Fungitoxic and Insecticidal Plant Polypeptides

Review Review Fungitoxic and Insecticidal Plant Polypeptides Fungitoxic and Insecticidal Plant Polypeptides Arlete Beatriz Becker-Ritt,1,2 Célia Regina Carlini1,2 1 Graduate Program in Cellular and Molecular Biology, Center of Biotechnology, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil 2 Department of Biophysics-IB and Center of Biotechnology, UFRGS, Porto Alegre, RS, Brazil Received 29 January 2012; revised 9 May 2012; accepted 23 May 2012 Published online 31 May 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22097 ABSTRACT: to increase the supply of plant-derived proteins and tackle According to the World Bank and FAO, the population the hunger in a global scale. # 2012 Wiley Periodicals, grows worldwide and the poorest countries are expected Inc. Biopolymers (Pept Sci) 98: 367–384, 2012. to double their population within the next decades, Keywords: fungitoxic; insecticidal; polypeptides reaching *7.2 billion in 2015. Moreover, the food and ﬁnancial crisis together with the global economic recession pushed the number of hungry and undernourished people in the world to unprecedented levels. The substitution of animal proteins by plant proteins in food and feed is a general trend because of the This article was originally published online as an accepted preprint. The ‘‘Published Online’’ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley. com lower cost and better production efﬁciency. Pathogens and pests can reduce the crop yields up to 30%. In some INTRODUCTION places, the losses can reach 80% due to climate A conditions, proliferation of insects, and fungal diseases. All together, the harvest and postharvest losses vary from 5% to 20% and depending on the commodity can be as high as 50%. Plants have a complex chemical armory for defense composed of low and high molecular mass compounds that can act over a variety of pests and pathogens, from micro-organisms to phytophagous insects or grazing animals. Among them, plant fungitoxic and insecticidal polypeptides represent promising alternatives Correspondence to: Arlete Beatriz Becker-Ritt, Universidade Luterana do Brasil— ULBRA, Biologia Celular e Molecular Aplicada à Saúde, Av. Farroupilha, 8001 Prédio 22, 58 Andar, Bairro São José Canoas/RS, CEP 92425-900, Brazil; e-mail: arlete.ritt@ufrgs.br or arlete.ritt@ulbra.edu.br C 2012 V Wiley Periodicals, Inc. PeptideScience Volume 98 / Number 4 vailable data from the World Bank and FAO show that the world population grows exponentially and some countries, especially the poorest ones are expected to double their population within the next decades, reaching *7.2 billion in 2015. Moreover, the food and ﬁnancial crisis together with the global economic recession pushed the number of hungry and undernourished people in the world to unprecedented levels. Plants serve as a major source of proteins and carbohydrates for humans and livestock.1 The substitution of animal proteins by plant proteins in food and feed is a general trend because of the lower cost and better production efﬁciency. Pathogens and pests can reduce the crop yields up to 30%. In some places, the losses can reach 80% due to climate conditions, proliferation of insects, and fungal diseases. All together, the harvest and postharvest losses vary from 5% to 20% and depending on the commodity can be as high as 50%. Thus, there is great interest in increasing the output and yield of agriculture by developing new strategies aiming to reduce crop losses to fungal diseases and insects as the 367 368 Becker-Ritt and Carlini farm land and water resources available worldwide for crop production are ﬁnite.2 About 80% of plant diseases can be traced to fungi and fungal-like organisms (FLO) (such as Pythium and Phytophora sp.) with over 8000 species shown to be phytopathogens. Fungi reproduce themselves both sexually and asexually, can grow on living or dead plant tissue, penetrating the tissue or at the plant’s surface, and can survive in a dormant stage until conditions become favorable for their proliferation. Like seeds, fungal spores are spread by many ways to other plants. Although some fungi are beneﬁcial to plants, especially by forming mycorrhizal associations with the plant’s roots, many others cause diseases in plants such as anthracnose, late blight, apple scab, club root, black spot, damping off, and powdery mildew. It is noteworthy to mention that plant pathogenic fungi and FLOs can be blamed for some of the world’s great famines and human suffering of historical importance.3 Wheat crops of the Middle Ages were commonly destroyed by the fungus (Tilletia spp.) causing bunt or stinking smut. The FLO Phytophthora infestans was responsible for the potato blight in Ireland and northern Europe consecutively in 1845–1846 and 1846–1847, causing death by starvation of more than 1 million people. The fungus Plasmopara viticola attacked the grape vineyards of central Europe in the 1870s, causing great losses to grape growers and wine makers.3 Antifungal proteins and peptides exert a protective activity against fungal invasion and play an important role in plant defense.4 A number of studies have been conducted to identify novel and potent antimicrobial proteins,1 specially because some of them have the potential to overcome antimicrobial resistance.1 Transgenic plants expressing antifungal proteins have been shown to display increased resistance to fungal diseases.5,6 To control fungal diseases, the approaches adopted can be divided into ﬁve categories.7 They involve the expression of gene products which: (i) are directly toxic to pathogens (e.g., chitinases, glycanases, osmotin, thaumatin, defensins, lectins, and phytoalexins)8,9 (ii) destroy or neutralize components of the pathogen (e.g., polygalacturonase, oxalic acid, and lipase); (iii) increase the structural defenses of the plant (e.g., elevated peroxidase and lignin levels); (iv) release regulatory signals related to the plant’s defenses (e.g., speciﬁc elicitors, hydrogen peroxide, salicylic acid, and ethylene); (v) are involved with the elicitation of a hypersensitive response, such as the production of reactiveoxygen species,10 programmed cell death,11 and, subsequently, a wide-spectrum systemic acquired resistance response.12 The methods available today for protecting plant crops against insect predation are still heavily dependent on environmentally aggressive chemicals and were estimated to reduce the losses in only *7%.13,14 Moreover, this type of chemical control caused negative impact on the environment as con- tamination of water and soil, toxicity to nontarget organisms and health problems on nearby populations.14,15 This fact justiﬁes the necessity for research and investments aiming the development of alternative approaches to this problem. The digestive tract of insects is their main food entry way. Therefore, the maintenance of its integrity has to be ensured at all costs, otherwise, their life cycles may be interrupted. Due to the decreased protein content of plant tissues, nitrogen acquisition is a factor that limits the nutrition of phytophagous insects. Hence, the digestive tract, or gut, is a potential target to control crop pests of economic interest. Possibly, the diversity of proteolytic enzymes described for phytophagous insects16 is a