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Antimicrobial peptides in the management of plant diseases.pptx
- 1. “UNLOCKING THE USE OFFUNCTIONAL PEPTIDES IN PLANT DISEASE CONTROL” Master’s Seminar on CSK Himachal Pradesh Krishi Vishvavidyalaya PalFunctional Peptidesur, H.P., India, 176062 Seminar In charge: Dr. Deepika Sud SMS(Plant Pathology) Speaker: Tanish Dhiman (A-2023-30-091) Msc. 2nd Year
- 2. OUTLINE •Introduction •History of Functionalpeptides •Sources of Functional peptides •Structure of Functional peptides •Classification Functional peptides •Mechanism of Functional peptides •Functional peptides in plant defense mechanism •Synthetic Functional peptides •Drawbacks of Functional peptides •Conclusion & future prospects
- 3. INTRODUCTION Functional peptides arepolypeptides having 12–50 amino acids sequence and also known as anti-microbial peptides. Part of natural immunity in plants First line of defense against phytopathogens (Tang et al., 2018; Zaslof, 2002) Positively charged and are diverse group of plant proteins Functional Peptidesipathic structure Signifcantly affect plant growth and development (Berrocal-Lobo et al., 2002; de Zélicourt et al., 2007; Fernandez et al., 1972).
- 4. HISTORY OF FUNCTIONALPEPTIDES 1939- Dubos extracted an anti microbial agent Bacillus from soil against Pneumococci 1940- Named this extract as Gramicidin 1942- First plant originated Functional Peptides was isolated by Balls et al., from wheat Named Purothionin and found effective against fungi and bacteria 1970- Okada and Yoshizumi isolated Hordothionin from endosperm of barley
- 5. SOURCE OF FUNCTIONALPEPTIDES Plants
- 6. Origin Source FunctionalPeptides Animal Cow, Frog skin Lactoferrin, Esculentin-1 Insect Moth haemolymph, Fruitfly Cercopin A,B Cercotoxin Plant Wheat Thionin (Fernandez et al,. 1972; Hughes et al., 2000) Barley, Radish Hordothionin, Rs-AFP2 Bacteria Pseudomonas, Pantoea Pseudophomins, Pantocins Human Epithelia Bombinin
- 7. STRUCTURE OF FUNCTIONALPEPTIDES Alpha helix Beta sheet Extended Loop
- 8. CLASSIFICATION OF FUNCTIONALPEPTIDES • Non disulfide bridged peptide • Peptides with disulfide group 1. Based on their structure • Synthetic non ribosomal peptides • Synthetic ribosomal/ Natural peptides 2. Based on their nature • Cationic peptides • Non cationic peptides 3. Based on their electrostatic charge 4. Based on their target microorganism • Antifungal • Antibacterial • Antiviral • Antiparasitic
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- 10. Antibacterial MECHANISMS OF FUNCTIONALPEPTIDES Xuan et al., 2020
- 11. Xuan et al.,2020
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- 13. Antiviral Surface target 1.Interaction aminoglycanswith virus 2.Blocking of viral entry into the cell 3.Suppression of cell fusion by interfering with the activity of ATPase protein. Intracellular target 4.Suppression viral gene expression 5.Inhibition of peptide chain elongation by inactivating the ribosome. Viral protein targets: 7.Binding of peptides to viral proteins causing inhibition of adsorption. TMV Mulder et al., 2013
- 14. FUNCTIONAL PEPTIDES INTHE PLANT DEFENSE MECHANISM
- 15. TYPES OF FUNCTIONALPEPTIDES
- 16. 1. THIONIS Balls etal. (1942) identified thionins for the first time in cereals and classified as plant toxins due to their toxic effect towards microbes. Thionins consist of 45-48 amino acids, 6 or 8 cysteine and 3 or 4 disulfde bonds (Stec 2006). Around 100 individual thionin sequences have been identified in more than 15 different plant species Hydrophobic Mostly Cationic Anti fungal and Antibacterial
- 17. CLASSIFICATION OF THIONINS Sixcysteine residue Three disulphide bridges Crambin Viscotoxins Phoratoxins Eight cysteine residue Four disulphide bridges /- purothionins / hordothionins Hellethionin-D
- 18. MODE OF INTERACTIONOF THIONINS thionins mechanism of action, by using glucosylceramides as receptors for fungi cell membrane insertion. Glucosylceramides
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- 20. SUPPORTIVE ARTICLE When flowersare inoculated with F. graminearum, the Thi2.4 protein had an antifungal effect on F. graminearum. They purified the Thi2.4 protein, conjugated it with glutathione-S-transferase (GST) . Total protein from F. graminearum was applied to GST-Thi2.4 and the fungal fruit body lectin (FFBL) of F. graminearum was identified as a Thi2.4- interacting protein. By contrast, FFBL-induced host cell death was effectively suppressed in transgenic plants that over expressed Thi2.4. Asano et al., 2013
- 21. Thi2.4 protein waspresent in flowers and flower buds, but not leaves or inflorescence stems. The molecular mass of the Thi2.4 protein was about 15 kD. 1. 2. 4. 3. F – FFBL M- Control
- 22. 2. DEFENSIN •In 1990,Mendez et al. isolated from barley and wheat and named as Defensin. •Small, cystine rich Functional peptides ranging from 45-54 amino acids. •Defensins consist of 3-5 disulfide bonds. •Abundantly present in the stomatal and peripheral cells. • Expression of plant defensins are also induced by abiotic stress and signalling molecules.
