Protease Inhibitors in Improvement of Plant Resistance to Pathogens and Insects

This review concerns the possibility of using plant inhibitors of proteolytic enzymes to improve plant resistance to insects and phytopathogens. The main argument in favor of this approach is that protease inhibitors are widespread in plant tissues and highly active with respect to various proteases of insects, bacteria, and fungi. Genetic engineering yields promising results in the field, as recent studies demonstrate. The main drawbacks of the approach and ways to improve its efficiency are discussed. Still, the approach has several advantages over the standard methods and is ecologically safe.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 4, 2005, pp. 702–708.Original Russian Text Copyright © 2005 by Dunaevsky, Elpidina, Vinokurov, Belozersky.     

Proteinase inhibitors in plant biotechnology: A review

Possible utilities for natural inhibitors of proteolytic enzymes in plant biotechnology have been reviewed. Among the potential areas of use of the inhibitors are (1) construction of transgenic plants with increased resistance to insects and other pests and (2) development of procedures for biosynthesis of recombinant proteins. In the latter case, the inhibitors will serve to prevent the protein degradation by proteinases.     

Proteinase inhibitors in plant biotechnology: a review

Possible utilities for natural inhibitors of proteolytic enzymes in plant biotechnology have been reviewed. Among the potential areas of use of the inhibitors are (1) construction of transgenic plants with increased resistance to insects and other pests and (2) development of procedures for biosynthesis of recombinant proteins. In the latter case, the inhibitors will serve to prevent the protein degradation by proteinases.     

昆虫对植物次生物质的代谢适应机制及其对昆虫抗药性的意义

植物次生物质(plant secondary metabolites)对昆虫的取食行为、生长发育及繁殖可以产生不利影响,甚至对昆虫可以产生毒杀作用。为了应对植物次生物质的不利影响,昆虫通过对植物次生物质忌避取食、解毒代谢等多种机制,而对寄主植物产生适应性。其中,昆虫的解毒代谢酶包括昆虫细胞色素P450酶系（P450s）及谷胱甘肽硫转移酶（GSTs）等,在昆虫对植物次生物质的解毒代谢及对寄主植物的适应性中发挥了重要作用。昆虫的解毒酶系统不仅可以代谢植物次生物质,还可能代谢化学杀虫剂,因而昆虫对寄主植物的适应性与其对杀虫剂的耐药性甚至抗药性密切相关。昆虫细胞色素P450s和GSTs等代谢解毒酶活性及相关基因的表达可以被植物次生物质影响,这不仅使昆虫对寄主植物的防御产生了适应性,还影响了昆虫对杀虫剂的解毒代谢,因而改变昆虫的耐药性或抗药性。掌握昆虫对植物次生物质的代谢适应机制及其在昆虫抗药性中的作用,对于明确昆虫的抗药性机制具有重要的参考意义。本文综述了植物次生物质对昆虫的影响、昆虫对寄主植物次生物质的代谢机制、昆虫对植物次生物质的代谢适应性对昆虫耐药性及抗药性的影响等方面的研究进展。     

植物耐虫性的研究方法

本文介绍了植物耐虫性的研究方法, 包括植物功能损失指数(耐虫指数)、产量损失率、植株被害率、存活率、根系体积(受害程度)、植株和害虫干重、叶片叶绿素荧光特性、保护酶活性和主茎伤流液量等生理生化指标以及害虫的种群发展和取食行为等方法, 并提出植物耐虫性机理的研究思路和方向。     

Proteolytic activation of canatoxin, a plant toxic protein, by insect cathepsin-like enzymes