response to the abundant presence of protease inhibitors in plant tissues. Transgenic plants have been built which express proteins that disorganize the digestive functions of insects, making the plants resistant to their phytophagous pests. Genes to be incorporated into such plants include those encoding digestive enzyme inhibitors, toxins capable of damaging the intestinal epithelium and antibodies raised against physiologically important proteins. Plants rely heavily on a chemical and biological armory for their defense against a variety of pests and pathogens ranging in size from microbes to insects or grazing animals.17 The screening of plant polypeptides that display toxic effects toward crop pests is worth investigating. The genetic engineering of plants aiming to increase resistance to insect predation or to phytopathogens may rely exclusively on the repertoire of genes found in plants, for example, by manipulating the expression of their endogenous defense proteins, or by introduction of an insect or fungal control gene derived from another plant.18 Such an approach may help reduce the problems of contamination and persistence in the environment, while providing a technological solution compatible with established models of licensing and legislation. These features can also minimize negative perception by the public that consume transgenic crop yields. To foster the development of a plant that is resistant to insects or fungi, multiple approaches must be considered having at least three well-deﬁned aspects. First, it is necessary to know the insect, or fungus to be controlled, its habitat and life cycle, as well as aspects of its physiology at a molecular level, to help identify potential control targets. A second approach addresses the structural characterization and the mechanism of action of toxic factors which, once introduced into plants, will control phytophagous pests or pathogenic micro-organisms. And, ﬁnally, a third approach, on which the previous ones depend, comprises the biochemical and molecular study of the host plant to be modiﬁed aiming pest-resistance, so that a suitable manipulation can be performed to the desired end. In this article, we reviewed this literature (up to Dec 2011) on plant fungitoxic proteins and peptides. Because many Biopolymers (Peptide Science) Fungitoxic and Insecticidal Plant Polypeptides fungitoxic polypeptides are also insecticidal, details on the mechanisms of entomotoxic activity of these molecules are also provided. The following sections are organized by classes or groups of polypeptides, with their physicochemical characteristics and description of their effects on fungi and/or insects. OVERVIEW OF PLANT FUNGITOXIC POLYPEPTIDES Plant seeds are usually scattered in environments rich in micro-organisms. To protect their seeds from the invasion of pathogens, plants have developed various defense systems during their evolution.19 The physical barriers of seeds, e.g., lower water content and hard seed shell, against bacterial and fungal invasion are destroyed during seed germination because the seed coat is ruptured by imbibition. The breakage of these physical barriers can allow the invasion of pathogens into seeds.20 Therefore, seed germination is a period vulnerable to pathogen attack in a plant’s life cycle.19,21 Phytopathogenic fungi produce enzymes that attack polymers of plant walls, such as cellulose and lignin, allowing them to infect roots, leaves, fruits, and seeds.22 The mechanisms of plant resistance to most fungal diseases are still poorly known. The elucidation of these mechanisms is a key step for the development of suitable control methods, as well as for understanding the pathogen-host interaction. Several families of plant fungitoxic polypeptides are described, based on spatial structure and biological activity: chitinases and b-1,3-glucanases, ureases, thionins, defensins, hevein- and knottin-like peptides, cyclotides, 2S albumins, and lipid transfer proteins (LTPs).23–30 The hydrolytic enzymes chitinases and b-1,3-glucanases are among the antifungal proteins that have been most explored for the generation of resistant transgenic plants as they are capable of degrading the main components of the fungal cell wall, chitins, and glucans, respectively.7 Other genes coding for proteins related to pathogenesis (PR) with antifungal activity include osmotin and proteins similar to thaumatin. Osmotin, a 24-kDa protein that belongs to the PR-5 family, presents homology to the thaumatin protein from Thaumatococcus danelli. Both proteins are expressed in plants under stress conditions and present antifungal activity in vitro, weakening the cell wall of the fungus by forming transmembrane pores. Osmotin, tested in combination with chitinase and b-1,3-glucanase, showed increased lytic activity.31,32 The expression of thaumatin in transgenic plants determined a delay in the development of diseases caused by several pathogens, including P. infestans Botrytis, Fusarium, Rhizoctonia, and Sclerotinia.7 Cotton (Gossypium hirsutum),30 Glycine max embryo-speciﬁc and Canavalia ensiformis seed ureases and bacterial urease Biopolymers (Peptide Science) 369 from Helicobacter pylori show fungicidal activity, regardless of the ureolytic activity, at submicromolar doses, affecting different phytopathogenic ﬁlamentous fungi and inducing injuries to the cell wall and/or cell membrane.29 Aspartic proteases (EC 3.4.23), one of the four super-families of proteolytic enzymes, are present in a wide variety of organisms.33 Solanum tuberosun aspartic proteases (StAPs) type 1 from tubers,34 type 2 and 3 from leaves35 are induced by both abiotic and biotic stress, have extracellular localization and have antimicrobial activity in vitro toward P. infestans and Fusarium solani, two potato pathogens.35–37 Table I summarizes some of the available data on antifungal plant proteins and peptides. OVERVIEW OF PLANT INSECTICIDAL POLYPEPTIDES Chitin, an N-acetylglucosamine polymer, is one of the main constituent elements of extracellular structures, such as the cuticle and peritrophic matrix of the insect gut and of the crustacean exoskeleton. This biopolymer is synthesized intracellularly by chitin synthase, a membrane-bound glycosyltransferase, carried through the plasma membrane, then coalescing into hard crystallites deposited on the insect cuticle. Synthesis of the cuticular chitin during insect development is coordinated by ecdysone, the molt hormone. The gut of insects from various orders, both in larvae and in adults, is lined with the peritrophic matrix, a structure that compartmentalizes the digestive process and protects the organism from parasitic and viral invasions.16 The peritrophic matrix is basically composed of chitin, in addition to proteins and proteoglycans.50 The contribution of the enzyme chitin synthase to the ﬁnal formation of the peritrophic matrix has not yet been fully elucidated.51 The digestive system of insects comprises the digestive tube (divided into the foregut, midgut, and hindgut), accessory organs, and Malpighian tubules.52 The midgut is the main organ for digestion and nutrient uptake. The diversity of insect food sources resulted in diversity in the gut architecture, and in the digestive enzyme proﬁles differing among insect orders,16 and the phase of the life cycle.53 