Chapter 2 Penetration of cuticles

1. Chapter 2 Penetration of cuticles

2. Cuticle- surface wax- cuticle proper- cuticle layer Preexisting defense structure

3. Cuticles • Cutin – • found on aboveground (aerial) parts of all herbaceous plants • a principal constituent of the cuticle • Suberin – • present on underground parts • differs from cutin in that it has dicarboxylic acids, long –chain components, and phenolic compounds. • Waxes – associated with both cutin and suberin

5. Surface wax deposits

6. Cutin, waxes, and suberin are made of hydrophobic compounds. • Cutin, suberin, and their associated waxes form barriers between the plant and its environment that function to keep water in and pathogen out. • However, they do not appear to be as important in pathogen resistance. • Fungi penetrate plant surface by mechanical means. • Others secrete cutinase to hydrolyze cutin.

7. The cuticle thickness of the New Mexican-type pepper increase from 12mm in immature green fruit to 24mm in mature red fruit. The susceptibility of unwounded fruit to infection of Phytophthora capsici decreases with increased ripening.

8. Contradictory results for the involvement of cutin in pathogen resistance • Basidiospores of Uromyces vignae penetrate the cuticle of broad bean, a nonhost, much faster and with higher efficiency than they penetrate the epidermis of cowpea, a host. • The infection, however, is stopped a few hours later within the cytoplasm of broad bean (Xu and Mendgen, 1991). • Thus, successful penetration of cuticle does not ensure disease development.

9. Penetration of cuticle

11. Cutinase produced by plant pathogens Alternaria alternata Bortrytis cinerea Cochliobolus heterostrophus Colletotrichum capsici Cryphonectria parasitica Fusarium solani f.sp. pisi Helminthosporium sativum Magnaporthe grisea Monilinia fructicola Phytophthora capsici Streptomyces scabies Ulocladium consortiale Venturia inaequalis

12. Molecular properties of cutinases • Cutinase is synthesized by many fungi and bacteria. • Cutinase is an esterase that initially hydrolyzes cutin to oligomers and further hydrolyze to fatty acid monomers. • Fungal cutinase genes are triggered by plant cutin monomers.

13. Molecular properties of cutinases • Cutinases are extracellcular enzymes. • Cutinases are glycoproteins containing of 3 ~ 16% carbohydrates. • MW 18 ~ 25 kD • Despite great similarities in amino acid compositions, immunological heterogeneity exists among cutinase from different fungi.

14. Evidence that cutinases are involved in penetration of cuticles • The cutinase is present at the site of penetration. • Specific inhibition of the enzyme prevents fungal penetration. • Supplementing a cutinase-deficient mutant with cutinase restores virulence. • Inserting the cutinase gene into an avirulent pathogen enables it to infect hosts.

15. Presence of cutinase at the site of penetration • Fusarium solani f.sp. pisi vs. pea (豌豆萎凋病) • Cutinase were detected at the penetration area of pea stems by ferritin-conjugated rabbit anti-cutinase antibody. • Erysiphe graminis f.sp. hordei vs. barley (大麥白粉病) • The esterase (cutinase) released in the first stage is constitutive and that the second one is synthesized after initial contact (Kuno et al., 1990) f.sp. = Formae specialis

16. Time course of morphogenesis of Blumeria graminis infection structures resistance disease

17. Insertion of the cutinase gene into a non-producing pathogen enables it to infect the host • Mycosphaerella sp. is a parasitic fungus that produces no cutinase. • Cutinase gene from F. solani f.sp. pisi was transferred into the Mycosphaerella sp. The transformants have the capacity to infect intact papaya fruit, and this infection was prevented by anti-cutinase antibodies (Dickman and Kolattukudy, 1989)

18. Inactivation of cutinase prevents fungal penetration • Infection of pea stems by spores of F. solani f.sp. pisi has been prevented completely in the presence of either anti-cutinase serum or cutinase inhibitor DIFP. • Cryphonectria parasitica vs. chestnut (西洋栗, Chestnut blight) • Virulent strains of the pathogen do not contain double-stranded RNA (dsRNA) in their cytoplasm. Hypovirulent strains contain one or more dsRNAs. • The virulent strains produced and secreted higher amounts of cutinase than the hypovirulent strains. • The presence of dsRNAs suppresses the expression of the cutinase gene (Varley et al. 1992).

