Optimisation and comparison of transient expression methods to express the green fluorescent protein in the obligate biotrophic oomycete Plasmopara viticola

Grape downy mildew is caused by Plasmopara viticola, an obligate biotrophic oomycete and a major pathogen of grapevine. Studying obligate biotrophic pathogens is difficult as they cannot grow without their host. We therefore attempted to develop a method where the pathogen could be visualized and quantified in planta without killing the host plant. To this end P. viticola was transformed with the marker gene gfp coding for the green fluorescent protein. Various transformation methods, namely electroporation, particle bombardment and transformation with Agrobacterium tumefaciens were applied. Although some methods yielded positive transformation events, no stable strain of P. viticola expressing gfp could be generated. Using the electroporation method, we obtained transient P. viticola transformants expressing gfp over 4 generations. In contrast, particle bombardment failed in transforming P. viticola. Transformation with A. tumefaciens had a low efficiency, only some structures were fluorescent and fluorescence was never observed in the subsequent generations.