- 23. Based on theireffect on pathogenic fungi Morphogenic plant defensins Inhibit the growth and branching of hyphae Non- morphogenic plant defensins Inhibit only hyphal growth
- 24. MODE OF ACTIONOF DEFENSIN Khan et al., 2019 The membrane permeabilization and ion leakage from the membrane occurs due to the interaction of serine residue with glycosyl part of the fungal cell membrane (de Paula et al. 2011). Antifungal activity of NAD1 involves in the membrane permeabilization of hyphae of the pathogen Fusarium oxysporum. Dimer formation was observed in NaD1 defensin.
- 25. Ex-DEFENSINS Functional Peptides Source Transgenic plant Pathogen testedReferences BrD1 Brassica rapa Rice Fusarium graminearum Choi et al., (2009) RsAFP2 Raphanus sativus Wheat/Rice Magnaporthe oryzae Rhizoctonia solani Jha and Chattoo et al., (2010) MsDef1 Medicago sativa Tomato Fusarium oxysporum Phytophthora parasitica Abdallah et al., (2010) NmDef02 N. megalosipho n Tobacco/ Potato Phytophthora infestans Alternaria solani Portieles et al., (2010) DEF2 Capsicum annum Tomato Botrytis cinerea Stoz et al., (2009)
- 26. SUPPORTIVE ARTICLE •Transgenic rice(Oryza sativa L. cv. Pusa basmati 1), overexpressing the Rs- AFP2 defensin gene from the Raphanus sativus was generated by Agrobacterium tumefaciens-mediated transformation. • It was observed that constitutive expression of Rs-AFP2 suppresses the growth of Magnaporthe oryzae and Rhizoctonia solani by 77 and 45%, respectively. Jha and Chattoo (2009)
- 27. ELISA of T2-transgenicrice plants expressing RsAFP2 Sub-cellular localization of Rs-AFP2 was determined by treating the transgenic leaf and root sFunctional Peptidesle with anti-Rs-AFP2 Leaf tissue Root tissue Transgenic Control
- 28. Significant changes inthe hyphal morphology Effects of the overexpression of RsAFP2 on resistance of transgenic plants to fungal pathogens. DLA : Diseased leaf area M. oryzae R. solani Rs-AFP2 Transgenic line Control
- 29. 3. LIPID TRANSFERPROTEINS (LTP) • Small cysteine rich peptides having molecular masses of lower than 10KDa. • It consists of 70-100 amino acid • Cationic peptides with a conserved pattern of four to five disulfide bridges having eight to ten cys-cys bonds. • LTPs having synergistic activity with thionins against Clavibacter spp. •Expression of lipid transfer proteins can be induced by abiotic stress.
