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  • Title: Agrobacterium tumefaciens mediated transformation of the oomycete plant pathogen Phytophthora infestans.
    Author: Vijn I, Govers F.
    Journal: Mol Plant Pathol; 2003 Nov 01; 4(6):459-67. PubMed ID: 20569405.
    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.
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