoding a variety of useful defense proteins

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can now be easily integrated into the genome of plants to complement the effect of naturally-occurring defense proteins, and thus improve their ability to remain healthy and productive in commercial agricultural systems. 12 Transgenic plants expressing δ-endotoxins from the soil bacterium Bacillus thuringiensis (Bt toxins), for instance appear particularly useful for the control of various insect and nematode pests.52 Bt toxins interact with specific receptors in the digestive tract of these organisms, causing major physiological disorders and subsequent death. This antibiotic effect confers to Bt toxin-expressing plants a remarkable resistance status, making them already suitable for commercialization. Effective biocidal proteins like Bt toxins, however are single chemical compounds like any other pesticide, and the large-scale use of Bt toxin-expressing plants in the coming years could exert a strong selection pressure on the target pests and favor the buildup of resistant populations.53 Several recent studies have demonstrated the ability of insects to develop resistance to Bt toxins,54 and eventual resistance to other biocidal proteins expressed in transgenic plants appears plausible. To minimize the development of such resistance in pest populations and to ensure their effectiveness over a long period, transgenic plants expressing a single resistance factor should be seen as one component of more elaborated, multicomponent control systems, representing one in several means to control the pests targeted.53 In this context, recombinant PIs appear of particular interest. Unlike toxic proteins, PIs do not kill the target pests. In most cases the ingestion of high doses of PI causes growth delays and alters reproductive functions, thereby reducing the fitness of the target organism. In contrast with antibiotic compounds, recombinant PIs usually provide the plant with only partial resistance, making this control mean particularly suitable in control strategies based on the deployment of several control means (see Chapters 13-15). Herbivorous pests showing reduced fitness after ingestion of PIs could be more suscep-

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tible, for instance to natural predators implemented in the field as biological control agents. Moreover, the inhibitory effect of PIs could improve the efficiency of defense proteins like Bt toxins or like the plant’s own defense proteins by preventing their degradation by the target pest proteases.36 Several PI-expressing plants have been developed in the last ten years, leading in some cases to promising developments. Considering the rapid developments in the field, it also appears likely that it is just a question of time before highly effective PIs are identified or designed for the inhibition of various pest protease systems (see for instance ref. 55). At this point continued efforts will still be needed, however to thoroughly understand protease/ PI interactions in the systems assessed. As noted above, extending the range of proteases susceptible to the action of recombinant PIs should allow the development of efficient PI-expressing plants, but this approach could have some drawbacks in the field. Maximizing the inhibitory spectrum of PIs should help improve their effect against target organisms, but it will at the same time decrease their specificity. Whereas biocidal compounds such as Bt toxins are interesting because of their high specificity toward the target pest, PIs with an extended inhibitory range could eventually interfere with nontarget organisms in the ecosystem (see Chapter 8). Alternatively, blocking a key process in the complex cascade of reactions leading to the degradation of proteins in specific biological systems could be sufficient to elude compensatory or PI-hydrolytic processes. The challenge, then, will be to define an ‘inhibitory equilibrium’ in the target environment allowing to alter specific physiological functions in the pest without affecting nontarget organisms. While at present considerable effort is made to maximize the binding capacity of recombinant PIs—a step which is essential to establish the usefulness of recombinant PIs in pest control, the next step could rather consist to optimize their inhibitory effects in the ecosystem.

Recombinant Protease Inhibitors in Plants

Acknowledgments I thank Line Cantin for helpful comments on the manuscript. This work was supported by an operating grant from the Natural Science and Engineering Research Council of Canada.

References 1. Wolf DH. Proteases as biological regulators— introductory remarks. Experientia 1992; 48:117-118. 2. North MJ. Comparative biochemistry of the proteinases of eucaryotic microorganisms. Microbiol Rev 1982; 46:308-340. 3. Branquinha MH, Vermelho AB, Goldenberg S et al. Ubiquity of cysteine- and metalloproteinase activities in a wide range of trypanosomatids. J Euk Microbiol 1996; 43:131-135. 4. Ryan CA. Proteinase inhibitors in plants: Genes for improving defenses against insects and pathogens. Annu Rev Phytopathol 1990; 28:425-449. 5. Koiwa H, Bressan RA, Hasegawa PM. Regulation of protease inhibitors and plant defense. Trends Plant Sci 1997; 2:379-384. 6. Henskens YMC, Veerman ECI, Nieuw Amerongen AV. Cystatins in health and disease. Biol Chem 1996; 377:71-86. 7. Vilcinskas A, Wedde M. Inhibition of Beauvaria bassiana proteases and fungal development by inducible protease inhibitors in the haemolymph of Galleria mellonella larvae. Biocontrol Sci Technol 1997; 7:591-601. 8. Dieguez-Uribeondo J, Cerenius L. The inhibition of extracellular proteinases from Aphanomyces spp. by three different proteinase inhibitors from crayfish blood. Mycol Res 1998; 102:820-824. 9. Polanowski A, Blum MS, Whitman DW et al. Proteinase inhibitors in the nonvenomous defensive secretion of grasshoppers: Antiproteolytic range and possible significance. Comp Biochem Physiol B 1997; 117:525-529. 10. Hilder VA, Gatehouse AMR, Boulter D. Proteinase inhibitor approach. In: Kung S-D, Wu R, eds. Transgenic Plants: Engineering and utilization, Vol. 1. New York: Academic Press, 1993:317-338. 11. Atkinson HJ, Urwin PE, Hansen PE et al. Designs for engineered resistance to rootparasitic nematodes. Trends Biotechnol 1995; 13:369-374. 12. Schuler TH, Poppy GM, Kerry BR et al. Insect-resistant transgenic plants. Trends Biotechnol 1998; 16:168-175.

