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Title:   Author  item
                                            Baumgartner, Kendraitem
                                            BAILEY, ANDY - UNIV OF BRISTOL, U.K.item
                                            FOSTER, GARY - UNIV OF BRISTOL, U.K.item
                                            KILARU, SREEDHAR - UNIV OF BRISTOL, U.K.
  Submitted to: Phytopathology
                                    Publication Type: Abstract Only
                                    Publication Acceptance Date: 3/13/2008
                                    Publication  Date: 7/1/2008
                                    Citation: Baumgartner, K., Bailey, A., Foster, G., Kilaru, S. 2008. Transgene expression in the basidiomycete root pathogen Armillaria mellea.. Phytopathology. 98:S19.Interpretive Summary:
                                Technical Abstract: Toward development of a genetic transformation system for Armillaria mellea, we used particle bombardment to identify promoters for driving transgene expression. The plasmid tested was pYES-hph-004iGFP, on which the green fluorescence protein gene, gfp, is linked to the Agaricus bisporus gpdII promoter and the Phanerochaete chrysosporium mnp1 terminator. Malt extract agar was inoculated with A. mellea (incubated at 25C, 24 d). YMGT agar (0.4% yeast extract, 1% malt extract, 1% agar, 0.01% tryptophan, 0.04% Dglucose) was inoculated with Coprinopsis cinerea (incubated at 37C, 5 d; positive control). Gene gun cartridges were prepared the day of use (6.25 mg 0.6-'m gold, 0.05M spermidine, 0.05 mg/ml PVP, 100 'g DNA per 25 in. tubing). Cartridges prepared with plasmid DNA or no DNA (negative control) were applied at 150 and 200 psi to each of 21 A. mellea and C. cinerea  colonies (Helios Gene Gun; Bio-Rad, Hercules, CA). Petri plates without lids were positioned 4 cm from the cartridge, with no screen between the barrel and agar surface. Each culture received two shots per cartridge. After 2 d, cultures were examined under fluorescent light on a compound microscope (Leica MZFL111; 100X, 450-490 nm excitation filters, 510 nm dichroic filter, 515 nm emission filter). The presence of a green dot among A. mellea hyphae bombarded with pYES-hph-004iGFP-coated particles verified that a single cell was penetrated by a particle, and that A. mellea is capable of translating gfp under control of the Agaricus bisporus gpdII promoter. This is the first instance of transgene expression in Armillaria. Expression was visible for at least one week, but subcultures did not express gfp after one month; expression was transient. Nonetheless, this is an achievement because plasmids for both Agrobacterium-mediated and protoplast/PEG mediated transformation that have been shown to give the highest transformation success among other Basidiomycetes with the Agaricus bisporus gpdII promoter.


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