Canatoxin is a protein isolated from jackbean (Canavalia ensiformis), seeds. Injected intraperitoneally, the toxin is lethal to mice but it is inactive if given orally. Canatoxin is also lethal when fed to insects with cathepsin-based digestion while insects with trypsin-based digestion are not affected. The hypothesis that canatoxin is proteolytically activated by cathepsins was investigated. Experiments were performed with 4(th) instar and adult Rhodnius prolixus fed meals containing canatoxin (2.5 microg/mg weight body). While 100% of nymphs died, no effect was observed in adults. Hemolymph taken from nymphs and adults showed the presence of canatoxin's proteolytic fragments. Reduced lethality was seen in R. prolixus 4(th) instars fed meals containing canatoxin and inhibitors of cathepsin enzymes, E-64 (2.0 microM) or Pepstatin-A (2. 0 microM). In another approach, canatoxin was digested in vitro with enzymes from the bruchid, Callosobruchus maculatus, and the resulting peptides were tested in R. prolixus. Three groups of toxic peptides (8,000-12,000 kD range) were separated by gel-filtration. When these peptides were fed to the insects simultaneously with the cathepsin inhibitors, no protective effect was seen. These results confirm the proteolytic activation of canatoxin by insect cathepsin-like enzymes to produce entomotoxic peptide(s). Furthermore, our data point towards overlooked differences in the digestive physiology of distinct life stages of R. prolixus. Arch.     

植物耐虫性的研究方法

本文介绍了植物耐虫性的研究方法,包括植物功能损失指数(耐虫指数)、产量损失率、植株被换害率、存活率、根系体积(受害程度)、植株和害虫干重、叶片叶绿素荧光特性、保护酶活性和主茎伤流液量等生理生化指标以及害虫的种群发展和取食行为等方法,并提出植物耐虫性机理的研究思路和方向.     

Elevated CO2 reduces the resistance and tolerance of tomato plants to Helicoverpa armigera by suppressing the JA signaling pathway

Both resistance and tolerance, which are two strategies that plants use to limit biotic stress, are affected by the abiotic environment including atmospheric CO(2) levels. We tested the hypothesis that elevated CO(2) would reduce resistance (i.e., the ability to prevent damage) but enhance tolerance (i.e., the ability to regrow and compensate for damage after the damage has occurred) of tomato plants to the cotton bollworm, Helicoverpa armigera. The results showed that elevated CO(2) reduced resistance by decreasing the jasmonic acid (JA) level and activities of lipoxygenase, proteinase inhibitors, and polyphenol oxidase in wild-type (WT) plants infested with H. armigera. Consequently, the activities of total protease, trypsin-like enzymes, and weak and active alkaline trypsin-like enzymes increased in the midgut of H. armigera when fed on WT plants grown under elevated CO(2). Unexpectedly, the tolerance of the WT to H. armigera (in terms of photosynthetic rate, activity of sucrose phosphate synthases, flower number, and plant biomass and height) was also reduced by elevated CO(2). Under ambient CO(2), the expression of resistance and tolerance to H. armigera was much greater in wild type than in spr2 (a JA-deficient genotype) plants, but elevated CO(2) reduced these differences of the resistance and tolerance between WT and spr2 plants. The results suggest that the JA signaling pathway contributes to both plant resistance and tolerance to herbivorous insects and that by suppressing the JA signaling pathway, elevated CO(2) will simultaneously reduce the resistance and tolerance of tomato plants.     

Some properties of proteolytic enzymes from Tribolium confusum

The digestive tract of Tribolium confusum Duv. larvae was studied for proteolytic enzymes properties. The pH optima are determined for the enzymes effect on various substrates. Proteases were partially purified by gel chromatography on Sephadex G = 100 and investigated for thyol compound influence on their activity. The activity of the enzymes is shown to increase considerably with addition of cystein, glutathione, 2-mercaptoethanol. dithiotreitol and EDTA. Dithiotreitol produces the strongest restoring effect and in concentration of 10(-6) M it activates the enzyme almost twice. Storage for 48 h at 4 degrees C induced a 2.5-fold decrease in the proteolytic enzyme activity; SH-groups in the catalytic action of enzymic solutions is shown. The maximum proteolytic activity is found in extracts from 14-day insects.     

Plant cytochromes P450: tools for pharmacology,plant protection and phytoremediation

Cytochromes P450 catalyse extremely diverse and often complex regiospecific and/or stereospecific reactions in the biosynthesis or catabolism of plant bioactive molecules. Engineered P450 expression is needed for low-cost production of antineoplastic drugs such as taxol or indole alkaloids and offers the possibility to increase the content of nutraceuticals such as phytoestrogens and antioxidants in plants. Natural products may serve important functions in plant defence and metabolic engineering of P450s is a prime target to improve plant defence against insects and pathogens. Herbicides, pollutants and other xenobiotics are metabolised by some plant P450 enzymes. These P450s are tools to modify herbicide tolerance, as selectable markers and for bioremediation.     