Protease/Inhibitor Interactions in Plant-Pest Systems 13. Gatehouse AMR, Gatehouse JA. Identifying proteins with insecticidal activity: Use of encoding genes to produce insect-resistant transgenic crops. Pestic Sci 1998; 52:165-175. 14. Jouanin L, Bonadé-Bottino M, Girard C et al. Transgenic plants for insect resistance. Plant Sci 1998; 131:1-11. 15. Michaud D, Vrain TC. Expression of recombinant proteinase inhibitors in plants. In: Cunningham C, Porter AJR, eds. Recombinant proteins from plants: Production and isolation of clinically useful compounds. Totowa NJ:Humana Press, 1998:49-64. 16. Burgess EPJ, Stevens PS, Keen GK et al. Effects of protease inhibitors and dietary protein level on the black field cricket Teleogryllus commodus. Entomol Exp Appl 1991; 61:123-130. 17. Chen M-S, Johnson B, Wen L et al. Rice cystatin: Bacterial expression, purification, cysteine proteinase inhibitory activity, and insect growth suppressing activity of a truncated form of the protein. Protein Expr Purif 1992; 3:41-49. 18. Oppert B, Morgan TD, Culbertson C et al. Dietary mixtures of cysteine and serine proteinase inhibitors exhibit synergistic toxicity toward the red flour beetle, Tribolium castaneum. Comp Biochem Physiol C 1993; 105:379-385. 19. Michaud D, Bernier-Vadnais N, Overney S et al. Constitutive expression of digestive cysteine proteinase forms during development of the Colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae). Insect Biochem Mol Biol 1995; 25:1041-1048. 20. Markwick NP, Reid SJ, Laing WA et al. Effects of dietary protein and protease inhibitors on codling moth (Lepidoptera: Tortricidae). J Econ Entomol 1995; 88:33-39. 21. Lorito M, Broadway RM, Hayes C et al. Proteinase inhibitors from plants as a novel class of fungicides. Mol Plant Microbe Interact 1994; 7:525-527. 22. Dunaevskii Y, Pavlyukova E, Belyakova G et al. Anionic trypsin inhibitors from dry buckwheat seeds: Isolation, specificity of action, and effect on growth of micromycetes. Biochemistry (Moscow) 1994; 59:739-743. 23. Joshi B, Sainani M, Bastawade K et al. Cysteine protease inhibitor from pearl millet: A new class of antifungal protein. Biochem Biophys Res Commun 1998; 246:382-387. 24. Barrett AJ. Classification of peptidases. Methods Enzymol 1994; 244:1-15. 25. Michaud D, Nguyen-Quoc B, Yelle S. Selective inhibition of Colorado potato beetle cathepsin H by oryzacystatins I and II. FEBS Lett 1993; 331:173-176.

7 26. Broadway RM. Are insects resistant to plant proteinase inhibitors? J Insect Physiol 1995; 41:107-116. 27. Jongsma MA, Bakker PL, Peters J et al. Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition. Proc Natl Acad Sci USA 1995; 92:8041-8045. 28. Bolter CJ, Jongsma MA. Colorado potato beetles adapt to proteinase inhibitors induced in potato leaves by methyl jasmonate. J Insect Physiol 1995; 41:1071-1078. 29. Michaud D, Cantin L, Vrain TC. Carboxyterminal truncation of oryzacystatin II by oryzacystatin-insensitive insect digestive proteinases. Arch Biochem Biophys 1995; 322:469-474. 30. Michaud D, Nguyen-Quoc B, Vrain TC et al. Response of digestive cysteine proteinases from the Colorado potato beetle (Leptinotarsa decemlineata) and the black vine weevil (Otiorynchus sulcatus) to a recombinant form of human stefin A. Arch Insect Biochem Physiol 1996; 31:451-464. 31. Michaud D, Cantin L, Raworth DA et al. Assessing the stability of cystatin/cysteine proteinase complexes using mildly-denaturing gelatin/polyacrylamide gel electrophoresis. Electrophoresis 1996; 17:74-79. 32. Broadway RM. Dietary proteinase inhibitors alter complement of midgut proteases. Arch Insect Biochem Physiol 1996; 32:39-53. 33. Broadway RM. Dietary regulation of serine proteinases that are resistant to serine proteinase inhibitors. J Insect Physiol 1997; 43:855-874. 34. Visal S, Taylor MAJ, Michaud D. The proregion of papaya proteinase IV inhibits Colorado potato beetle digestive cysteine proteinases. FEBS Lett 1998; 434:401-405. 35. Jongsma MA, Bolter C. The adaptation of insects to plant protease inhibitors. J Insect Physiol 1997; 43:885-895. 36. Michaud D. Avoiding protease-mediated resistance in herbivorous pests. Trends Biotechnol 1997; 15:4-6. 37. Bown DP, Wilkinson HS, Gatehouse JA. Differentially regulated inhibitor-sensitive and insensitive protease genes from the phytophagous insect pest, Helicoverpa armigera, are members of complex multigene families. Insect Biochem Mol Biol 1997; 27:625-638. 38. Jongsma MA, Stiekema WJ, Bosch D. Combatting inhibitor-insensitive proteases of insect pests. Trends Biotechnol 1996; 14:331-333. 39. Christeller JT, Shaw BD. The interaction of a range of serine proteinase inhibitors with bovine trypsin and Costelytra zealandica trypsin. Insect Biochem 1989; 19:233-241.

