Tomatinase from Cladosporium fulvum is required for full virulence by degrading a-tomatine during tomato infection

PO-29 Tomatinase from Cladosporium fulvum is required for full virulence by degrading a-tomatine during tomato infection Bilal Ökmen, Desalegn Woldes Etalo, Jérôme Collemare, Ioannis Stergiopoulos, Ric CH de Vos, Matthieu HAJ Joosten and Pierre JGM De Wit a) Laboratory of Phytopathology, Wageningen University,Droevendaalsesteeg 1, 6708 PB, Wageningen, The Netherlands b) Centre for BioSystems Genomics, P.O. Box 98, 6700 AB Wageningen, The Netherlands c) Plant Research International Bioscience, PO Box 16, 6700 AA, Wageningen, The Netherlands Plants produce several secondary metabolites with antimicrobial activity to protect themselves against fungi. a-tomatine is a steroidal glycoalkaloid presents in tomato up to 1 mM concentration. It is hypothesized that a-tomatine forms a complex with fungal membrane sterols , resulting in pore formation and loss of membrane integrity. However, Fusarium oxysporum is able to fully infect tomato due to the secretion of tomatinase. This enzyme cleaves a-tomatine into lycotreaose and tomatidine, which are non-toxic compounds. Cladosporium fulvum is a biotrophic fungus that causes tomato leaf mold. During infection C. fulvum secretes proteins to successfully colonize the apoplast of tomato leaves. Analysis of C. fulvum genome showed the presence of a single homologue (TOM) of the F. oxysporum tomatinase gene that is predicted to be secreted. Also, in vitro biochemical assay showed that C. fulvum culture filtrate could cleaves a-tomatine into lycotreaose and tomatidine. To study the role of the TOM gene in the virulence of C. fulvum, targeted gene disruption was performed for C. fulvum TOM. In the mutants, activity of tomatinase for cleavage of a-tomatine into lycotreaose and tomatidine was abolished in vitro. Also, spores germination assay showed that ¿-tom mutants were more sensitive to a-tomatine than the wild type. Tomato plants infected with the ¿-tom mutants showed a delay in the disease process, indicating that tom gene is required for the full virulence of C. fulvum. Our results indicate that detoxification of a-tomatine by C. fulvum is required for full virulence on tomato.