Molecular Plant Pathology Lecture 5

Molecular Plant Pathology: Lecture 5 - Biochemical Methods: Cell Wall Degrading Enzymes

• Learning Outcomes: Students should be able to describe primary plant cell wall and middle lamella components, list cell wall-degrading enzymes used by plant pathogens, explain the role of these and pectic enzymes in pathogenesis, and give pathogen examples that use these enzymes.

Plant Cell Wall Structure

• Primary Cell Wall: Contains cellulose microfibrils, hemicellulose, and pectin as major components.
• Middle Lamella: Contains pectin, acts as a glue between adjacent plant cells.

Cell Wall-Degrading Enzymes

• Types: Cellulases, hemicellulases, pectinases, ligninases, endo- and exo-acting enzymes, and proteases are used by plant pathogens.
• Functions: Degrade plant cell wall components, facilitating pathogen entry and colonization. This enables the release of carbohydrates for pathogen growth and reproduction.

Hemicellulose

• Classification: Based on the main sugar in the polymer backbone: xylan (β-1,4-linked D-xylose), mannan (β-1,4-linked D-mannose), and xyloglucan (β-1,4-linked D-glucose).
• Structure Variation: Hemicellulose structure varies by plant species.

Pectin

• Backbone: Primarily composed of alpha-1,4-linked D-galacturonic acid residues, which can be methyl-esterified or substituted with acetyl groups.
• Classification: Pectins are categorized into homogalacturonan (HG), xylogalacturonan, and rhamnogalacturonan (RG-I or RG-II) based on their structure.
• Role: HG is responsible for plant cell wall rigidity, while RG-I potentially influences cell wall plasticity.

Pathogens and CWDEs

• Specific Pathogens: Many vascular wilt and root pathogens (e.g., Verticillium alboatrum, Verticillium dahlia, N. haematococca, Fusarium oxysporum) often exhibit higher pectinase activity.
• Effect on Vascular Bundle: Pathogens might block or collapse vascular bundles during disease development.

Surface Layer Degradation

• Cutinase: Involved in cutin degradation and penetration in some pathogens (e.g., Botrytis cinerea, Fusarium, Colletotrichum).
• Cutin: Hydrolyzes the ester bonds of the plant polymer cutin.
• Lipases: Used by some pathogens to degrade waxy layers, crucial for germination, exemplified by Sclerotinia sclerotiorum.

Cutinase Mechanism

• Active Site: Serine, aspartic acid, and histidine residues comprise the highly conserved catalytic triad of cutinases.
• Fungal Spores: Recognising cutin monomers and initiating cutinases production is vital for spores in order to successfully infect plants.

Cellulose Degradation

• Cellulose: Composed of glucose linked by β-(1,4') glycosidic bonds. Degraded through sequential cleavage by different classes of enzymes, including endoglucanase, exoglucanase, and β-glucosidase.
• Classes of Cellulases: Exoglucanases (cellulose 1,4-β-cellobiosidases) , endoglucanases, and β-glucosidases.

Hemicellulose Hydrolysis

• Fungi: Use xylanases to degrade the linear β-1,4-xylan polysaccharide into xylose.
• Enzymes: Additional enzymes facilitate the process (e.g., arabino-furanosidases, acetylxylan esterases, ferulic acid esterases, and a-glucuronidases)

Hydrolysis of Hemicellulose Backbone

• Xylan, Xyloglucan, Mannan: Specific endoglucanases for xylan, β-1,4-endoglucanases for xyloglucan, and β-1,4-endomannanases for mannan.
• Other Enzymes: Xylosidases, glucosidases, or mannosidases may complete the degradation process.

Pectin Hydrolysis

• PGs or Pectin Depolymerases (PG), pectin lyase and pectate lyase : Involved in cleaving a-1,4 glycosidic linkages between D-galacturonic acid, crucial for their degradation.

Pectinases

• Polygalacturonidases (PGs): Typically part of the GH28 family, they specifically hydrolyze a-1,4-glycosidic linkages within a-galacturonic acid chains.
• Pectin Lyases: Preferentially hydrolyze heavily methyl-esterified substrates, optimal pH around 5.5.
• Pectate Lyases: Favor lower degrees of esterification, have optimal pH around 8.5, and require calcium ions for activity.

Pectate Degradation

• Symptoms (Fusarium oxysporum): Cause wilting of shoots and leaves, chlorosis of vascular system, and decrease in transpiration in infected plants.

Other Important Enzymes and Concepts

• CAZymes: Carbohydrate-active enzymes, instrumental in degrading plant polysaccharides by fungi during infection, essential for pathogenicity.
• PGIPs: Polygalacturonase-inhibiting proteins, produced by plants, which restrain microbial CWDEs in a defense mechanism.
• Necrotrophs and CWDEs: Necrotic pathogens can display a broad host range due to their extensive CWDE repertoire.

Pathogen Erwinia chrysanthemi

• Wide Host Range: Causes tissue rots in many fruits and vegetables; high pectinase production enables widespread tissue maceration.
• Operon Required for Pathogenicity: Operons encode critical pectin-degrading enzymes (PG, PMEs, PLs) for effective pathogenicity.

Bacterial Infection of Plant Tubers

• Spread Mechanisms: Bacteria from infected tubers can spread to new plants through various means (insects, soil, etc). Infection often through wounds.