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8 40. Urwin PE, Atkinson HJ, Waller DA et al. Engineered oryzacystatin-I expressed in transgenic hairy roots confers resistance to Globodera pallida. Plant J 1995; 8:121-131. 41. Michaud D, Cantin L, Bonade Bottino M et al. Identification of stable plant cystatin/nematode proteinase complexes using mildlydenaturing gelatin/polyacrylamide gel electrophoresis. Electrophoresis 1996; 17:1373-1379. 42. Girard C, Bonadé-Bottino M, Pham-Delegue M-H et al. Two strains of cabbage seed weevil (Coleoptera: Curculionidae) exhibit differential susceptibility to a transgenic oilseed rape expressin oryzacystatin I. J Insect Physiol 1998; 44:569-577. 43. Szardenings M, Vasel B, Hecht H-J et al. Highly effective protease inhibitors from variants of human pancreatic secretory trypsin inhibitor (hPSTI): An assessment of 3-D structure-based protein design. Protein Eng 1995; 8:45-52. 44. Urwin PE, McPherson MJ, Atkinson HJ. Enhanced transgenic plant resistance to nematodes by dual proteinase inhibitor constructs. Planta 1998; 204:472-479. 45. Le-Nguyen D, Mattras H, Coletti-Previero MA et al. Design and chemical synthesis of a 32 residues chimeric microprotein inhibiting both trypsin and carboxypeptidase A. Biochem Biophys Res Commun 1989; 162:1426-1430. 46. Jongsma MA, Bakker PL, Stiekema WJ et al. Phage display of a double-headed proteinase inhibitor: Analysis of the binding domains of potato proteinase inhibitor II. Mol Breeding 1995; 1:181-191. 47. Koiwa K, Shade RE, Zhu-Salzman K et al. Phage display selection can differentiate insecticidal activity of soybean cystatins. Plant J 1998; 14:371-379.

48. Zhao Y, Botella MA, Subramanian L et al. Two wound-inducible soybean cysteine proteinase inhibitors have greater insect digestive s inhibitory activities than a constitutive homolog. Plant Physiol 1996; 111:1299-1306. 49. Visal S, Michaud D, Yelle S. Identification of a gamma-linolenic acid-induced tomato leaf cystatin-like protein with potential for biocontrol of the phytophagous pest Colorado beetle. Plant Physiol 1996; 111s:40. 50. Thomas JC, Adams DG, Keppenne VD et al. Manduca sexta encoded protease inhibitors expressed in Nicotiana tabacum provide protection against insects. Plant Physiol Biochem 1995; 33:611-614. 51. Taylor MAJ, Lee MJ. Trypsin isolated from the midgut of the tobacco budworm, Manduca sexta, is inhibited by synthetic pro-peptides in vitro. Biochem Biophys Res Commun 1997; 235:606-609. 52. Peferoen M. Progress and prospects for field use of Bt genes in crops. Trends Biotechnol 1997; 15:173-177. 53. Brattsen LB. Bioengineering of crop plants and resistant biotype evolution in insects: Counteracting coevolution. Arch Insect Biochem Physiol 1991; 17:253-267. 54. Tabashnik BE. Evolution of resistance to Bacillus thuringiensis. Annu Rev Entomol 1994; 39:47-79. 55. Gruden K, Strukelj B, Popovic T et al. The cysteine protease activity of Colorado potato beetle (Leptinotarsa decemlineata Say) guts, which is insensitive to potato protease inhibitors, is inhibited by thyroglobulin type-1 domain inhibitors. Insect Biochem Mol Biol 1998; 28:549-560.

CHAPTER 2

Control of Phytophagous Insect Pests Using Serine Proteinase Inhibitors John A. Gatehouse, Angharad M.R. Gatehouse and David P. Bown

2.1. Serine PIs and Their Role in Plant Protection 2.1.1. Introduction

T

he role of proteinase inhibitors (PIs) in plant defense against predators and pathogens is now well established. Although diverse endogenous functions for these proteins have been proposed, ranging from regulators of endogenous proteinases to storage proteins, evidence for many of the roles is partial, or confined to isolated examples. On the other hand, many PIs have been shown to act as defensive compounds by direct assay or by expression in transgenic plants, and a body of evidence consistent with their role in plant defense has been accumulated. The case for serine PIs as defensive compounds against predators is particularly clear-cut, since the major proteinases present in plants, used for processes such as protein mobilization in storage tissues, contain a cysteine residue as the catalytically active nucleophile in the enzyme active site. Serine proteinases are apparently not used by plants in processes involving large-scale protein digestion, and thus the presence of significant quantities of inhibitors with specificity towards these enzymes in plants cannot be for the purposes of regulating endogenous proteinase activity. In contrast, a major role for serine PIs in animals seems to be to block

the activity of endogenous proteinases in tissues where this activity would be harmful, as is the case with the pancreatic trypsin inhibitors found in mammals. The presence of significant amounts of serine PIs in plant tissues therefore suggests not an endogenous role, either protective or regulatory, but instead suggests that the targets of these inhibitors are the digestive proteinases of phytophagous animals. Further evidence for a role of serine PIs in plant defense is provided by considering the sites of synthesis and accumulation of these proteins. They are normally accumulated in storage tissues, both in seeds and vegetative storage tissues such as potato tubers, and can reach concentrations as high as 2% of total protein. Since plant survival depends on the protection of storage tissues against predators, this pattern of accumulation supports the defensive role. There is little evidence that PIs accumulated in these tissues function as a storage reserve by being broken down on germination or sprouting. Direct evidence for a defensive role is shown by the synthesis of serine PIs in wounded tissue, as described below.

