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

Grape downy mildew is caused by <i>Plasmopara viti-cola</i>, an obligate biotrophic oomycete and a major path-ogen of grapevine. Studying obligate biotrophic patho-gens 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 <i>P. viticola</i> was transformed with the marker gene gfp coding for the green fluorescent protein. Various transformation methods, namely electroporation, particle bombard-ment and transformation with <i>Agrobacterium tume-faciens</i> were applied. Although some methods yielded positive transformation events, no stable strain of <i>P. viticola</i> expressing gfp could be generated. Using the electroporation method, we obtained transient <i>P. viti-cola</i> transformants expressing gfp over 4 generations. In contrast, particle bombardment failed in transform-ing <i>P. viticola</i>. Transformation with <i>A. tumefaciens</i> had a low efficiency, only some structures were fluorescent and fluorescence was never observed in the subsequent generations.