The hindgut and the Malpighian tubules of insects are reabsorption and excretion organs. ATPases and eicosanoid metabolism are involved with the control of ﬂuid secretion and electrolyte balance in the Malpighian tubes in some insects and other arthropods.54,55 Hematophagous insects, such as Rhodnius prolixus, ingest blood meals equivalent to up to 10–12 times their initial body weight. The rapid diuresis in these insects is a key process, accounting for a discharge of 40% of the blood meal weight56 in a short time period, so as to allow the insect to move away from the feeding site, 370 Becker-Ritt and Carlini Table I Plant Proteins and Peptides with Antifungal Properties Protein/Peptides C-FKBP Chitinase Potide-G (serine protease Inhibitor) Cucurmoschin Pr-1 Pr-2 Thaumatinlike proteins (TLPs) PDC1 (corn defensin) VuD1 (defensin) StAPs (Solanum tuberosum aspartic proteases) Plant Source Fungi Brassica campestris L. ssp. pekinensis Candida albicans, Botrytis cinerea, Rhizoctonia solani, and Trichoderma viride Leucaena leucocephala Collectotrichum sp 1 Collectotrichum sp 2 Pestalestiopsis sp 1 Anthanose collectotrichum Fusarium sp 1 Fusarium monoliforme Fusarium oxysporum Pestalestiopsis sp 2 Pestalestiopsis sp 3 Pestalestiopsis sp 4 Cercospora sp Drechslera sp Sclerotium sp Rhizoctonia solani Potato tubers (Solanum tuberosum L. cv. Golden Valley) Black pumpkin seeds Botrytis cinerea Fusarium oxysporum Mycosphaerella oxysporum Pumpkin rinds Aspergillus ﬂavus (Cucurbita) Botrytis cinérea Colletotrichum coccodes Fusarium moniliforme var. subglutinans Fusarium oxysporum Fusarium solani Rhizoctonia solani Trichoderma harzianum Botrytis cinérea Pumpkin rinds Aspergillus fumigates (Cucurbita) Aspergillus parasiticus Botrytis cinerea Colletotrichum coccodes Didymella bryoniae Fusarium oxysporum Fusarium solani Trichoderma harzianum Trichoderma viride French bean Phaseolus Pleurotus ostreatus vulgaris cv. Kentucky Fusarium oxysporum wonder Coprinus comatus Corn (Zea mays) Fusarium graminearum Cowpea (Vigna unguiculata) Potato (Solanum tuberosum) Fusarium oxysporum Aspergillus ﬂavus Fusarium solani Doses Test References 16 lM 4 lM 8 lM 8 lM 0.5 lg 0.5 lg 0.5 lg 1 lg Radial growth inhibition 237 Hyphal extension inhibition 238 Turbidity 239 Radial growth inhibition 140 Radial growth inhibition 240 Radial growth inhibition 1 Radial growth inhibition 241 Microplate assay— hyphal growth Radial growth inhibition Germination of spores 242 1 lg 1 lg 1 lg 1 lg 1 lg 2 lg 2 lg 2 lg 100 lg/lL 375 lg 375 lg 375 lg 80 lM 20 lM 20 lM 20 lM 10 lM 10 lM 20 lM 20 lM 20 lM 40 lM 80 lM 20 lM 10 lM 40 lM 10 lM 10 lM 20 lM 20 lM 60 lg 60 lg 60 lg 5–50 lg/mL 100 lg/mL 4 lM 181 37 Biopolymers (Peptide Science) Fungitoxic and Insecticidal Plant Polypeptides Table I 371 Plant Proteins and Peptides with Antifungal Properties (continued) Protein/Peptides Ec-LTP Plant Source Weed barnyard grass (Echinochloa crusgalli (L.) Beauv Fungi Doses Phytophthora infestans Helminthosporium sativum Lipid transfer proteins Chilli pepper (Capsicum C. musae (LTPs) annuum L.) seeds F. oxysporum Candida albicans, Saccharomyces cerevisiae Schizosaccharomyces pombe Urease Soybean (Glycine max (L) Colletotrichum musae Merril Curvularia lunata Penicillium herguei Fusarium oxysporum Urease Jackbean (Canavalia Fusarium solani ensiformis) Colletotrichum musae Curvularia lunata Penicillium herguei Fusarium oxysporum Urease Cotton (Gossypium Colletotrichum musae hirsutum) Curvularia lunata Penicillium herguei Cysteine protease Sugarcane (Saccharum) Trichoderma reesei inhibitor Lectin Talisia esculenta Fusarium oxysporum Colletotrichum lindemuthianum Saccharomyces cerevisiae reducing the risk of its own predation. The Malpighian tubules and the hindgut are under neuroendocrine control and account for a ﬁne balance of diuretic and antidiuretic hormones, which use cyclic nucleotides and/or eicosanoids as secondary messengers.57 The insecticidal effect of the ureases from C. ensiformis seems to involve disorders in the secretory system, manifested in inhibition of diuresis in the insect.58 Table II summarizes some of the available data on insecticidal plant proteins and peptides. FOCUSING ON THE POLYPEPTIDES Plant Ureases Ureases (EC 3.5.1.5) are nickel-dependent enzymes that catalyze urea hydrolysis resulting in ammonia and carbon dioxide.78 They are widely distributed in plants, fungi and bacteria, but are not synthesized by animals. In plants and fungi, the ureases are hexamers or trimers of one subunit of *840 amino acids.79 Bacterial ureases possess 2 or 3 smaller subunits and present 50%–60% homology with plant ureases. It Biopolymers (Peptide Science) Test References IC50: 27.4 lg/mL (4–6 8C) IC50: 54.8 lg/mL (RoomTemp) IC50: 164 lg/mL Radial growth inhibition 195 70 and 150 lg/mL 70 and 150 lg/mL 9 to 70 lg/mL 9 to 70 lg/mL 9 to 70 lg/mL Microplate assay 197 0.81 6 0.04 lM 0.61 6 0.02 lM 0.27 6 0.02 lM 0.61 6 0.03 lM 0.57 lM 0.57 lM 0.57 lM 0.57 lM 0.57 lM 10 lg 10 lg 10 lg 50 lg/mL Turbidimetric evaluation 29 Turbidimetric evaluation 29 Turbidimetric evaluation 30 Growth inhibition assay Turbidimetric evaluation 243 280 lg/mL 280 lg/mL 244 280 lg/mL has been postulated that these enzymes are involved in both, nitrogen bioavailability and defense processes in plants.80 Pires-Alves et al.81 identiﬁed a family of urease genes induced by abscisic acid in the legume C. ensiformis (jackbean), coding for at least three protein isoforms: (a) jbure-I, accession M65260, the major classic urease JBURE-I; (b) jbure-IIb, accession AF468788, with 86% similarity to the classic urease82; and (c) jbure-III, or canatoxin (CNTX), a third member of the family, a toxic protein.83 CNTX-like proteins,84 and ureases accumulate in the mature seed, suggesting a defense role associated with both insecticidal85 and fungicidal properties.29 Fungitoxic Activity of Ureases. CNTX was the ﬁrst urease shown to inhibit the radial growth of several ﬁlamentous fungi and later, we29 demonstrated that the embryo-speciﬁc urease from Glycine max (soybean), the major urease from C. ensiformis and a bacterial urease from H. pylori show fungicidal activity, regardless of their ureolytic activity, towards different phytopathogenic ﬁlamentous fungi. More recently, the fungicidal activity of the cotton (G. hirsutum) seed urease30 372 Becker-Ritt and Carlini Table II Plant Proteins and Peptides with Insecticidal Properties Protein Plant Source Insect Doses Test References Urease Jackbean (Canavalia ensiformis) Palo Fierro (Olneya tesota) Common bean, Phaseolus vulgaris L Amaranthus Oncopeltus fasciatus 0.01% (w/w) 55 Zabrotes subfasciatus 88 lg Artiﬁcial cotton seeds Enzymatic activity 116 Hypothenemus hampei Ferrari Hypothenemus hampei Ferrari Callosobruchus maculatus 1 mg Enzymatic activity 112 1 mg Enzymatic activity 112 10 lg Enzymatic activity 245 Callosobruchus maculatus 4 lg Enzymatic activity 248 Tribolium castaneum Diabrotica virgifera virgifera Callosobruchus chinensis Riptortus clavatus Callosobruchus chinensis Riptortus clavatus Dysdercus peruvianus 4 lg 85 lM Artiﬁcial diet 246 0.3% 0.5% 0.5% 1.0% 0.02% (w/w) Artiﬁcial diet 249 Artiﬁcial diet 249 Artiﬁcial diet 56 Dysdercus peruvianus 0.05% (w/w) Artiﬁcial diet 56 Acanthoscelides obtectus 6.0 units of a-amylase Bernfeld method 108 Callosobruchus maculatus Zabrotes subfasciatus