Recombinant Protease Inhibitors in Plants, edited by Dominique Michaud. ©2000 Eurekah.com.

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2.1.2. Inhibitors of Serine Proteinases 2.1.2.1. Inhibitors from Plant Sources The serine proteinases trypsin, chymotrypsin and elastase, which belong to a common protein superfamily, are responsible for the initial digestion of proteins in the gut of most higher animals. In vivo they are used to cleave long, essentially intact polypeptide chains into short peptides which are then acted on by exopeptidases to generate amino acids, which are the end-point of protein digestion. The three types of digestive serine proteinases are distinguished by their specificity, trypsin specifically cleaving C-terminal to residues carrying a basic side chain (lys, arg), chymotrypsin showing a preference for cleaving C-terminal to residues carrying a large hydrophobic side chain (phe, tyr, leu), and elastase showing a preference for cleaving C-terminal to residues carrying a small neutral side chain (ala, gly). Inhibitors of these serine proteinases have been described in many plant species, and are probably universal throughout the plant kingdom, with inhibitors of trypsin by far the most common type. At least part of this bias can be accounted for by the fact that (mammalian) trypsin is readily available, is the easiest of the proteinases to assay using synthetic substrates, and thus is used in screening procedures. At least seven distinct families of serine PIs have been described in plants. These proteins have a common mechanism of action: the inhibitor binds to the active site on the enzyme to form a complex with a very low dissociation constant (10-7 to 10-14 M at neutral pH values), thus effectively blocking the active site. A binding loop on the inhibitor, usually “locked” into conformation by a disulphide bond, projects from the surface of the molecule and contains a peptide bond (“reactive site”) cleavable by the enzyme. This peptide bond may be cleaved in the enzymeinhibitor complex, but cleavage does not affect the interaction, so that a hydrolyzed inhibitor molecule is bound just as well as an unhydrolyzed one. The inhibitor thus directly mimics a normal substrate for the

Recombinant Protease Inhibitors in Plants

enzyme, but does not allow the normal enzyme mechanism of peptide bond cleavage to proceed to completion (dissociation of the product). Specificity of the inhibitor-enzyme interaction is primarily determined by the specificity of proteolysis determined by the enzyme. For example, trypsin cleaves C-terminally to a basic amino acid residue, so the reactive site in a trypsin inhibitor will comprise an arg-X or lys-X dipeptide. However, the strength of interaction, and thus the effectiveness of the inhibitor is not solely determined by the reactive site, since other residues in the reactive site region of the inhibitor are also important in stabilizing the enzyme-inhibitor complex. Extensive structurefunction studies have been carried out on certain inhibitors. The most widely studied types of serine PIs in plants are both typified by inhibitors isolated from soybean seeds, and named after their discoverers: the Kunitz and BowmanBirk families of inhibitors. Kunitz inhibitors are typically monomeric proteins containing a polypeptide of approximately 190 amino acid residues, with two intra-chain disulphide bridges. Each molecule contains a single binding site which interacts strongly with the proteinase against which the inhibitor is directed (usually trypsin or chymotrypsin). Generally the distribution of proteins of this type seems to be sporadic across a range of plant families (legumes and cereals), with certain plant species such as winged bean (Psophocarpus tetragonolobus) accumulating Kunitz-type inhibitors in their seeds to a significant proportion of total protein, whereas many others contain only small amounts or no detectable proteins of this type. The Kunitz inhibitors themselves belong to a superfamily which includes proteins such as the sweet-tasting protein thaumatin and proteins induced by pathogens (PR proteins), which have sequence homology, but are functionally distinct since they do not inhibit serine proteases. Bowman-Birk inhibitors are common in seeds of legume species, and are found in other families (such as cereals) also. These PIs are proteins based on a polypeptide of 70-80

Control of Phytophagous Insect Pests Using Serine Proteinase Inhibitors

amino acids, which can form oligomers. The basic polypeptide unit contains a high proportion of cysteine residues, and forms multiple intra-chain disulphide bridges (typically 7 per monomer), leading to a very tightly folded and rigid conformation. The monomeric unit contains two binding loops with reactive sites, and can thus inhibit two molecules of proteinase per molecule of inhibitor. The two binding loops can have similar or different inhibitory specificities. These proteins can be quite abundant in seeds, making up as much as 1% of total seed protein. A further family of serine PIs is found in seeds of barley and other cereals, and is termed the barley trypsin inhibitor family. These inhibitors are proteins of 11-14,000 Mr which contain a single enzyme inhibitory site. Like Bowman-Birk inhibitors, they have a high cysteine content. Potato and other members of the Solanaceae contain two families of PIs, which are described as the potato inhibitors I and II families (PI-I, PI-II), both being originally characterized from potato tubers, where they are accumulated. These are again based on relatively small polypeptides. PI-I is an oligomeric protein containing subunits of approx. Mr 8,000; the major form is a tetramer, with an indicated Mr of 39,000.1 PI-I has a low cysteine content, and, unusually for serine PIs, disulphide bonds are not essential for activity. PI-II is a dimer, with a polypeptide size of 12 kDa; it has two reactive sites and five intra-chain disulphide bonds per monomer.2 Finally, members of the family Cucurbitaceae contain a further group of PIs in their seeds, noteworthy for their very small size (29-32 amino acid residues); these are again relatively cysteine-rich. It seems likely that further families of PIs will be identified as more plant sources are screened for inhibitory activity, and the proteins responsible are purified and characterized. PIs in plants appear to form an example of what has been termed “convergent evolution,” where proteins with similar functions have arisen from different precursors, but share a similar functional mechanism. Further details of these inhibitors can be found in review articles 3-5.

