Lecture 5 - Cell Wall Degrading Enzymes - BIOL 3025

BIOL 3025: Molecular Plant Pathology: Lecture 5 – Biochemical methods: Cell wall degrading enzymes 1  LEARNING OUTCOMES At the end of this lecture, you should be able Describe the main structural and chemical components of the primary plant...

BIOL 3025: Molecular Plant Pathology: Lecture 5 – Biochemical methods: Cell wall degrading enzymes 1  LEARNING OUTCOMES At the end of this lecture, you should be able Describe the main structural and chemical components of the primary plant cell wall and middle lamella List the various types of cell wall-degrading enzymes used by plant pathogens Explain the role of wall-degrading enzymes and pectic enzymes in pathogenesis Give examples of pathogens which use such enzymes 2 4  Hemicellulose Hemicelluloses are classified by the main sugar in its polymer backbone: 1. Xylan (β-1,4- linked D-xylose), 2. Mannan (β-1,4-linked D- mannose), 3. Xyloglucan (β-1,4-linked D- glucose) 5  Hemicellulose Hemicelluloses structure may vary by plant species Hemicelluloses include xyloglucans, xylans, mannans and glucomannans, and β- (1→3,1→4)-glucans 6  Monomers in Hemicellulose http://www.rsc.org/images/Gross_Box_hemicellulose_tcm18-150454.jpg 7  Pectin The pectin backbone consists mainly of alpha-1,4-linked D-galacturonic acid residues They may be methyl-esterified or substituted with acetyl groups. 8  A polymer of polyglacturonic acid with side chain = Pectin http://www.frontiersin.org/files/Articles/66284/fphar-04-00128-HTML/image_m/fphar-04-00128- 9 g001.jpg  Pectin sugars Pectins are classified in three general groups HG or homogalacturonan (linear polymer), Xylogalacturonan (branched by β- 1,3-linked D-xylose) RG-I or RG II-or rhamnogalacturonan 10  HG is responsible for the wall rigidification RG-I may play a role in cell wall plasticity -for example, by preventing HG chains to interact with Ca2+ ions. 11 Vascular wilts and Roots pathogens Many vascular wilt and root pathogens, such as Verticillium alboatrum, Verticillium dahlia, N. haematococca, and Fusarium oxysporum, tend to have more pectinases. They may block or collapse vascular bundles during disease Fusarium wilt of tomato can cause a vascular development. discoloration on one side of the stem. fusarium wilt tomato 12  Surface layer degradation Cutinase- involved in the degradation and penetration of of cutin - Used by some pathogenic fungi eg Botrytis cinerea , Fusarium species, Colletotrichum spp. Cutinases can hydrolyze the ester bonds of the plant polymer cutin Lipases- involved in germination of some pathogens by degrading waxy layers eg used by Sclerotinia sclerotiorum in bean leaves Kuonoh et. al. Cutin= C16 and C18 fatty acids attached to a glycerol backbone 14 (1988)  Cutinase The active site of cutinases consists of a catalytic triad of Ser, Asp, and His and is highly conserved in fungal pathogens viz. biotrophs, hemibiptrophs, and necrotrophs. Fungal spores sense that they have landed on the host plant surface and remain dormant until they sense suitable conditions for infection. Cutin monomers are recognized by the spore surface, leading to the production of cutinases Constitutive-type cutinases alter the host plant surface to allow successful spore adhesion and release cutin monomers from the cuticle layer that are essential for subsequent stages of infection. Some fungi e.g. Erysiphe graminis f. sp hordei and Blumeria gramini use these monomers during early-stage infection to promote germ tube assembly and appressoria differentiation. 15  Cell Wall degrading enzymes (CWDE’s) Cell Wall Many pathogenic fungi actively kill and degrading degrade plant tissue and utilize liberated ezymes carbohydrates for growth and reproduction. Cellulases Hemicellulases Pectinases Examples Ligninases Endo- and exo-acting enzymes Proteases 16  Cellulose β C1-C4 bonds Basidiomycetes tend to have more cellulolytic enzymes than Ascomycetes Major Classes of Cellulases 1. Exoglucanases (cellulose 1,4-β-cellobiosidases) glucose linked by a β– (1,4′) glycosidic bond 2. endoglucanases 3. β-glucosidases cellobiose The final step of cellulose degradation is that β-glucosidase cleaves cellobiose into two molecules of glucose 18 Cellulose degradation β1,4-endoglucanase (EGL) Exoglucanase / Cellulose degradation Cellobiohydrolase (CBH) β-glucosidase (BGL) 19  Hydrolysis of Hemicellulose Fungi use xylanases to degrade the linear polysaccharide β-1,4-xylan into xylose Enzymes such as: arabino furanosidases , acetylxylan Esterases, ferulic acid esterases, and α-glucuronidases remove side groups from the xylose xylose backbone. 