Involvement of Proteolytic Enzymes and Their Inhibitors in Plant Protection (Review)

The literature data on possible ways of involvement of proteolytic enzymes and their inhibitors in protection of plants from pests and disease are analyzed. Certain practical applications of natural protease inhibitors to plant protection are discussed.     

Dietary proteinase inhibitors alter complement of midgut proteases

Plant serine proteinase inhibitors (Pls) have the potential to restrict the growth and/or development of herbivorous insects. However, there are limitations to the efficacy of these Pls. An insect's susceptibility to a Pl is determined, at least in part, by the relative proportion of proteolytic enzyme activity in the midgut that is suppressed by that inhibitor. Insects adapt to dietary trypsin inhibitor in their host plant by secreting “inhibitor-resistant” trypsin(s). These “inhibitor-resistant” enzyme(s) may be the standard proteinase(s) secreted into the midgut (e.g., Pieris rapae), or may be enhanced following ingestion of proteinase inhibitor (e.g., Helicoverpa zea). In addition, insects may be pre-adapted to specific Pl(s), following adaptation to a Pl from the same family. For example, Pieris rapae is a crucifer specialist that is resistant to cabbage Pl, but is also resistant to Kunitz soybean trypsin inhibitor, a Pl in the same family as cabbage Pl, but from a non-host plant. The ultimate value of this pre-adaptation to herbivory by a species of insect will be determined by the number of different families of Pl in host plant(s) to which the species has adapted, and the distribution of those families among other species of plants. Thus, it is possible that the presence of a plant Pl limits herbivory by insect(s). However, multiple inhibitors, matched to the complement of enzymes in the insect's midgut, may be required to enhance this resistance of plants to herbivorous insects. © 1996 Wiley-Liss, Inc.     

The role of proteolytic enzymes and their inhibitors in plant protection (review)

Literature data on possible ways of involvement of proteolytic enzymes and their inhibitors in protection of plants from pests and diseases are analyzed. Certain practical applications of natural protease inhibitors to plant protection are discussed.     

Potential physiological role of plant glycosidase inhibitors

Carbohydrate-active enzymes including glycosidases, transglycosidases, glycosyltransferases, polysaccharide lyases and carbohydrate esterases are responsible for the enzymatic processing of carbohydrates in plants. A number of carbohydrate-active enzymes are produced by microbial pathogens and insects responsible of severe crop losses. Plants have evolved proteinaceous inhibitors to modulate the activity of several of these enzymes. The continuing discovery of new inhibitors indicates that this research area is still unexplored and may lead to new exciting developments. To date, the role of the inhibitors is not completely understood. Here we review recent results obtained on the best characterised inhibitors, pointing to their possible biological role in vivo. Results recently obtained with plant transformation technology indicate that this class of inhibitors has potential biotechnological applications.     

Identification and characterization of digestive serine proteases from inhibitor-resistant Helicoverpa zea larval midgut

Protease inhibitors mediate a natural form of plant defence against insects, by interfering with the digestive system of the insect. In this paper, affinity chromatography was used to isolate trypsins and chymotrypsins from Helicoverpa zea larvae, which had been raised on inhibitor-containing diet. Sensitivity of the fractions to inhibition by plant proteinase inhibitors was tested, and compared to the sensitivity of proteinases found in insects raised on diet to which no inhibitor had been added. The isolated chymotrypsin activity was found to be less sensitive to plant protease inhibitors. The sensitivity of the isolated trypsin activity was found to be intermediate between completely sensitive trypsins and completely insensitive forms that have been previously described. Mass spectrometry was used to identify one trypsin and two chymotrypsins in the partially purified protease fraction. The sequence features of these proteases are discussed in relation to their sensitivity to inhibitors. The results provide insight in the enzymes deployed by Helicoverpa larvae to overcome plant defence.     