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2.1.2.2. Inhibitors from Other Sources Inhibitors of serine proteinases are also found in animal and bacterial sources, and are often involved in the regulation of endogenous proteinases. For example, the pancreas in mammals produces large quantities of a trypsin inhibitor which prevents activation of the inactive zymogen precursors of digestive enzymes, synthesized in the pancreas, until they have passed into the gut. Insect haemolymph also contains significant amounts of PIs, which belong to a superfamily of proteins designated serpins, and may regulate the activities of proteinases involved in moulting although they also inhibit digestive proteinases. A summary of these inhibitor families is provided in a recent review by Reeck and coworkers.3

2.2. Effects of Serine PIs on Insect Digestive Proteolysis 2.2.1. Introduction Four main classes of peptidases have been distinguished on the basis of the catalytically active amino acid residue or other functional group they contain: serine proteases, cysteine proteases, aspartyl proteases and metalloproteases. All four classes have been identified as playing roles in protein digestion in different species within the insect kingdom,6 and inhibitors of all four classes have been identified in different plant species. The roles these different protease types play can be divided into primary digestion (by endopeptidases, or proteinases) and secondary digestion (by exopeptidases). Metalloproteases, such as carboxypeptidases and Aminoaeptidases, are employed as exopeptidases, and thus are usually not important in the initial stages of digestion. The other classes of enzyme all function as endopeptidases. By analogy with higher animals, and on the basis of some early characterization work in species such as locust, it was assumed that serine proteinases were the major digestive enzymes in all insects. However, many coleopteran species and some Hemiptera contain both cysteine and aspartyl proteinases,

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with little or no detectable serine proteinase activity. It is tempting to view this switch in the nature of the major digestive enzymes as an adaptation by the insect to a diet of plant tissues rich in serine PIs,6 and the fact that many phytophagous Coleoptera, which feed on seeds or other storage tissues, contain cysteine proteinases supports the hypothesis. However, cysteine proteinases are also found as major digestive enzymes in carnivorous coleopterans such as ladybirds7 and blood- sucking hemipterans such as Rhodnius prolixus,8 which are not directly exposed to plant serine PIs, and thus the causal connection cannot be proved. Even in those insects which do use serine proteinases as their major digestive enzymes, the insect enzymes differ from their mammalian counterparts in some biochemical properties. The best-studied systems are the digestive enzymes of lepidopteran larvae, particularly some of the more significant crop pests. In these insects, the gut pH is highly alkaline9 and serine proteases have correspondingly high pH optima (typically 10.5-11, as compared to approx. 9 for the mammalian enzymes). The use of substrates and chemical inhibitors specific for the different proteinases has allowed the identification of enzymes with activities typical of trypsins, chymotrypsins and elastases in lepidopteran larvae,10 although their substrate specificities and sensitivity towards PIs may show differences from the mammalian enzymes. For example, the chymotrypsins in several noctuid larvae show very low activity towards chymotrypsin substrates or inhibitors which contain only a single amino acid residue (e.g., N-benzoyl-tyrosine p-nitroanilide, BTpNA; N-tosyl- phenylalanine chloroketone, TPCK), but readily hydrolyze substrates containing short peptides such as succinylalaninealanine-proline-phenylalanine-p-nitroanilide (SAAPFpNA), and are strongly inhibited by the specific peptide inhibitor chymostatin.10 The relative contributions of the trypsin, chymotrypsin and elastase activities to overall protein digestion vary from one species to another, with trypsin and chymotrypsin being normally the major contributors (Fig. 2.1).

Recombinant Protease Inhibitors in Plants

2.2.2. Effects of Purified Serine PIs on Insects and Insect Digestion Many studies have been carried out, both in vivo and in vitro, to determine the effects of PIs on insect survival and development. In general, assays carried out in vivo have used artificial diets, into which inhibitors can be incorporated at known concentration, as the basis for bioassays which follow insect development and survival. Relative to a control diet with no added inhibitor, an “effective” PI can be expected to lower insect survival, to decrease insect biomass, and to decrease the rate of insect development in terms of time taken to pass through defined stages of the life cycle (Fig. 2.2). PIs do not usually show toxic effects, which would produce high levels of insect mortality in a short period of assay. Instead, their effects are produced in the long term, and are thus termed antinutritional, antinutritive or antimetabolic rather than toxic. Unfortunately, some bioassays have been carried out with protein fractions of questionable purity. For example, in an early investigation reported by Lipke,11 it was shown that purified soybean Kunitz and Bowman-Birk trypsin inhibitors were not toxic to larvae of the flour beetle Tribolium confusum, but a partially purified protein fraction which inhibited proteolytic activity of both insect and mammalian gut extracts was toxic. The undefined nature of such fractions has led to questioning the role of PIs in the effects observed, although in the example given the failure of soybean Kunitz and Bowman-Birk inhibitors to affect Tribolium larvae is not surprising since these insects use cysteine proteinases for digestion.12 The inhibition of proteolysis observed was possibly due to an inhibitor of cysteine proteases also present in soybean, albeit at low level.13 In general, there can now be no doubt that PIs are antimetabolic to insects, but there is a specificity in the effect observed, both from inhibitor to inhibitor and from insect species to insect species. The effects of PIs on insect digestive enzymes can be measured directly, by means of enzyme assays carried out in vitro.14 Most workers have measured the effects of PIs on