Zabrotes subfasciatus 0.25 lg Bernfeld method 115 a-amylase inhibitor amylase inhibitors amylase inhibitors cysteine protease inhibitor cystatin inhibitor Soybean (Glycine max (L) Merril Soybean (Glycine max (L) Merril Soyacystatin N Oryzacystatin I Soybean (Glycine max (L) Merril Rice (Oryza sativa) Oryzacystatin II Rice (Oryza sativa) Urease Urease a-amylase inhibitors a-amylase inhibitors II a-amylase inhibitor Jackbean (Canavalia ensiformis) Soybean (Glycine max (L) Merril Wheat (Triticum aestivum) Common bean (Phaseolus vulgaris L.) Common bean (Phaseolus vulgaris cv. Magna) Acanthoscelides obtectus Cryptolestes ferrugineus Cryptolestes pusillus C. pusillus Oryzaephilus surinamensis O. surinamensis Sitophilus granarius Tribolium castaneum T. castaneum Drosophila melanogaster Sarcophaga Aedes aegypti Monomorium pharaonis Apis melliﬁca Venturia canescens Ephestia cautella Ephestia elutella Ephestia kuehniella Manduca sexta Ostrinia nubilalis Blattella germanica pH 4.5 pH 6.0 >1000 10.9 88.6 359.9 44.9 134.1 >1000 2.4 4.5 N.A. N.A. N.A. N.D. 33.8 N.A. N.A. N.A. N.A. N.A. N.A. N.A. 448.0 4.2 N.I. N.D. >1000 695.6 >1000 4.8 7.5 14.0 82.0 41.1 29.6 39.4 22.7 N.I. N.I. N.I. N.I. N.I. N.I. Enzymatic 122 activity Biopolymers (Peptide Science) Fungitoxic and Insecticidal Plant Polypeptides 373 Table II Plant Proteins and Peptides with Insecticidal Properties (continued) Protein Soyacystatin N M a-amylase inhibitors I Plant Source Soybean (Glycine max (L) Merril Common bean (Phaseolus vulgaris) Insect Liposcelis decolor Acheta domesticus Eurydema oleracea Graphosoma lineatum Callosobruchus maculatus Doses Test N.A. N.I. N.I. >1000 N.I. N.I. N.I. >1000 0.2% Bruchus pisorum 0.2 lg inhibitor/100 lg of seed protein Canatoxin Jackbean (Canavalia ensiformis) Dysdercus peruvianus 0.003% Jaburetox-2Ec Jackbean (Canavalia ensiformis) Dysdercus peruvianus 0.01% (w/w) Spodoptera frugiperda 16.3 lg Dysdercus peruvianus 0.01% (w/w) Rhodnius prolixus Triatoma infestans 5th instar Triatoma infestans adults Aulacothum solani 0.026 mg/g 0.1 mg/g Jaburetox-2Ec Jackbean (Canavalia ensiformis) Lectin (agglutinin GNA) Lectin Gufanthus nivulis References Artiﬁcial seed Transgenic peas (Pisum sativum) Artiﬁcial cotton seeds Artiﬁcial cotton seeds Discs of Phaseolus vulgaris leaves Artiﬁcial cotton seeds Injection Injection 247 111 65 58 46 0.1 mg/g 0.1% (w/v) Injection Artiﬁcial diet 100 Feeding on 103 leaves from transgenic papaya plants Artiﬁcial 149 seed Snowdrop (Galanthus nivalis) Tetranychus cinnabarinus 0.74% of total soluble protein Phaseolin Lima bean (Phaseolus lunatus) Callosobruchus 1.4% (w/w) Vicilin Embryo of Phaseolus vulgaris, WAF-9 Maculatus Callosobruchus 2.22% 6 0.64% Artiﬁcial seed 150 Vicilin Testa of Phaseolus vulgaris, WAF-9 Maculatus Callosobruchus 0.84% 6 0.10% Artiﬁcial seed 150 Maculatus Callosobruchus 6.3% Artiﬁcial seed 150 Maculatus Callosobruchus 1.07% 6 0.32% Artiﬁcial seed 150 Vicilin Embryo of Vigna unguiculata (EPACE10) Vicilin Embryo of Vigna unguiculata (IT81D1045) Maculatus Biopolymers (Peptide Science) 374 Becker-Ritt and Carlini and the recombinant JBURE-IIb apourease from C. ensiformis82 against the maize phytopathogen P. herguei were described. The fungitoxic activity of ureases occurs at submicromolar doses, making these proteins 2–3 orders of magnitude more potent than any other known antifungal proteins of plant origin, producing injuries to the cell wall and/or cell membrane and plasmolysis.29 In yeast, such Pichia membranifasciens, Saccharomyces cerevisae and the human pathogens Candida albicans and C. tropicalis, there is an impairment of glucose fermentation and formation of pseudo-hiphae when the cells are exposed to C. ensiformis major urease for 30 min (Postal et al., unpublished data). Entomotoxic Activity of Ureases. CNTX is lethal when ingested by bruchids (e.g., Callosobruchus maculatus) and bugs (Hemiptera, e.g., Nezara viridula, Dysdercus peruvianus, R. prolixus), but it does not affect other insects (Lepidoptera, e.g., Manduca sexta, Anticarsia gemmatalis; Orthoptera, e.g., Schistocerca americana; Diptera, e.g.: Drosophila melanogaster, Aedes aegypti).58 Recently, we reported the insecticidal activity of C. ensiformis major urease on nymphs of the milkweed bug Oncopeltus fasciatus.59 Comparison of two plant ureases (C. ensiformis and G. max) and the bacterial urease from Bacillus pasteurii revealed that only plant ureases exhibited insecticidal activity, which is independent of their ureolytic activity.66 Jaburetox-2, a recombinant peptide obtained from a truncated cDNA of a C. ensiformis urease gene, showed potent insecticidal activity when added at 0.01% w/w to the diets of sensitive insects, and it also killed insects that are not sensitive to native (full-length) ureases.58,73,86 After ingestion, intact enzymatically active urease can be found in the hemolymph of R. prolixus and contributes to the entomotoxicity by interfering on several physiological systems of the insect.87,88 The insecticidal action mechanism of ureases and derived peptides is not yet fully elucidated. The insecticidal effect of urease on bugs is stage-dependent, affecting nymphs, but not adults, and is related to the digestive enzyme proﬁle of the insect.53,89 The insecticidal effect of CNTX on R. prolixus suggested the impairment of diuresis and electrolyte balance.89 Stanisçuaski et al.87 demonstrated that both the native C. ensiformis major urease and the recombinant peptide Jaburetox-2Ec affect serotonin-induced secretion of R. prolixus isolated Malpighian tubules. Although the antidiuretic effect of Jaburetox-2Ec is cGMP-dependent and accompanied by changes of the transepithelial potential of the tubules, the effect of native C. ensiformis urease involves eicosanoid metabolites and mobilization of calcium ions. CNTX promoted a similar effect on R. prolixus tubules secretion, decreasing the secretion rate, whereas the trichain urease from H. pylori had no signiﬁcant effect.87 Native C. ensiformis urease displays entomotoxic properties not shared by the jaburetox-2 recombinant peptide, such as impairment of water secretion and potentiation of serotonin-induced contractions of the R. prolixus crop.88 Other studies have suggested that both, the native urease as well as the recombinant peptide, have the ability to insert themselves into lipid membranes, resulting in permeability changes.90 Proteinase Inhibitors Proteinase inhibitors are expressed by the plant in response to attacks by predators and pathogens.91,92 As primary products of gene expression, proteinase inhibitors are great candidate tools for plant biotech programs aimed at resistance. Proteinase inhibitors are considered antimetabolite agents, because they reduce the availability of amino acids essential for the synthesis of proteins that drive growth, development and reproduction of the affected organism. Several review articles describe proteinase inhibitors as multifunctional monomers or homo-multimers with different reactive sites within the polypeptide.72 The major differences among those inhibitors are their molecular mass, content of cysteine residues, and number of reactive sites. In general, these polypeptides have 18 to 26 kDa, and two Cys-Cys bonds in a single or double-chain polypeptide.93 Inhibitors that are members of the Kunitz family have in