Secreted serine and metalloproteases are required for virulence in Verticillium dahliae

PO-38 Secreted serine and metalloproteases are required for virulence in Verticillium dahliae Parthasarathy Santhanam, Mojtaba Keykhasaber, and Bart P H J Thomma Laboratory of Phytopathology, Wageningen University, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands. Vascular wilt diseases caused by soil-borne pathogens are among the most devastating plant diseases worldwide. The fungus Verticillium dahliae causes vascular wilt diseases in over 200 dicotyledonous species. An effective way to identify genes that are required by the pathogen to cause disease on its host(s) is random mutagenesis followed by pathogenicity testing. Agrobacterium tumefaciens-mediated gene transfer (ATMT) has been widely used for large scale insertional mutagenesis in fungi, where the Agrobacterium transfers a part of its plasmid (T-DNA) into the host genome. Resulting mutants can be analyzed for altered pathogenicity or virulence. As mutants are tagged by the T-DNA, subsequent cloning of the gene of interest is possible. Through ATMT, we have generated 900 random insertional mutants in V. dahliae that are analysed for reduced pathogenicity or virulence on susceptible tomato. This has resulted in the identification of 80 transformants that are severely compromised in pathogenicity. The T-DNA integration site of the selected transformants were identified using Inverse PCR. We found that the T-DNA in one of transformants was inserted in the coding region of a secreted serine protease, while in another transformant it was inserted near a gene that encodes a secreted metalloprotease. Functional characterization of the two genes will be carried out to identify their contribution to Verticillium wilt.