19. Contradictory results for the involvement of cutinase gene in cuticle penetration • No correlation was found between cutinase activity of Botrytis cinerea isolates and production of lesions on young tomato fruits (灰霉病gray mold, leaf blight). • Cutinase genes is not required for the fungal pathogenicity on pea. (Stahl and Schafer. 1992. Plant Cell) • Cutinase-deficient mutants were obtained from the isolate of F. solani f.sp. pisi by transformation-mediated gene disruption. These mutants showed no difference in pathogenicity and virulence on pea compared to the wild-type. These observations indicate that cutinase is not a virulence factor in F. solani f.sp. pisi (Stahl et al., 1994. MPMI).

21. Figure 6. Pathogenicity Tests of the Wild-Type of N. haematococca and a Cutinase-Deficient Mutant. (A) Symptoms on peas grown 20 days in soil infested with wild-type strain 77-2-3 and cutinase-deficient mutant 77-102 are shown in pot 2 and pot 3, respectively. Each pot was infested with 5 x 106 conidia. Yellowing of basal foliage and stunted growth of the above-ground plant parts were caused to the same extent by both fungi. Uninoculated control plants are shown in pot 1. Ten to 15 replicate plants were used per treatment. The experiment was repeated three times with results similar to those shown here. (B) Detailed picture of the root and the lower stem of plants shown in (A). The root and lower stem of an uninoculated control plant are shown at left. The dark brown of upper tap root and the below-ground epicotyl at center is caused by infection with the wild-type strain. The same symptoms are caused by infection with the cutinase-deficient mutant 77-102, as shown at right. (C) Lesions on etiolated pea segments, caused by germinating conidia of wild-type strain 77-2-3 (left) and null mutant 77-102 (right). Uninoc. Mutant WT Uninoc. WT Mutant Mutant WT

22. Correlation between cutinase production and virulence of the pathogen • F. solani f.sp. pisi mutants with reduced cutinase activity are less virulent (Rogers, Flaishman, and Kolattukudy. Plant Cell 1994).

24. Figure 6. Infection of Pea Seedlings by F s. pisi 77-2-3 and Cutinase Gene-Disrupted Mutant 77-102. (A) Representative intact seedlings of an uninoculated control and seedlings inoculated with 77-2-3 or its gene-disrupted mutant 77-102. (B) All seedlings from one experiment in which seedlings were grown on vermiculite inoculated with 77-2-3 or its mutant 77-102. (C) The stems of seedlings inoculated with 77-2-3 (left) or its mutant 77-102 (right). Stems inoculated with 77-2-3 show severe lesions, and those inoculated with mutant 77-102 exhibit a limited number of lesions.

25. The drawbacks of Sthal & Schaffer’s experiment • Multiple spore levels were not tested. • Microscopic examination of the progression of lesions.

26. Mechanical force as a means of direct penetration • Many plant-pathogenic fungi form appressoria prior to penetration of plant tissues. • Appressoria first adhere to the host surface and then produce infection pegs that pierce the underlying plant cuticle and cell wall.

27. Main function of appressoria • To anchor the fungus firmly to the plant • To penetrate the surface either directly through the cuticle or through natural openings

28. The methods for penetration • Physical force • Hydrophobins • Melanisation (by DHN-melanin) • Turgor pressure • Cell wall-degrading enzymes e book, P.38-42

29. Hydrophobins • A pathogenicity gene, MPG1, is highly expressed during appressorium formation has been identified in Magnaporthe grisea (稻熱病). • The gene encodes a 15 kD hydrophobin (96-187 aa) that contains 8 cysteine residues. • Hydrophobin interacts with the hydrophobic rice surface and undergoes polymerisation which provides protection against desiccation. • acts as a developmental sensor for appressorium formation. • However, B. cinerea has no hydrophobin, whilst C. fulvum has hydrophobins but does not form appressorium.

30. Melanisation • Dihydroxynaphthalene (DHN)-melanin is a dark fungal pigment that deposites between fungal cell membrane and the appressorium cell wall and binds to chitin. • The role of melanin is to seals the appressorium and to retard the efflux of glycerol from the appressorium, allowing hydrostatic turgor to build up. • By 24-31 h after development, appressorium maturation and turgor generation occur. • A penetration peg emerges and extend from the pore into the cuticle.

31. Time course of morphogenesis of Blumeria graminis infection structures resistance disease

32. END