their structure a b trefoil formed by 12 interconnected antiparallel b strands that are stabilized by numerous hydrogen and disulﬁde bonds.93 Proteinase inhibitors are usually classiﬁed according to the proteinase class on which they act: serine, cysteine, aspartic, or metalloproteinases. Plant tissues, in particular seeds, are rich in serine proteinase and cysteine proteinase inhibitors, far less abundant, metalloproteinase and aspartic proteinase inhibitors have also been described in plants. Some plant serine proteinase inhibitors are bifunctional, having antitryptic and anti-a-amylase activities that are associated with two different protein domains.91 The activity of the low-molecular weight trypsin inhibitors in seeds and in tubercles was correlated with defense against herbivorous animals.94 These proteins also inhibit the development of nematodes,95 mollusks,96 fungi,97 as well as insects.98–100 Usually, proteinase inhibitors are induced by pathogen attacks suggesting a role in plant defense.93,101,102 The strategy of exploiting the biotechnological potential of seed cystatins from wild, and related nonagricultural plants as defense proteins could be used in breeding programs of cultivated legumes. This strategy is part of the socalled genomics-guided transgenes initiative,103 which is Biopolymers (Peptide Science) Fungitoxic and Insecticidal Plant Polypeptides expected to be better accepted by the public and to face less strict government regulations. The central tenet to this strategy is that the transfer of homologous genes from related species would entail metabolic changes in the transgenic organism that would be similar to natural or induced mutations in the host genome. Fungitoxic Activity of Proteinase Inhibitors. Corn trypsin inhibitors inhibit the a-amylases of Aspergillus ﬂavus,104 increasing the potential for application of these proteins to fungal diseases. Some trypsin inhibitors inhibit fungal growth and take part in the set of proteins associated with plant defense.105 A 16-kDa protein exhibiting antitrypsin activity was isolated from Helianthus annus ﬂowers and was shown to inhibit spore germination and mycelial growth of the fungi Sclerotinia sclerotiorum and F. solani, at concentrations of 3–5 lg/mL.106 The 18-kDa antitrypsin protein, obtained from Psoralea corylifolia seeds, inhibited the growth of the fungi Alternaria brassicae, Aspergillus niger, Fusarium oxysporum, and Rhizoctonia cerealis fungi.105 The microbiocidal activity of phytocystatins has been reported in a number of studies. Recombinant strawberry cystatin, FaCPI-1, inhibited the growth of the fungi Botritis cinerea and F. oxysporum at micromolar concentrations.107 Abraham et al.108 demonstrated that six out of seven barley cystatins inhibited the growth of B. cinerea and F. oxysporum, with EC50 < 1.5 lM for both fungi. The antifungal activity mechanism of the phytocystatins, however, is not fully understood. Martinez et al.109 demonstrated, by site-directed mutagenesis, that the inhibition of B. cinerea by barley cystatin is not associated with the proteinase inhibitory property of cysteine proteinases. Changes in membrane permeability were detected when oryzacystatin (OC-I) was expressed in the cytosolic compartment of tobacco leaf cells. However, in this case, growth inhibition in Sclerotium rolfsii by a cystatin from Colocasia esculenta tubercles was directly related to the inhibition of a cysteine proteinase in the fungus.110 Additional studies are, therefore, needed to elucidate the antifungal action mechanisms of each phytocystatin on different pathogens. Entomotoxic Activity of Proteinase Inhibitors. Transgenic plants expressing serine or cysteine proteinase inhibitors show an increased resistance to some lepidopterans and coleopterans, respectively, conﬁrming the participation of these proteins in plant defense.111 For instance, the poplar plant, expressing orizacystatin-I (OC-I) genes, was shown to be resistant to Chrysomela tremulae.112 Corn cystatin genes expressed in rice plants showed repressive effects on cysteine proteinases in Sitophilus zeamais.98 An extensive list of transBiopolymers (Peptide Science) 375 genic plants expressing proteinase inhibitors is reviewed by Ref. 72, all of which have shown resistance to different pest, including insects and fungi.72,113 Some of these plants have been approved for market by the U.S. FDA (http://www.epa.gov/pesticides/biopesticides/ pips/index.htm). Selection pressure on insects made them coevolve with plants and thus most acquired the ability to escape the defense mechanisms of the plants they feed on.113 Examples of this adaptation are some Hemiptera that have residual levels of digestive enzymes because their main nitrogen source are amino acids.113 Phytophagous insects usually present gene families encoding isoforms of digestive proteinases that enable rapid adaptation to the presence of proteinase inhibitors in their diets by induction of the expression of inhibitorinsensitive enzymes.113,114 Coleopterans illustrate this type of adaptation: while cysteine proteinases are their main digestive enzymes, some species display an alkaline region containing serine proteinase(s) in their midgets.113,115 Pea albumin PA1b, which belongs to the inhibitory cystine knot family or knottin family, is toxic to the weevils Sitophilus oryzae, S. zeamais, and S. granaries.116 The human disease vector dipteran Culex pipiens is also sensitive to PA1b.117 Lectins Lectins are proteins of nonimmune origin, displaying one or more noncatalytic domains able to recognize and reversibly bind to free- and/or conjugate carbohydrates, without modiﬁcation of their covalent structures.118 They are found in all kingdoms and are particularly abundant in plants.119 These proteins are able to interact selectively with several cell types, a characteristic that underlies a myriad of biotechnological applications, such as analytical, preparative and separation techniques, cell typing, characterization of macromolecules, studies of immune function.93 Lectins puriﬁed from different plants can display different biochemical and biophysical properties.120 Lectins are able to interact with cell membranes of several organisms, like: fungi, bacteria and insects, specially glycoconjugates, glycolipids, glycoproteins, and polysaccharides, thus, working as defense proteins.93 Physiological functions proposed for plant lectins include participating in the colonization and nodulation of legume roots by rhizobial bacteria and participating in defense mechanisms against phytopathogens, nematodes, and phytophagous insects.121 Fungitoxic Activity of Lectins. Among lectins with inhibitory action on phytopathogenic fungi, are those found in potatoes, tomatoes, Urtica dioica, wheat germ agglutinin 376 Becker-Ritt and Carlini (WGA), peas, among others.122,123 Ciopraga et al.122 observed that the binding of WGA to the cell wall of Fusarium graminearum and F. oxysporum affected the growth of the germ tubes as revealed by the dramatic changes observed in fungal morphology: lysis of hyphal tips, swelling and vacuolation of cell content. A chitin-binding lectin from the corm of Gastrodia elata (Orchidaceae) displayed a strong in vitro inhibitory activity on the hyphal growth of the phytopathogenic fungi Valsa ambien, Rhizoctonia solani, Gibberella