Paxillus involutus was transformed using gold particle mediated gene transfer. Transformation was determined using the hygromycin B phosphotransferase gene (hph) as the selectable marker and the β-glucuronidase gene (GUS) as a reporter gene. Southern blot analysis confirmed that the vector DNA for both hph and GUS genes was integrated into the fungal genome. Variations in the number of multiple gene copies and insertions were found in the transformants. The hygromycin resistant transformants were mitotically stable maintaining both the hph and GUS genes in the fungal genome six months following transformation. Western blot analysis determined that the GUS gene was capable of transcribing and translating its protein product in the transformed fungus. Enzyme assays of GUS extracts determined that β-glucuronidase was active in the transformed fungi. Pure culture synthesis experiments showed that the ability of P. involvutus to form ectomychorrhizae with Pinus resinosa was not altered by transformation. These results provide the first report of a successful transformation of an ectomycorrhizal fungus using particle bombardment.
Financed by the National Centre for Research and Development under grant No. SP/I/1/77065/10 by the strategic scientific research and experimental development program:
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