Control of Phytophagous Insect Pests Using Serine Proteinase Inhibitors

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Fig. 2.1. Proportion of proteolytic activity (azoalbumin hydrolysis) attributable to different serine proteinases in gut extracts from two lepidopteran larvae, shown by inhibition of proteolysis with chemical inhibitors specific to different proteinase activities. the proteases present in insect gut contents, which usually contain mixtures of digestive enzymes. However a few assays have employed purified enzymes. Provided that the synthetic substrates used are specific for particular enzymes, the use of gut contents for enzyme assays does not cause problems in interpretation of data, and allows parameters measuring the effectiveness of different inhibitors (e.g., I50, the concentration of inhibitor to cause a 50% decrease in enzyme activity, or Ki, the

Michaelis-Menten Inhibition constant) to be determined. The use of gut contents is also advantageous when the effects of inhibitors on total proteolytic activity in the insect gut are measured. However, for detailed study of the parameters of interaction between inhibitors and proteolytic enzymes, purified insect enzymes are necessary. In vitro inhibition assays have shown that plant PIs are as effective against insect digestive proteases as against those of higher animals.

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Recombinant Protease Inhibitors in Plants

Fig. 2.2. Typical effects of a serine PI (soybean Kunitz trypsin inhibitor, SKTI) on survival, growth and development of lepidopteran larvae (Lacanobia oleracea, tomato moth) when delivered in artificial diet (2.0% of total s). Slower growth is shown by the inhibitor causing a significant and progressive decrease in mean larval weight, and retarded development by slower progression through the larval instars, as shown by the instar distributions at three time points. See Gatehouse et al56 for complementary data. However, the strengths of interaction (i.e., the effectiveness of inhibition) for a given inhibitor with insect and mammalian enzymes of similar substrate specificities differ. Further, when different insect species are compared, a given inhibitor will be found to be more effective at inhibiting proteolysis in some species than others. Serine PIs in many phytophagous insect species will inhibit the enzymes of primary protein digestion, and might be thought to be antimetabolic simply by virtue of preventing efficient utilization of protein in the diet. In support of this idea, supplementation of

limiting amino acids in the diet can overcome the antimetabolic effects of inhibitors.15 Prevention of efficient recycling of the amino acids used to synthesize proteinases may be a further significant factor in the antimetabolic effects observed, since the digestive proteinases in animals are normally subject to autodigestion, and this can be blocked by PIs.

2.3. The Wounding Response in Plant Defense The clearest evidence for the role of serine PIs in the defense of plants against insect pests is the induced synthesis of these

Control of Phytophagous Insect Pests Using Serine Proteinase Inhibitors

proteins that occurs when many plant species are wounded, which can be caused by insect feeding, or mimicked by mechanical damage. The wound response in plants has been extensively investigated over recent years. Wounding has been shown to result in a variety of changes in the physiological state of the tissue, and can either result in a local reaction when it is restricted to a defined region in the near vicinity of the original wound such as in the case of the production of phytoalexins, or a systemic reaction, when the response occurs in tissues some distance from the wound site. One of the best studied cases of a systemic reaction induced in higher plants was the demonstration that wounding of the leaves of either potato or tomato plants by adult Colorado potato beetles or their larvae induced a rapid accumulation of serine PIs.16 This accumulation was not confined to just wounded leaves, but was also found to be present in leaves which had not been attacked. Mechanical damage was also found to cause an accumulation of these inducible PIs.17 Interestingly, it has recently been shown that the timing of transcript accumulation of several wound-induced genes in insectdamaged leaves is different to that in mechanically damaged leaves, with transcripts for the inhibitor PI-II accumulating more rapidly in potato leaves which had been damaged by insects than those damaged mechanically. It would appear that insect regurgitant, in this case from Manduca sexta larvae, causes the transcript accumulation profiles to shift to parallel those in insect damaged tissue. On the basis of their findings, Korth and Dixon18 suggested the presence of a heat-stable, insect derived elicitor which functions to induce the rapid accumulation of transcripts that may be involved in plant defense against herbivores. A factor, or wound hormone, called the PI-inducing factor (PIIF) was found to be released from the damaged leaves and transported to other leaves within 2-3 hours after wounding where it initiates synthesis and accumulation of the two potato serine PIs, PI-I and PI-II.17 Transport appears to take place in the phloem and is directed predomi-