zeae, Ganoderma lucidum, and B. cinerea.124 The cotyledonary Lutzelburgia auriculata agglutinin, which is released to the surrounding medium during seed germination, inhibited in vitro the fungal growth of Colletotrichum lindemuthianum, F. solani, and A. niger.125 Entomotoxic Activity of Lectins. The deleterious effect on insects from the orders Coleoptera (beetles), Homoptera (plant lice and aphids), and Lepidoptera (butterﬂies and moths) has been documented for several lectins,18,126 amongst them the lectins of the WGA and the snowdrop Galanthus nivalis (GNA). Down et al.74 observed that GNA inhibited the growth of the aphid Aulacorthum solani and reduced its fecundity by 65%. Among other insecticidal lectins are those from pitomba (Talisia esculenta) and Annona coriacea.127,128 Transgenic plants expressing different entomotoxic lectins are being tested in ﬁeld (http://www.epa.gov/pesticides/biopesticides/pips/index.htm). McCafferty et al.75 reported that a papaya tree transformed with the GNA (Galanthus nivalis L. agglutinin) gene expressed a biologically active lectin and presented acceptable control levels for the aphid Tetranychus cinnabarinus. Maize plants, expressing snowdrop lectin (GNA) demonstrated resistance to aphids (Rhopalosiphum maidis Fitch).129 Allium sativum leaf agglutinin expressed in transgenic rice enhanced the resistance against to the major sap-sucking insects: brown plant hopper (BPH), green leafhopper (GLH), and white backed plant hopper (WBPH).130 insect-resistant transgenic plants that express heterologous aamylase inhibitors as they show insecticidal effect on several agricultural pests,61,71 specially storage insects.135 In the genus Phaseolus, the a-amylase inhibitors are members of a family of insecticidal proteins that includes phytohemagglutins (lectins) and arcelins.136 The aAI-1 isoform, present in the most cultivated varieties of black beans, inhibits mammalian a-amylases and the enzymes of the insects C. chinensis, C. maculates, and B. pisorum, but it does not inhibit the a-amylase ZSA of the weevil Z. subfasciatus, an important pest of stored black beans.131 The variant aAI-2, with 78% homology compared with aAI-1, is found only in wild accessions of the common bean and inhibits speciﬁcally the a-amylase ZSA of Z. subfasciatus larvae.68,131 These inhibitors, however, do not protect black beans against the bruchid A. obtectus, which is insensitive to arcelins and the inhibitors aAI-1 or aAI-2. On the other hand, the a-amylase of this bruchid is affected by inhibitors extracted from wheat and barley.67 An a-amylase inhibitor with high homology with aAI-1 was puriﬁed from Palo Fierro seeds, and just like the latter, it did not present inhibitory activity against ZSA.60 The 3D structure of some a-amylases complexed with inhibitors have been determined: with a-amylase inhibitors from black beans,137 with a bifunctional a-amylase/trypsin inhibitor from ﬁnger millet,132 with an a-amylase/subtilisin inhibitor from Amaranthus (knottin type),138 with an aamylase/subtilisin inhibitor from barley (BASI, Kunitz-like type),139 and the crystal structure of the inhibitor complex 0.28 from wheat (cereal type) with a-amylase from the insect Tenebrio molitor.140 In the case of black bean a-amylase inhibitor aAI-1 complexed with an a-amylase from T. molitor, it was demonstrated that Tyr186 and Tyr37 strongly bind to residues within the catalytic cleft.141 Studies on the speciﬁcity of aAI-1 (isolated from Phaseolus vulgaris cv. Magna) against a-amylases from 30 species that are important crop pests revealed a selective inhibition of a-amylases from three insect orders (Coleoptera, Hymenoptera, and Diptera) and also inhibition of a-amylases from annelids.69 a-AMYLASE INHIBITORS a-amylases (a-1,4-glucanohydrolases), enzymes present in animals, plants, and micro-organisms, catalyze the polysaccharide hydrolysis with a-1,4 type bonds, such as starch and glycogen, producing smaller oligosaccharides. In insects feeding on seeds during the adult and/or larval stage, these are the most important digestive enzymes.131,132 a-amylase inhibitors occur in cereals67,133 and leguminous plants.131,134 a-amylases inhibitors selectively affect a-amylase from insects, animals and micro-organisms. These inhibitors present an interesting potential for the development of Chitin-Binding Proteins Chitin, a polymer of N-acetylglucosamine residues, is found in the exoskeleton of invertebrates, such as nematodes, crustacean carapaces, cuticles, and midguts of insects,16,142 and also on cell walls in fungi and green algae. The family of chitin-binding proteins contains members with different activities and origins.143 These proteins possess a 30–43 amino acid domain, with conserved cysteines and glycines. This domain confers speciﬁcity for chitin binding; Biopolymers (Peptide Science) Fungitoxic and Insecticidal Plant Polypeptides however, binding is not restricted to chitin but encompasses other glycoconjugates containing N-acetyl-D-glucosamine or N-acetyl-D-neuraminic acid residues.144,145 Chitinases (EC 3.2.1.14) are present in different plant species and plant organs and may be constitutive or induced by biotic and abiotic factors.146,147 Their action leads to the formation of oligosaccharides which work as elicitors, triggering the induction of other plant defense proteins.148 Chitinases may act in synergism with other defense proteins, among them b-1,3-glucanases, ribosome-inactivating proteins and osmotins.149 Plant chitinases show nematicidal activity when placed on nematode eggs.7 Class III chitinases hydrolyze the N-acetylmuramic acid linkages and the N-acetylglucosamine residues from peptidoglycans on the bacterial wall.150 The Chia4 chitinase (ATCHIT4) of Arabidopsis thaliana splits chitin from the pathogenic protozoon Phytomonas françai.151 Vicilins, members of a large class of seed storage proteins, and chitin-binding lectins are examples of chitin-interacting proteins that show deterrent effect against phytophagous insects and phytopathogenic fungi. Fungitoxic Activity of Chitin-Binding Proteins. Cowpea and other legume vicilins exhibit antifungal properties probably related to their ability to bind chitin, inhibiting spore germination, and interfering with the development of hyphae from phytopathogenic fungi.152 Chitinases, like alpha-glucanases, degrade the cell wall and inhibit the growth of fungal pathogens.153 In the early stage of pathogenesis, apoplastic chitinases release elicitor molecules that activate the defense mechanisms of plants.147 Some studies have indicated that chitin hydrolysis is not necessary for the antifungal activity, suggesting that the chitin-binding domain possesses an intrinsic antifungal activity.154,155 Brassica napus plants constitutively expressing a tomato chitinase were less sensitive to the fungi Cylindrosporium concentricum and S. sclerotiorum.156 Chrysanthemum157 and cucumber158 expressing a rice chitinase gene became less susceptible to the grey mold, B. cinerea. The cotransfer and expression of rice chitinase and of an alfalfa glucanase in the grass Agrostis palustris conferred resistance against the fungi S. homoeocarpa and R. solani.41 A recombinant papaya chitinase (CpCHI) inhibited, at a 76 nM level, the germination of Alternaria brassicicola spores.159 Expression of the