title: Agrobacterium tumefaciens mediated transformation of the oomycete plant pathogen Phytophthora infestans.
abstract: SUMMARY Agrobacterium tumefaciens is widely used for plant DNA transformation and, more recently, has also been used to transform yeast and filamentous fungi. Here we present a protocol for Agrobacterium-mediated DNA transformation of the oomycete Phytophthora infestans, the causal agent of potato late blight. Binary T-DNA vectors containing neomycin phosphotransferase (npt) and beta-glucuronidase (gus) fused to oomycete transcriptional regulatory sequences were constructed. Seven days of co-cultivation followed by transfer to a selective medium containing cefotaxim to kill Agrobacterium and geneticin to select for transformants, resulted in geneticin resistant colonies. Under optimal conditions with Agrobacterium supplemented with a ternary plasmid carrying a constitutive virG gene and in the presence of acetosyringone as inducer, up to 30 transformants per 10(7) zoospores could be obtained. The majority of these transformants contained a single T-DNA copy randomly integrated at a chromosomal locus. Using a similar protocol, geneticin resistant transformants of two other oomycetes species were obtained, Phytophthora palmivora and Pythium ultimum.
authors.affiliation: Laboratory of Phytopathology, Wageningen University, and Graduate School Experimental Plant Sciences, Binnenhaven 5, NL-6709 PD Wageningen, the Netherlands.
authors.forename: Irma
authors.initials: I
authors.lastname: Vijn
authors.forename: Francine
authors.initials: F
authors.lastname: Govers