15

nantly upwards to younger leaves,19 although evidence for hydraulic signals in the xylem and electrical signals have also been presented. The levels of PIIF released in response to wounding depends upon both the severity and location of the wound. Subsequent work revealed that the PIIF was a highly methylated polysaccharide containing galacturonic acid, rhamnose, galactose, arabinose and fucose, and that it was probably a fragment of the plant cell wall, at least in tomato.20 Within two to three days after attack the inhibitors can account for over 10% of the soluble proteins in leaves throughout the plant, where they can remain for long periods of time stored in the central vacuoles of the cells.21,22 The accumulation requires light and is temperature-dependent. These wound-induced inhibitors have been well characterized. Both PI-I and PI-II are potent inhibitors of chymotrypsin and subtilisin, and both inhibit trypsin, but less strongly. More recently, a wound-induced trypsin inhibitor has been isolated and characterized from alfalfa leaves. This particular inhibitor was identified as a member of the Bowman-Birk inhibitor family.23 Following these initial reports, genes encoding the wound-inducible inhibitors from both potato24 and tomato25 have been isolated and characterized. Transformation of tobacco plants with a gene encoding potato PI-II resulted in a systemic induction of the transgene expression after wounding, 26 showing that the signal inducing PI gene expression was similar in the two species. Several plant-derived chemicals, including methyl jasmonate, jasmonic acid4 and an 18-amino acid polypeptide called systemin were found to regulate the expression of wound-inducible PI genes. Growth regulators such as abscisic acid and auxin were similarly shown to regulate expression of these genes. A currently accepted model for the expression of wound-inducible PIs is that systemin is released by wounding and activates a membrane-derived lipid signal. This signal is thought to be linolenic acid, which is subsequently converted to jasmonic acid4 via the octadecanoid pathway.27 Further informa-

16

tion can be found in a recent review by Bergey et al.28 A signal cascade then leads to the activation of jasmonate-responsive genes (jrgs), and their products are responsible for the physiological response. The involvement of systemin in the wound response and subsequent accumulation of wound-inducible PIs has been elegantly demonstrated by exposing transgenic tomato plants expressing an antisense prosystemin gene to lepidopteran larvae. The plants containing the antisense construct were significantly more attacked than the corresponding control plants, with the insects on the transgenic plants being significantly larger, due to suppression of the normal wounding response.29 An alternative model for jasmonate signalling in barley has been put forward recently, whereby there are several distinct signal-transduction pathways. Contrary to what occurs in tomato and potato, in barley the abscisic acid signal is not mediated through a jasmonate signal cascade, but is independent of it. In the model put forward by Lobler and Lee,30 induction of jrgs 9 (a “wounding-induced” gene) is caused by the presence of products resulting from activation of expression of gene families, some of which are individually induced by either abscisic acid or jasmonate. Jasmonate signalling is mediated through two pathways, one sensing extracellular jasmonate and the other sensing intracellular jasmonate. It has also been suggested that two distinct signal transduction pathways that can distinguish between insect damage and abiotic damage may be involved in the wound-induced responses in potato.18

2.4. “First-Phase” Use of Foreign Serine PI Genes Although the possibility that plant PIs were insecticidal compounds had been suggested as early as 1947,31 a clear demonstration that these proteins could be significant factors in protecting plants against insect attack was not put forward until 1972, when Ryan and coworkers16 demonstrated their accumulation in the leaves of certain species of solanaceous plants as a result of wounding

Recombinant Protease Inhibitors in Plants

(see above). These authors suggested that insect damage might act as a trigger for the synthesis of PIs, which would then act as defensive proteins by blocking insect digestion, leading to starvation and death. Evidence that this protective role could be observed in plant tissues which had accumulated PIs was first provided by our group,32 after we observed a correlation between high levels of trypsin inhibitor and resistance to a seed storage pest, the bruchid Callosobruchus maculatus, in seeds of cowpea (Vigna unguiculata), and showed that addition of the cowpea PI to a semiartificial diet inhibited development and decreased survival of larvae of this insect. Although subsequent work has shown that other factors are also involved in resistance of cowpea varieties to the seed weevil,33 our initial finding supported the hypothesis put forward by Ryan and colleagues, and opened the way for the subsequent exploitation of such compounds in crop protection by genetic manipulation.

2.4.1. Identification of Antimetabolic PIs In order to identify suitable PIs and their encoding genes for transfer to plants, systematic studies of a wide range of inhibitors have been carried in an attempt to find a kinetic parameter which could be useful in predicting the potential of any inhibitor to act as a resistance factor to a given pest. The dissociation constant of the enzyme/inhibitor complex was suggested as such a parameter (see Chapter 10, this volume).34 Although this strategy has been successfully adopted in specific cases, there are in fact limitations on relying solely upon information obtained from in vitro tests, as will be discussed in detail elsewhere (see Section 2.5). To this end, testing the inhibitor in artificial diet against the target insects is more reliable, although this too has its limitations, as was found to be the case for SKTI when tested against the tomato moth Lacanobia oleracea in artificial diet and on SKTI-expressing transgenic potato plants (see Section 2.5).

Control of Phytophagous Insect Pests Using Serine Proteinase Inhibitors

2.4.2. Insect-Resistant Transgenic Plants Expressing Serine PIs The first gene of plant origin to be transferred to another plant species to produce enhanced insect resistance was isolated from cowpea, and encoded a Bowman-Birk type serine PI with two inhibitory sites active against bovine trypsin (CpTI). 35 We produced a full-length cDNA clone of CpTI, and placed its coding sequence under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter in the final construct prepared for transfer to plants. Transgenic tobacco plants were produced by a standard Agrobacterium tumefaciensmediated transformation protocol, using a binary vector system. Transformants were screened for CpTI expression, which showed that many of the resulting plants expressed CpTI at levels greater than 0.1% of total soluble protein, a general observation for expression of proteins of this type in transgenic plants. Plants expressing CpTI at the highest levels (approximately 1% of total soluble protein) were clonally propagated, and used for insect bioassays. Initially bioassays were carried out using first-instar larvae of the tobacco budworm, Heliothis virescens, a pest of major economic importance attacking tobacco, cotton and maize. With these clonal plants and subsequent generations derived from their self-set seed, the CpTI-expressing plants showed reduced damage by up to 50% compared to the control plants, and reduced insect survival and biomass, again by as much as 50%. The antimetabolic effects of CpTI expressed in transgenic tobacco have also been observed with other lepidopteran pests including H. zea, Spodoptera littoralis and Manduca sexta. Subsequent trials carried out in California showed that the expression of CpTI in tobacco afforded significant protection in the field against H. zea.36 Following on from the study using tobacco as a model system, the gene encoding CpTI has been expressed in a range of different crops (Table 2.1). Constitutive expression of CpTI in potato showed significant antimetabolic effects on larvae of the tomato moth (Lacanobia oleracea), with