CHIT42 enzyme of Trichoderma harzianum in tobacco and potato plants made them tolerant to the leaf pathogens Alternaria alternata, B. cinerea and to the systemic pathogen R. solani.160 Tobacco expressing the CHIT42 enzyme of Metarhizium anisopliae161 showed considerable resistance to the fungus R. solani.162 Biopolymers (Peptide Science) 377 Entomotoxic Activity of Chitin-Binding Proteins. In the case of the cowpea (Vigna unguiculata), a legume of socioeconomic importance in the Northern and Northeastern regions of Brazil, the resistance of some genotypes to the bruchid C. maculatus was ascribed to the presence of variant vicilins.163 These vicilins interact more strongly than the ordinary vicilins to the chitin present in the guts of bruchid larvae and are less available for use by the insect.164 Vicilins of other legumes,165 and proteins homologous to vicilins present in seed integuments from C. ensiformis,166 P. lunatus,76 and P. vulgaris77 have also been shown to be toxic to C. maculatus. Chitin-binding lectins are entomotoxic, as shown for rice and nettle lectins and for WGA; the latter is detrimental to C. maculatus larvae and to the corn pests, Ostrinia nubilalis and Diabrotica undecimpunctata.167 Antimicrobial Peptides: Defensins Plants protect themselves against microbial attack producing many defensive antimicrobial peptides (AMPs), either constitutively or induced upon infection challenge, among which are defensins.147,168 Plant AMPs display considerable structural and functional diversity across the plant kingdom.169 Genome sequence analysis of Arabidopsis and rice revealed hundreds of genes encoding potential AMPs, indicating a considerable redundancy in AMP-based defense systems.170 Plant defensins, or c-thionins, are basic peptides, with 45– 54 amino acids residues, and eight conserved cysteine residues, forming disulﬁde bridges. They are resistant to adverse conditions, such as extreme pH (pH *2), high temperature (*858C), oxidation, and proteolytic action.23,27,171 Defensins share some structural features, as revealed mostly by nuclear magnetic resonance: a cysteine-stabilized ab motif, with three antiparallel a-strands and one a-helix,172 except for the PhD1 protein from Petunia hibrida,173–175 which is a plant defensin with ﬁve disulﬁde bonds.176 Other residues are variable, leading to a wide biological diversity of defensins so that there are antifungal23,27 and bactericidal defensins.177,178 Others have no antimicrobial activity, but inhibit a-amylases or protein synthesis,177,179,180 and can play a role in plant defense against insects.134 Despite the antimicrobial activity of some defensins, they do not appear to affect human or plant cells in vitro.24 Plant defensins are present in leaves, ﬂoral organs, pods, and seeds.22,27 Following fungal infection defensin expression is induced locally, in some cases systemically21,181 and also by external application of methyl jasmonate, but not of salicylic acid,182 hormones that are involved in signal transduction pathways associated with different pathogen-plant interactions. 378 Becker-Ritt and Carlini Fungitoxic Activity of Defensins. Plant defensins may have a morphogenic antifungal action, reducing hyphal elongation and branching, or nonmorphogenic, i.e., causing a delay in hypha elongation without morphological distortions.22 Plant defensins cause Ca+2 inﬂow and K+ outﬂow through the plasma membranes of living hyphae and alkalinization of the extracellular environment.183 Unlike insect and mammalian defensins, plant defensins do not form ion-permeable pores in membranes but rather change the electrical properties of the lipid double layer. The antifungal activity of defensins requires speciﬁc binding to the fungal membrane,184 but, contrary to mammalian and insect defensins, plant defensins do not interact directly with membrane phospholipids.185 The details of the mode of action of plant defensins, as well as possible intracellular targets, are as yet, unknown. Almeida et al.186 characterized, structurally and functionally, two pea seed defensins Psd1 e Psd2. Both exhibited a high antifungal activity against several phytopathogenic isolates, as well as against fungi that usually infect immunosuppressed patients. Some fungi that are sensitive to the action of Psds are resistant to Amphotericin B and Benomyl, agents that are traditionally used for ﬁghting opportunistic mycoses in humans, and fusariosis in the agricultural sector, respectively.187 A defensin-like antifungal peptide was puriﬁed from seeds of P. vulgaris ‘‘Cloud Bean’’ and the peptide exerted antifungal activity against Mycosphaerella arachidicola and was also active against F. oxysporum.188 Many studies of plant–pathogen interactions in gymnosperms have demonstrated a potential role for AMP family members in defense against fungal pathogens.189 In the past few years, countless defensins have been expressed in plants to increase resistance to phytopathogens. Peanut plants expressing mustard defensins were reported to have considerably increased resistance to fungi.190 Similar results were obtained with transgenic rice expressing Dahlia defensins.191 These and other studies clearly demonstrate the possibility for using defensins as resistance factors against phytopathogens. Among the many plant AMPs described so far, MiAMP1 (isolated from Macadamia integrifolia) expressed in canola plants provided protection against attack by the fungal phytopathogen Leptosphaeria maculans, cause of blackleg disease.192 In vitro, MiAMP1 had antimicrobial activity against many phytopathogenic fungi, Oomycetes and gram positive bacteria193 with a concentration range of 0.2–2 lM generally required for a 50% inhibition of growth (IC50). LJAMP1, a small heat-stable protein puriﬁed from seeds of the medicinal herb motherwort (Leonurus japonicus Houtt), inhibits in vitro the growth of an array of fungi and bacteria, with more evident effects against hyphomycete fungi, such as A. alter- nata, Cercospora personata, and A. niger. Transgenic tobacco overexpressing LJAMP1 showed signiﬁcantly enhanced resistance not only the fungal pathogen A. alternata but also the bacterial pathogen Ralstonia solanacearum, while no visible alteration in plant growth and development was observed.194 Entomotoxic Activity of Defensins. VrD1 (VrCRP) from a bruchid-resistant mungbean was the ﬁrst defensin from plant exhibiting in vitro and in vivo insecticidal activities.183 Some defensins are inhibitors of a-amylases and proteinases179,195 and can play a role in plant defense against insects. Defensins isolated from cowpea seeds display inhibitory activity toward amylases and proteinases.45,196,197 Structural analyses of cowpea defensins have shown a dependence on the high density of surface ationic residues to facilitate interaction with the catalytic site of target enzymes45,197 as well as their toxicity against bacteria pathogenic to plants and to humans.198 The essentiality of arginine and lysine residues for biological activities of defensins underlies attempts to improve the insecticidal or bactericidal properties of these molecules by site-directed mutagenesis.198 AMPs: LTPs LTPs, one of the plants AMP, are low-molecular weight (9– 10 kDa) basic (pI around 8) peptides with diverse activities and ubiquitous distribution throughout the plant