CYTOLOGICAL AND MOLECULAR CHARACTERIZATIONOF NECROSIS INDUCING cDNAS FROM PHYTOPHTHORAINFESTANS GERMINATING CYSTS

Potato virus X (PVX)-based vectors have emerged as robust and reliable systems to express virulence and avirulence genes from microbial and viral pathogens and to express heterologous genes from plants. We currently use this technology to carry out functional screens of Phytophthora infestans cDNAs in plants. Polyadenylated mRNA isolated from P. infestans cysts germinated for 16 hours in water was used to synthesize cDNAs. The cDNAs were cloned unidirectionally in pSPORT1 vector, and then subcloned into the PVX binary vector pGR106 using a PCRbased approach. The ligation mixture was transformed directly into Agrobacterium tumefaciens. We performed functional screens of 2500 clones by toothpick inoculation onto the lower leaves of Nicotiana benthamiana plantlets. Starting from 7 to 15 days post inoculation, most of the inoculated plants showed systemic mosaic symptoms typical of PVX infection. However, 5 clones showed localized and systemic necrosis, and an additional 5% showed stunting and/or no-symptoms. Sequencing of the five inserts revealed four different cDNAs, three of which encode predicted proteins with no significant homology in public databases. These necrogenic inserts were also cloned into the binary vector pCB301 and transformed into A. tumefaciens to confirm the response without the PVX vector. Diverse host and non-host plants were tested. The phenotypes induced by those clones were characterized for induction of PR proteins and accumulation of phenolic compounds and callose.