Submitted to: Fungal Genetics Conference Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 1/19/2001
Publication Date: N/A
Technical Abstract: 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 <£>nptll£> 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 2500V/cm at 50 microfarad capacitance reproducibly produced transformants, albeit at low efficiency (0.1-0.4 transformants from ~100,000 regenerable protoplasts per microgram of DNA). Twenty seven independent transformants exhibited wild-type growth on potato dextrose agar amended with geneticin at 50 microgram per ml, a concentration that near completely inhibited the growth of untransformed fungus. Southern blot analysis indicated that transforming DNA was integrated into the fungal genome as a tandem array of plasmid monomers. Co-electroporation of pHAMT35N/SK with pEGFP encoding enhanced green fluorescent protein (EGFP) under the control of the immediate early promoter from the mammalian cytomegalovirus produced 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 are discussed.