- 30. Possible mechanisms behindthe roles of non-specific lipid-transfer proteins (nsLTPs) during plant–pathogen interactions Mode of action of LTP
- 31. Lipid transfer proteins Source Targetpathogen Reference Ca-LTP Capsicum annum Canida albicans, Saccaromyces cerevisiae Cruz et al., (2010) Ps-LTP1-3 Pisum sativum Fusarium solani, Fusarium oxysporum Bogdanov et al., (2016) Gt-LTP2 Gentiana triflora Botrytis cinerea Akinori kiba et al., (2011) Ace-Functional Peptides1 Allium cepa Fusarium oxysporum Cammue et al., (1995) Cc-LTP-1 Coffea canephora seeds Candida albicans Zottich et al., (2011) Ex- LIPID TRANSFER PROTEINS
- 32. 4. HEVEIN likepeptides •Archer isolated hevein like plant Functional Peptides from rubber tree latex. •Heveins are small Functional proteins of 42-45 amino acids and of 4.7 Kda with conserved residues of glycine and aromatic acids. •They are cationic proteins having 3- 5 disulfide bonds. •The N-terminal region contains a chitinbinding hevein domain
- 33. Based on thenumber of cysteine residues Three disulfide bridge Six cysteine residue 6C- hevein Four disufide bridge Eight cysteine residue 8C- hevein Five disulfide bridge Ten cysteine residue 10C- hevein
- 34. MODE OF ACTIONOF HEVEIN Chitinase Fungalysin WAMP’S
- 35. Hevein Source Againstpathogen Reference M-hevein Mulberry Trichoderma viride Zhao et al., (2011) GAFP G. biloba Fusarium graminearum Alternaria alternata Huang et al., (2000) WjFunctional Peptides1 Wasabia japonica Fungi: Botrytis cinerea Fusarium solani Magnaporthe grisea Alternaria alternata Bacteria: Escherichia coli Agrobacterium tumefaciens Kiba et al., (2003) Ex- HEVEIN like peptides
- 36. 5. KNOTTIN typepeptides ‑ • Nguyen et al. (1990) isolated it from Mirabilis jalapa. • The typical structure of knottins involves conserved disulfde bonds between multiple cysteine pairs, forming a cystine knot. • Generally, knottin-type peptides are the smallest in size among plant Functional Peptidess. • Promote resistance to biotic and abiotic stresses, stimulating root growth, acting as signaling molecules, and enhancing symbiotic interactions
- 37. Knottins Source Against pathogen Reference Cy-Functional Peptides1 Cycasrevoluta Fungi and bacteria Yokohama et al., (2009) Wr-A11 Wr-A12 Wrightia religiosa Tenebrio molitor Nguyen et al., (2014) Ex- KNOTTIN-like Peptides
- 38. OTHER FUNCTIONAL PEPTIDES PuroindolinesSnakins Cyclopeptides • Contain a unique tryptophan-rich domain • These proteins were isolated from wheat endosperm. • PIN-1 is able to form ion channels in membrane. Ca2+ ions modulate channels formation and/or opening (Charnet et al. 2003) • Membranotoxin • Isolated from potato tubers. They comprise the cell wall- associated peptide snakin-1 (StSN1) and snakin-2 (StSN2). • Do not interact with artificial lipid membranes. • StSN1 gene does not respond to abiotic stress. •Most of Snakin genes are regulated by plant hormones (Nahirñak et al., 2012). •Cyclic proteins of about 28-37 aminoacids. • Composed of amino acid residues arranged in a cyclic ring, usually without disulphide bridges. • Lipidic cyclopeptides (LCPs) are produced by several plant-associated and soil-inhabiting bacteria.