20  Hydrolysis of the Hemicellulose backbone xylan xyloglucan Mannan xyloglucanactive β-1,4- β-1,4- β-1,4- endoxylanase endomannanase endoglucanase β-1,4- β-1,4- β-1,4-xylosidase glucosidase mannosidase The three types of hemicellulose backbones are hydrolysed by a specific set of enzymes. 21 22  Pectin hydrolysis The degradation of pectin backbones requires two classes of enzymes: glycoside hydrolases and polysaccharide lyases fungal glycoside hydrolases involved in the degradation of the pectin backbone belongs to GH family 28 Endo- and exo-polygalacturonases of GH28 cleave the α-1,4-glycosidic bonds between the α-galacturonic acids. Pectin and pectate lyases both cleave, via a β-elimination mechanism, the α- 1,4-linked D-galacturonic acid residues within the smooth regions of pectin 23 24 Hydrolysis of Pectin Polygalacturonase also known as pectin depolymerase (PG) or pectinase; pectin lyase and pectate lyase can cleave the glycosidic bonds between D-galacturonic acid residues in polygalacturonan of pectin 25  Pectinases Polygalacturonidases (PGs) belonging to GH28 family, can hydrolyse of the α1,4-glycosidic bonds between the α-galacturonic acid Pectin lyases attack preferentially heavily methyl-esterified substrates and have their optimum pH around 5.5 (Mayans et al. 1997). In contrast, pectate lyases favor lower degrees of esterification, have their optimum pH around 8.5, and require Ca2+ for their activity 26  Pectinases Enzyme Substrate Pectinases Pectin matrix Pectin lyase (PnL) splits α-1,4-linkage between methylgalacturonides Pectin methyl pectin and pectinic acid to form pectate esterase (PME) Pectate lyase ( PL) split the α-(1-4) linkage between galacturonosyl residues in pectate (homogalacturonan, HGA). Polygalacturonase attacks α-1,4-glycosidic bonds of pectate by hydrolytic (endo- & exo- PG) cleavage. 27 http://opencourse.ndhu.edu.tw/pluginfile.php/962/mod_resource/content/0/Ch_3_Cell_wall_degradation.pdf 28 http://opencourse.ndhu.edu.tw/pluginfile.php/962/mod_resource/content/0/Ch_3_Cell_wall_degradation.pdf 29  Pectate degradation Pectate degrading enzymes in culture filtrates of Fusarium oxysporum f.sp. lycopersici can cause the following disease symptoms ; ̶ wilting of shoot and leaves, ̶ chlorosis of vascular system, ̶ decrease of transpiration in infected plants. 30 31  Some fungal CWDE’S Cellulase Pectinase Hemicellulase Pectin methyl esters http://www.frontiersin.org/files/Article s/87127/fpls-05-00228- HTML/image_m/fpls-05-00228- g002.jpg 32 Carbohydrate-active enzymes (CAZymes) Fungi produce several CAZymes for the degradation of plant polysaccharide materials to facilitate infection and/or gain nutrition. They play a role in pathogenicity. Four functional classes of CAZymes : 1) Glycoside hydrolases (GHs), - hydrolyze the glycosidic bond between two or more carbohydrates, or between a carbohydrate and a noncarbohydrate moiety, such as a protein, or a lipid 2) Glycosyltransferases (GTs), 3) Polysaccharide lyases (PLs), - mainly degrade glycosaminoglycans and pectin 4) Carbohydrate esterases (CEs)- catalyze the de-O or de-N-acylation of 33 esters or amides Necrotrophs and CWDE’s CWDE’s are found in a wide range of necrotrphic fungi eg Botrytis and Erwinia These pathogens have a wide host range Erwinia causes tissue rots of many fruits and vegetables Botrytis soft rot on apples 34  Erwenia (Dickeya) chrysyanthemi Wide host range; ― maceration of plant tissue ― many pectin-degrading enzymes, ― PME, pectin acetyl esterase, PL, PG out operon – required for Soft rot of tomato by E.chrysanthemi Stems hollow and often collapse, with a pathogenicity dark external discoloration Source: http://erec.ifas.ufl.edu/tomato-scouting- guide/diseases/erwinia-soft-rot.shtml 35  36 http://www.apsnet.org/publications/apsnetfeatures/Article%20Images/12-34.jpg http://cdn.c.photoshelter.com/img-get/I0000Pmk3QpqeGjM/s/600/600/3050514.jpg 37  Polygalacturonase inhibiting proteins, (PGIPs) Plants produce proteins to inhibit microbial CWDE as one mechanism of disease resistance Microbial CWDEs inhibiting proteins such as xylanase inhibitor protein (XIP), or xyloglucan endoglucanase inhibiting protein (XEGIPs) Extracellular leucine-rich repeat (LRR) proteins that recognize and inhibit fungal polygalacturonases (PGs). 38 REFERENCES Jafra, S., Figura, I., Hugovieux-Cotte-Pattat, N., and Lojkoswka, E. (1999). Expression of Erwinia chrysanthemi pectinase genes pelI, pelL and pelZ during infection of potato tubers. MPMI vol. 12: 845-851 Lucas (1998) Plant Pathology and Plant Pathogens 3rd Edition Blackwell Publishing Bellen, T., Van Campenhout, S., Robben, S. and Volckaert, G (2007). Microbial Endoxylanases: Effective Weapons to Breach the Plant Cell-Wall Barrier or, Rather, Triggers of Plant Defense Systems. MPMI Vol. 19, No. 10, 2006, pp. 1072– 1081. DOI: 10.1094 / MPMI -19-1072 39