Colorado potato beetles show differential digestive compensatory responses to host plants expressing distinct sets of defense proteins

Herbivorous insects fed plants expressing proteinase inhibitors (PIs) compensate for the loss of digestive proteolytic functions by producing novel proteinases. We assessed here whether such compensatory responses represent a general, non-specific adaptation to defense-related proteins in host plant tissues, or if distinct responses occur depending on the stress exerted on the plant. As a model, growth, development, and digestive proteases of the Colorado potato beetle (Leptinotarsa decemlineata Say) were monitored after feeding larvae with plants pre-treated with either methyl jasmonate or arachidonic acid, two compounds inducing different sets of defense genes in potato. In brief, larvae fed plants treated with jasmonate or arachidonate were negatively affected compared to larvae fed non-treated plants, suggesting the potency of both molecules to induce partial resistance to potato beetles in potato. On the other hand, larvae fed treated plants partially compensated for the presence of defense-related proteins by adapting their digestive proteolytic system, both quantitatively and qualitatively. These compensatory processes varied depending on the treatment, the larvae fed arachidonate-treated plants showing the most dramatic response. Compensation to jasmonate and arachidonate was also influenced by a cysteine PI from rice expressed in the plant, pointing out the possible indirect effects of recombinant defense proteins on naturally-occurring plant-insect interactions. These observations, while showing the potential of jasmonate and arachidonate as inducers of partial resistance to the potato beetle in potato, also suggest that digestive compensation in herbivorous insects is determined, at least in part, by defense-related compounds found in the plant in response to different stress stimuli or as a result of ectopic expression in transgenic plants.     

Viral protein cleavages as a drug design target.

Many animal and plant viruses encode proteolytic enzymes which are involved in regulation of viral replication and assembly. The proteases are becoming well-characterized, and offer attractive features for the design of selective antiviral agents. Approaches to the assay of viral protease inhibitors and the various classes identified are described.     

Effect of blood plasma inhibitors on activity of serine and cysteine proteinases]

Data on study of action plasma inhibitors on activity of pancreatic proteolytic enzymes (trypsin, chymotrypsin) and plant proteinases (papain, bromelain), included in composition of enzyme mixes, used for orally application are submitted. It is established, that serine proteases are more sensitive to inactivation of plasma inhibitors, than cysteine enzymes. Main inhibitor of the papain and bromelain is alpha-2-macroglobulin in complex with which they preserve significant part of initial activity. A high-sensitivity method of determination of activity enzyme combinations, enabling to detect nanograms of them in presence of plasma inhibitors is offered. It can be used for study pharmacokinetic and optimization of enzyme mixes application in clinical practice.     

Heterogeneous production of metallo-type peptidases in parasites belonging to the family Trypanosomatidae

Proteolytic enzymes play a central role in the physiology of all living organisms, participating in several metabolic pathways and in different phases of parasite-host interactions. We have identified cell-associated peptidase activities in 33 distinct flagellates, including representatives of almost all known trypanosomatid genera parasitizing insects (Herpetomonas, Crithidia, Leishmania, Trypanosoma, Leptomonas, Phytomonas, Blastocrithidia and Endotrypanum) as well as the biflagellate kinetoplastid Bodo, by using SDS-PAGE containing gelatin as co-polymerized substrate and proteolytic inhibitors. Under the alkaline pH (9.0) conditions employed, all the flagellates presented at least one peptidase, with the exception of Crithidia acanthocephali and Phytomonas serpens, which did not display any detectable proteolytic enzyme activity. All the proteolytic activities were completely inhibited by 1,10-phenanthroline, a zinc-chelating agent, putatively identifying these activities as metallo-type peptidases. EDTA and EGTA, two other metallopeptidase inhibitors, E-64 (a cysteine peptidase inhibitor), pepstatin A (an aspartyl peptidase inhibitor) and PMSF (a serine peptidase inhibitor) did not interfere with the metallopeptidase activities detected in the studied trypanosomatids. Conversely, Bodo-derived peptidases were resistant to 1,10-phenanthroline and only partially inhibited by EDTA, showing a distinct inhibition profile. Together, our data demonstrated great heterogeneity of expression of metallopeptidases in a wide range of parasites belonging to the family Trypanosomatidae.