Future Perspectives

Currently, two principal biotechnology strategies are proposed to engineer effective pest control in plants ectopic expression of pesticidal proteins, and induction or poten-tiation of the plant natural defensive response. At present, screening gene pools without taxo-nomic constraint can help identify novel insecticidal determinants, but in the future this approach will be augmented by directed in vitro molecular evolution (see Chapter 11, this volume). Phage display has been reported for the...

References

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 metallo-proteinase activities in a wide range of trypanosomatids. J Euk Microbiol 1996 4. Ryan CA. Proteinase inhibitors in plants Genes for improving defenses against insects and pathogens. Annu Rev...

Soichi Arai and Keiko Abe 31 Introduction

Cystatins were initially defined as proteins that specifically inhibit cysteine proteinases in general,1 but this understanding has recently undergone certain changes. Recent advances in enzymol-ogy revealed the existence of a variety of cysteine proteinases, resulting in their classification into several families, namely the papain, the calpain, the caspase and the asparagine-specific processing enzyme families. It is now understood that cystatins are specific inhibitors of enzymes belonging...

Conclusion

In summary, the use of recombinant PIs may represent an attractive way to protect plants from a variety of fungal, bacterial and viral pathogens. At this point the protease inhibitor interactions taking place in vivo between plant pathogens and their hosts remain poorly understood, but it clearly appears that proteolytic events may play a critical role in several key plant pathogenic processes. It also appears plausible, however that pathogens, like herbivorous insects possess the metabolic...

Insect Responses to Dietary Serine PIs

Despite successes in enhancing the resistance of transgenic plants towards insects by the expression of foreign PIs, other examples of this technology have given disappointing results, with little or no protection observed. For example Jongsma et al50 produced transgenic tobacco plants expressing the chymotrypsin trypsin-specific potato inhibitor II (PI-II) constitutively. In contrast to results reported above in Section 2.4, the growth of Spodoptera exigua larvae fed with detached leaves of...

Exogenous Targets

Along with their role in the regulation of endogenous proteolysis, plant cystatins may have exogenous targets originating from invaders such as viruses, bacteria, and insects. In recent years the potential of plant cystatins in inhibiting cysteine proteinases of insect origin has been evaluated and the importance of studying pest digestive proteases, in particular, has been stressed. Historically, a paper by Houseman43 is probably the first describing the existence of a cysteine proteinase...

Bacterial Proteases

Proteases are also secreted by plant pathogenic bacteria. Erwinia chrysanthemi, for instance secretes several extracellular enzymes to macerate plant tissues, including pectinases, cellulases, phospholipases, xylanases and proteases.15 Investigations on the pathogenicity of this organism, however suggested that the secreted proteases were not essential during the maceration process. Knockout strains were created by the deletion of certain genes, such that the relative contribution of the...

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 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...

Resistance to PIs

Finally a fourth factor to consider is the risk that the target organism can develop resistance to the inhibitor. As noted above (see Section 5.1.), recombinant PIs should not exert a selection pressure on the target pathogens as high as that exerted by toxic Fig. 5.2. Interactions between soybean Kunitz trypsin inhibitor (SBTI) and the extracellular proteases of the plant fUngal pathogen Pythium ultimum. The fUngus was grown at room temperature in a liquid medium containing 0.35 (w v) casein....

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 metallo-proteases. 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...

The Inhibition of Pest Extracellular Proteases

In reaction to these hydrolytic processes, several organisms also developed strategies to counteract the adverse effects of exogenous 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...

Extracellular Proteases in Plant Pathogens

In this perspective, the use of protease inhibitor (Pl)-encoding cDNA sequences may appear of particular interest in the development of plants which are partly resistant to pathogens. Because they can inhibit a wide variety of proteolytic enzymes, PIs were proposed as a tool to control pest and pathogenic organisms as diverse as herbivorous insects, parasitic nematodes and human microbial pathogens (see Chapters 2, 3, 4 and 17, this volume).4-6 Until now little is known about the potential of...

Risks Associated with Pollen and Honeybees

Figure 8.2 shows the changes in PI-expressing plants that may affect pollinating honeybees, the possible fate of pollen expressing PIs and the risks that may be associated with it. A similar scheme may be applied to other bee species, with some minor variations depending on the degree of sociality and obviously without the risks to humans from ingestion of pollen or honey. A transgenic plant expressing a PI may affect the bee indirectly via changes in flower attractiveness due to pleiotropic or...

The OD Pathway Regulates Host Plant Defense Against Herbivores

Inhibitors of the OD pathway were shown to block wound-induced PI synthesis and cellulysin p-glucosidase-induced volatile emission.32,50,51 Both Arabidopsis fad3-2 fad7-2 fad8 triple mutant plants, that are deficient in LA synthesis, or tomato defl mutant plants, that are defective for the OD pathway were susceptible to insect attack.14,52 Exogenously supplied JA restored resistance of these mutants. Transgenic plants over-expressing allene oxide synthase constitu-tively accumulated JA but not...