- 39. Puroindoline Susceptible speciesReferences PINA and PINB from wheat Fungi: Alternaria brassicicola Marino et al., (2009) Botrytis cinerea Verticillium dahliae Cochliobolus heterostrophus Zhang et al., (2011) PINA from wheat Bacteria: Erwinia amylovora Jing et al., (2003) Staphyllococcus aureus Dhaliwal et al,. (2009)
- 40. •Pina and Pinbwere introduced into corn. •Pina⁄Pinb expression–positive transgenic events were evaluated for resistance to Cochliobolus heterostrophus, the corn southern leaf blight (SLB) pathogen. SUPPORTIVE ARTICLE Zhang et al., (2010)
- 41. Positive lines Mo17: Resistant,A188: Susceptible, A679: Moderately resistant, B73: Moderately susceptible,
- 42. Snakins Susceptible speciesReferences StSN1 and StSN2 from Solanum tuberosum Fungi: Botrytis cinerea Fusarium solani Fusarium oxysporum F sp conglutinans Bacteria Clavibacter michiganensis Ralstonia solanacrarum Berrocal Lobo et al., (2002) Ex- SNAKINS
- 43. Cyclopeptides Origin Target pathogen Reference Syringomycins andsyringopeptins Pseudomonas sp Botrytis cinerea Lavermicocca et al., (1997) Pseudomonas syringae Venturia inaequalis Burr et al., (1996) Tolaasins Pseudomonas sp Rhizoctonia solani, Rhodococcus fascians Bassarello et al., (2004) Pseudophomins Pseudomonas fluorescens BRG100 Sclerotinia sclerotiorum Pedras et al., (2003) Massetolide Pseudomonas fluorescens SS101 Phytium intermedium de Souza et al., (2003) Ex-Functional Peptides of CYCLOPEPTIDES
- 44. PSEUDOPEPTIDES Amide of anamino acid that does not occur in natural peptides. Having few peptide bonds and complex amino acid modification. Produced by bacteria.
- 45. Ex- PSEUDOPEPTIDES Pseudo peptides Source Against pathogens Reference BlasticidinsBacillus cereus K55-S1 Pyricularia oryzae Copping et al., (2000) Mildiomycin Streptovertticillium rimofaciens B-98891 Powdery mildew pathogen Copping et al., (2000) Pantocines Pantoes agglomerans Erwinia amylovora Jin et al., (2003)
- 46. SUPPORTIVE ARTICLE The bioactivityof MIL-C against powdery mildew disease in vivo and in vitro were examined systematically and compared to MIL and triadimefon. Huang et al., (2010)
- 47. MIL- C Tridiamefon Eradicating effect of MIL,MIL-C and triadimofon against powdery mildew (Sphaerotheca fuliginea) on leaves of greenhouse grown cucumber. (MIL: 150 mg/L; MIL-C: 50 mg/L; Triadimefon: 200 mg/L) 1 week after first spray; 1 week after second spray; 2 weeks after second spray 1. 2. 3.
- 48. BACTERIOCINS • Ribosomal synthesized Functionalpeptides. • Produced by bacteria. • Kill or inhibit closely related bacterial strains. PEPTAIBOLS • Non ribosomal synthesized Functional linear peptides. • Produced by fungi • Affect fungi and plant pathogenic gram positive bacteria. SYNTHETIC FUNCTIONAL PEPTIDES
- 49. EX- Synthetic FunctionalPeptides Functional Peptides Source Against pathogen 1. Bacteriocins Galtrol Agrobacterium radiobacter Agrobacterium tumefaciens Nogall Agrobacterium radiobacter K1026 Agrobacterium tumefaciens Histick N/T Bacillus subtilis St MB1600 Fusarium, Rhizoctonia, Aspergillus 2. Peptaibols Trichokonins Trichoderma koningii Clavibacter michiganensis,Fusarium, Botrytis, Bipolaris Harzianins and Trichorzins Trichoderma harzianum Sclerotium cepivorum
- 50. NOGALL COMPANION SERENADE SUBTILE COMMERCIAL PRODUCTSOF FUNCTIONAL PEPTIDES
- 51. Resistance toFunctional Peptidess by pathogens such as Pectobacterium carotovorum, Dickeya dadantii (PhoH, PhoP, PhoS genes) In vitro testing of the leaf extracts from the plants expressing cationic peptides shows that the expressed peptides are unstable or degraded by proteases (Cary et al., 2000; Li et al., 2001). Lack of the strategies needed to optimize expression and stability of expressed peptides in transgenic plants. High extraction cost DRAWBACKS
- 52. •The best strategyfor providing enhanced and broad-spectrum resistance using cationic peptides is the expression of different molecules at different stages of disease development. •Combinations of potential peptides may be a successful strategy for generating broad-spectrum disease resistance, including resistance against viruses in plants. •Strategies for a regulated and/or inducible, and tissue-specific expression of peptides, may prove to be effective for better performance in the greenhouse as well as in the field •Successful applications of a transgenic approach using these peptides to control plant diseases, particularly viruses, will likely help eradicate certain plant diseases, reduce the environmental impact of intensive agricultural practices. Conclusion and Future Prospects
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