17

mean larval weight being decreased by nearly 50% in plants expressing CpTI at approx. 0.5% of total soluble protein in the leaves,37 although little effect on larval survival was observed and plant damage was also little reduced (Fig. 2.3). A better example is the use of this gene to protect strawbery against damage by vine weevil Otiorhynchus sulcatus, which is the major pest of strawbery in Europe, where it is endemic. The larvae damage root systems resulting in severe loss of yield or plant death, and recent reports suggest that damage is no longer confined to the root systems but is also occurring on the vegetative tissues. Since the withdrawal of persistent organochlorines, at least in parts of Europe, the control of adults and larvae has become difficult as few of the currently available products are effective under field conditions and no genetic resistance in commercial germplasm has been identified. The CpTI construct described above has been inserted into strawbery cultivars by Agrobacterium-mediated gene transfer for control of this pest.38,39 Results from two independent glasshouse trials showed that expression of the CpTI gene in transgenic strawbery plants was effective in protecting roots from vine weevil larval feeding.40 Control plants inoculated with vine weevil eggs had significantly less root mass than equivalent non-inoculated plants (see Fig. 2.4), and showed a substantial and significant reduction in weight compared with all but one of the transgenic lines; similar results have been obtained in recent field trials (Graham, pers. comm.). Although this inhibitor is at present being constitutively expressed, work is underway to identify a promoter which will not be active in the fruit of this plant. Similarly, encouraging results have also been obtained in rice where the constitutive expression of CpTI conferred significantly enhanced levels of resistance in the field towards two species of rice stem borer, Sesamia inferens and Chilo suppressalis.41 Despite CpTI being insecticidal against a wide spectrum of insect pests, mammalian feeding trials incorporating the purified protein at levels of 10% of total protein content in the diet showed no acute toxic effects,42 possibly

Recombinant Protease Inhibitors in Plants

18

Table 2.1. Transgenic plants expressing plant serine PIs with enhanced resistance towards insects Inhibitor

Plant

Pest

CpTIa

tobacco rice

Heliothis virescens(L) Chilo suppressalis (L) Sesamia inferens (L) Lacanobia oleracea (L) Otiorynchus sulcatus (C)

35 41 41 37 40

Manduca sexta (L) Chrysodeixis eriosoma (L) Chilo suppressalis (L) Sesamia inferens (L)

43 45 46 46

potato strawbery PI-II

tobacco rice

Reference

Barley TI

tobacco

AgrotisIpsilon (L) Spodoptera littoralis (L)

59 59

Na PI

tobacco

Helicoverpa punctigera (L)

60

aAbbreviations: CpTI, cowpea trypsin inhibitor; PI-II, potato wound inducible protease inhibitor II;

NA PI, NIcotiana alata multi-functional proteinase inhibitor; Barley TI, barley trypsin inhibitor; L, lepidoptera; C, Coleoptera

reflecting differences in the organization of insect and mammalian digestive systems. Other serine PI-encoding genes have also been tested as protective agents for crops. For example, the tomato PI-II gene (which encodes a trypsin inhibitor with some chymotrypsin inhibitory activity), when expressed in tobacco, was shown to confer insect resistance43 when expressed constitutively using the CaMV 35S promoter, but, interestingly, not when expressed with a wound-inducible promoter. The bioassays showed that the decrease in larval weight in insects reared on transgenic plants was roughly proportional to the level of PI-II being expressed. Several of the transgenic plants were shown to contain inhibitor levels over 200 µg/g tissue, which is within the range that is routinely induced by wounding leaves of either tomato or potato plants.44 However, tobacco plants expressing tomato PI-I at similar levels had no deleterious effects upon larval development, showing the specificity

of interactions between inhibitors and insect species. McManus et al45 obtained similar results with potato PI-II expressed in tobacco against the noctuid lepidopteran Chrysodeixis eriosoma, the green looper. The woundinducible potato PIs (PI-I and PI-II) have now been constitutively expressed in a range of crops where they have been shown to confer resistance (Table 2.1). As with CpTI, expression of PI-II in rice conferred significant levels of protection in the field towards rice stem borers.46 Serine PIs from animal sources have also been tested as potential protective agents. For example, the serpin-type serine PIs from insect haemolymph have been identified as possibly being targeted against insect proteinases, since they may play an endogenous regulatory role. Transgenic alfalfa, cotton and tobacco expressing insect haemolymph inhibitors have been produced,47-49 and some protection against insect pests has been observed. The most interesting result has been

Control of Phytophagous Insect Pests Using Serine Proteinase Inhibitors

19

Fig. 2.3. Effects of the serine PI cowpea trypsin inhibitor (CpTI) on larvae of tomato moth (Lacanobia oleracea) when expressed in transgenic potato plants at approx. 0.5% of total soluble leaf protein. Eight neonate larvae were placed onto each of 12 control and 19 transgenic plants, and allowed to develop for 22 days, when the most developed larvae were in the final instar. The transgenic plants showed marginally reduced survival (but not significant at p