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Transformation of Pythium aphanidermatum to geneticin resistance

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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 β-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 μF capacitance reproducibly produced transformants, albeit at low efficiency (0.1–0.4 transformants from ~105 regenerable protoplasts per microgram of DNA). Thirty-two independent transformants exhibited wild-type growth on potato dextrose agar amended with geneticin at 50 μg/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 β-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.

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Fig. 1A–D.
Fig. 2A–E.
Fig. 3A, B.
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Fig. 5A–D.

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Acknowledgements.

The author wishes to acknowledge the efforts of Brant B. Bigger and Lisa M. Voll for technical expertise and creative input in the study and contributions from Gary E. Nielsen, Jeff Miller, and Roger Effertz. We are indebted to David Johnson (Michigan State University–East Lansing) for taxonomic appraisal of the P. aphanidermatum used in the study. This project was supported by USDA-ARS CRIS 0015442090. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA or imply approval to the exclusion of other products that may also be suitable.

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  1. Sugarbeet and Potato Research Unit, USDA–Agricultural Research Service, Northern Crop Science Laboratory, Fargo, ND 58105-5677, USA

    John J. Weiland

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  1. John J. Weiland
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Correspondence to John J. Weiland.

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Communicated by S. Hohmann

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Weiland, J.J. Transformation of Pythium aphanidermatum to geneticin resistance. Curr Genet 42, 344–352 (2003). https://doi.org/10.1007/s00294-002-0359-y

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