Transformation of Pythium aphanidermatum to geneticin resistance

Conditions for the production of protoplasts and gene transfer in Pythium aphanidermatum were investigated. Efficient protoplast generation was possible after culture of mycelium in potato dextrose broth followed by digestion with 0.5% (w/v) each of cellulase and beta- d-glucanase. Plasmid pHAMT35N/SK encoding the nptII gene under control of the Ham34 promoter from the oomycete Bremia lactucae was used to define electroporation parameters for gene transfer. A square-wave electroporation pulse of 2500 V/cm at 50 microF capacitance reproducibly produced transformants, albeit at low efficiency (0.1-0.4 transformants from approximately 10(5) regenerable protoplasts per microgram of DNA). Thirty-two independent transformants exhibited wild-type growth on potato dextrose agar amended with geneticin at 50 microg/ml, a concentration that near completely inhibited the growth of untransformed P. aphanidermatum. Southern blot analysis indicated that transforming DNA was integrated into the oomycete genome and that the DNA was stably inherited through sporogenesis. Growth on geneticin-free media, the ability to form zoospores or oospores, and the ability to cause disease in sugarbeet seedlings in the laboratory were indistinguishable between a subset of the transformed isolates and the progenitor isolate 898B. Co-electroporation of pHAMT35N/SK with plasmid pACT-GUS encoding the Escherichia coli gusA gene controlled by oomycete transcriptional promoter and terminator sequences or with pEGFP encoding enhanced green fluorescent protein under the control of the immediate early promoter from the mammalian cytomegalovirus produced, respectively, stable beta-glucuronidase and transient expression of blue-green fluorescence. Application of the technique to studies on the biochemical basis for pathogenesis in this agriculturally important group of fungi is discussed.