kingdom.199 Structurally, LTPs are formed by four a-helices, present eight conserved cysteine residues forming four disulphide bridges, and a hydrophobic cavity capable of accommodating phospholipid, acyl CoA or fatty acid molecules of different sizes.200 As the extracellular location of a number of these proteins has been conﬁrmed,201 which makes a role in the trafﬁc of lipids across organelles unlikely, other biological functions have been proposed for LTPs in plants such as: cutin and suberin deposition, embryogenesis, adaptation to the environment, and defense against pathogens.199 Genes coding for LTPs are induced in response to pathogen infection.202 The tissue-speciﬁc expression and apoplast localization of LTP and an increase in the resistance against bacterial infection in transgenic plants expressing these peptides are in agreement with their potential role in pathogen defense.199,203–205 Nonspeciﬁc LTPs (nsLTPs) are small (91 to 95 amino acids residues) basic (pI 8–10) proteins, with no tryptophan residues and with eight conserved cysteines forming four disulﬁde bridges.206 nsLTPs also have high resistance to denaturants, heat and proteases.207 Plant nsLTP can bind a broad range of lipid molecules and as such can be used as potential drug carriers.208 In vitro antimicrobial activities of nsLTP1 Biopolymers (Peptide Science) Fungitoxic and Insecticidal Plant Polypeptides suggest a possible role in the defense of plants against bacterial and fungal pathogens.207,209 Fungitoxic Activity of LTPs. In vitro assays have demonstrated the ability of LTPs to inhibit the growth of pathogenic bacteria and fungi.20,209 LTP isolated from resting caryopsis of the weed barnyard grass Echinochloa crusgalli inhibited signiﬁcantly the development of phytopathogenic fungi P. infestans and Helminthosporium sativum, agents of the late blight of potato and tomato and the root rot of herbs, respectively.46 LTPs from sunﬂower and cucumber seeds are able to make fungal membranes permeable.47,210 The LTP of cowpea (V. unguiculata) seeds, present on the cell wall of cotyledonary and embryo cells, and in intracellular organelles, inhibited the growth of the phytopathogenic fungi F. oxysporum and F. solani.200,211 The LTPs from chilli pepper (Capsicum annuum L.) seeds exhibited strong fungicidal activity against C. albicans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, causing morphological changes with pseudohyphae formation and permeabilization of yeast plasma membrane.47 A new class of LTPs associated with the Ace-AMP1 from onion (A. cepa L.) was recently isolated from three related Allium plants: garlic (A. sativum L.), Welsh onion (A. ﬁstulosum L.), and Nanking shallot (A. ascalonicum L.).212 Nonspeciﬁc lipid transfer proteins (nsLTPs) are major lipid binding proteins in plants representing as much as 4% of the total soluble proteins.199 These proteins have been isolated from a large variety of plants, such as maize,213 castor bean,214 barley,215 wheat,216 rice,217 Amaranthus hypochondriacus,218 mung bean,219 motherwort,194 Medicago truncatula,220 pomegranate,221 sesame,222 and Narcissus tazetta.223 A nonspeciﬁc lipid-transfer peptide (nsLTP) isolated from carrot (Daucus carota sativa) seeds showed fungicidal activity against the pathogenic fungus Verticillium dahliae.224 Cumin (Cuminum cyminum) is an aromatic plant used as ﬂavoring and seasoning agent in foods. Cumin seeds have been found to possess signiﬁcant biological activities that could be ascribed to a LTP such as antibacterial,225 antifungal,226 anticarcinogenic,227 antidiabetic,228 antioxidant,227,229 and antithrombotic.226 379 residues connected in a knotted topology,232 which combined with the cyclic backbone form a structural motif known as the cyclic cystine knot,231,233 conferring to these molecules an exceptional stability.234 Cyclotides can be classiﬁed into two subfamilies, Möbius, bracelet, according to the presence or absence of a twist formation in the backbone of the peptide and the presence of a cis-Pro motif.231,235–237 In plants, cyclotides derive from precursor proteins which undergo cleavage and cyclization to produce mature molecules containing one, two or three cyclotide domains.230 This class of proteins, a group of 200 different cyclotides isolated from plants, include kalata B1,237 kalata B2,236,237 the circulins,238 cyclopsychotride,239 and several peptides from Viola species240 and recently also found in Fabaceae species.241 Cyclotide-like sequences, at the nucleic acid level, encoding six Cys residues in a similar spacing to that in known cyclotides, have been reported for variety of important monocotyledonous plants, including wheat (Triticum spp.), rice ans maize (Zea maize)242 and from the leguminous species Clitoria ternatea (Butterﬂy pea).243 Entomotoxic and Antifungal Properties of Cyclotides. The function of cyclotides in plants is so far yet poorly elucidated. The high level of expression in leaves233 and production of several isoforms within a single plant are consistent with a role in recognition or defense.230,244 Cyclotides display many biological properties that may be related to a role in plant defense, such as proteinase inhibition, cytotoxicity to tumor cells, antiviral effects, and insecticidal activities.230,236,237,245,246 Consistent with a potential role in plant defense, a cyclotide-like sequence in maize was reported to be upregulated in response to fungal infection.247 Initial assays with insect pests of the Lepidoptera family fed on artiﬁcial diets containing kalata B1 and B2, at concentrations similar to those naturally occurring in plants (0.15% w/v), demonstrated their insecticidal properties.236 Helicoverpa punctigera caterpillars fed on kalata B1-containing diets had 50% mortality rate and those surviving failed to progress past the ﬁrst instar stage.230,236 Electron micrographs showed that this insecticidal activity involved disruption of the midgut membranes of the insects.248 Cyclotides CONCLUDING REMARKS Cyclotides are small circular polypeptides composed of 28 to 37 amino acid residues with a head-to-tail cyclic backbone in which the N and C termini are linked via a peptide bond.230– 232 First reports on these proteins appeared in the early 1970s after studies on the medicinal properties of kalata-kalata, an African plant.233 Cyclotides contain six conserved cysteine Pathogens and pests can dramatically reduce crop yields. Exploring the armory of antifungal and insecticidal polypeptides that plants have developed to protect themselves against fungal pathogens and herbivorous insects represent promising alternatives to tackle hunger on a global scale by increasing the supply of plant-derived proteins. In this review, we Biopolymers (Peptide Science) 380 Becker-Ritt and Carlini give an updated overview on plant polypeptides that may be used for genetic engineering of plants aiming increased resistance to insect predation or to phytopathogens, either by overexpression of their endogenous defense proteins, or by introduction of an insect or fungal control gene derived from another plant. Several successful reports on transgenic plants with enhanced protection and yield will further develop and hopefully will appear as additions to the list of GM crops licensed to market. 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