How Did Agric Fawe Lea Their Eggs

Engineering of crop plants with resistance to herbivores and pathogens An approach using primary gene products. Plant Mol Biol 1983 2 223-233. 2. Ryan CA. Proteinase Inhibitors. In Stumpf PK, Conn EE, eds. The Biochemistry of Plants A comprehensive treatise. New York Academic Press 1981 351-370. 3. Alfonso J, Ortego F, Sanchez-Monge R et al. Wheat and barley inhibitors active toward alpha-amylase and trypsin-like activities from Spodoptera frugiperda. J Chem Ecol...

Contents

Protease Inhibitor Interactions in Plant-Pest Systems A Brief 1 1.2. The Inhibition of Pest Extracellular 1.3. Plant Pest Control with PIs After the Hits the Misses 3 1.4. Future 2. Control of Phytophagous Insect Pests Using Serine Proteinase John A. Gatehouse, Angharad M.R. Gatehouse and David P. Bown 2.1. Serine PIs and Their Role in Plant 2.2. Effects of Serine PIs on Insect Digestive Proteolysis 11 2.3. The Wounding Response in Plant 14 2.4. First-Phase Use of Foreign Serine PI 2.5. Insect...

Thierry C Vrain 41 Introduction

Nematodes are the most successful multicellular form of animal life on earth. They colonize all trophic milieux with enough water to sustain their aquatic origin, the bottom of the seas, rivers and lakes, all continents, the deserts and the toundra included, usually in billions per acre. Nematodes are important pests of global agriculture, debilitating crops and farm animals. They cause many crippling human diseases such as river blindness or elephantiasis in tropical areas of Africa, and many...

Regeneration of Cystatin Expressing Transgenic Plants

The discussion above confirms the potential of incorporating PI-encoding genes in plants for insect control. To this end, Duan et al62 carried out experiments to introduce the gene of the serine-type inhibitor potato PI-II into several Japonica rice varieties, and regenerated a large number of transgenic rice plants. Wound-inducible expression of this gene driven by its own promoter resulted in high-level accumulation of the inhibitor protein in transgenic plants. The introduced gene was stably...

Research on Direct Effects of PIs on Bees

A number of studies have now been published describing the effects of PI ingestion on adult worker honeybees in terms of toxicity and changes in bee gut protease activity levels.14-17 There has also been some unpublished work carried out on the effects of PI ingestion on olfactory learning behavior, which is a significant component of foraging behavior in adult bees.18,19 However there have been no published studies of the direct effects of PIs on larvae or reproductive adult bees, or on their...

Multilevel Interactions The Ecosystem

A strategy recently proposed to improve the effect of recombinant PIs in pest control consists in using inhibitors with a broad spectrum of activity against the target pest proteases, thereby limiting the number of insensitive proteases available to the pest to hydrolyze the inhibitor or to compensate for inhibited proteolytic functions.3,10 In such a case an effective PI or combination of PIs could inhibit, for instance both P1a and P1b (see the model above), thus leaving to the pest only P2...

Oryzacystatin and Other Plant Cystatins

The rice grain contains a cysteine proteinase inhibitor (PI) that is protein in nature.7 Extensive studies carried out by our group confirmed that this protein inhibits papain in a stoichiometric manner and that, interestingly, it is very heat-stable, even under cooking conditions. The use of an antibody raised against the inhibitor showed that it occurs in a number of rice cultivars at average concentrations of 2-3 mg kg seed.9,10 Of possible physiological significance is the fact that...

Control of Insects with Cystatins

Antifeedant Effects of Cysteine Pis While some pests invade plant seeds to consume their storage proteins, plant seeds have acquired the ability to defend themselves against insect invasion, notably by producing PIs active against pest digestive proteinases. In fact, it is thought that seeds and insects have been co-evolving over a long period of time, and that when an insect adapts to the defenses of a given plant, it has the potential to become a pest.6 Actually, there are a number of...

Plant Parasitic Nematodes

Plant parasitic nematodes procure their food by drawing in the content of individual cells of many plant tissues. Secretions from the esophageal glands are injected through the stylet in the cytoplasm presumably to achieve an enzymatic partial liquefaction of cytoplasmic and organelle proteins, before the nematode draws in the digested content through its stylet.30,31 The root-knot and cyst nematode species stimulate the formation of a dedicated feeding site. The juvenile nema-todes penetrate...

Effects of Recombinant PIs on Nematodes in Plants

Natural PIs are an important element of the plant defense response to herbivory,58,59 and they may also act to restrict infection by nematodes. Methodologies to express recombinant PIs in plants were reviewed recently.60 Serine and cysteine recombinant PIs expressed in plants may effectively suppress plant parasitic nematodes. CpTI, a serine PI from an insect-resistant breeding line of cowpea, was shown to suppress growth of the tobacco budworm Heliothis virescens feeding on transgenic tobacco...