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United States Department of Agriculture

Agricultural Research Service

Title: Transformation of Pythium Aphanidermatum to Geneticin Resistance.

item Weiland, John

Submitted to: Current Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 19, 2002
Publication Date: March 1, 2003

Interpretive Summary: Oomycete pathogens of sugarbeet belonging to the genus Pythium and Aphanomyces cause severe, annual destruction to the crop in the U.S. The fundamental biology and biochemistry behind the mechanisms used by these pathogens to gain access to the hypocotyl of sugarbeet seedlings and the root of mature plants remains poorly characterized. Defined mutants could be generated using gene transfer technology that would yield important information regarding the genes and enzymes needed by the pathogens in order to infect sugarbeet plants. As a first step in this approach for analyzing the biochemistry of infection, a gene transfer procedure for the oomycete pathogen Pythium aphanidermatum was developed. The procedure now renders possible the transfer of DNA and genes into the genome of P. aphanidermatum so that the role in pathogenesis played by these genes might be determined. This is the first report of gene transfer in any member of the genus Pythium, which comprises pathogens of nearly all crops world-wide.

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 b-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 2500V/cm at 50 mF capacitance reproducibly produced transformants, albeit at low efficiency (0.1-0.4 transformants from ~105 regenerable protoplasts per microgram of DNA). Twenty seven independant transformants exhibited wild-type growth on potato dextrose agar amended with geneticin at 50 mg/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 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.

Last Modified: 4/21/2015
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