Genetic manipulation of arbuscular mycorrhizal (AM) fungi has been hindered by their obligate biotrophism that narrows the cultivation under axenic conditions. In order to establish a stable transformation methodology for these fungi, we took advantage of the Agrobacterium tumefaciens mediated transformation and of an in vitro mycorrhizal system where large amounts of extraradical mycelium can be produced under axenic conditions. We carried out sensitivity screenings to different fungicides (hygromycin, benomyl and phosphinothricin) to be used as positive selection markers in this system. We found that hygromycin and benomyl were very effective at low concentrations (25 and 0.01 ?g/ml respectively) against both spore germination and hyphal growth of G. intraradices in contrast to the herbicide phosphinotricin. A binary vector was constructed containing the GFP reporter gene and hygromycin as selection marker, both under the control of the heterologous gpd promoter from Aspergillus nidulans. Extraradical hyphae of G. intraradices were transformed with this construct by co-cultivation with A. tumefaciens. Fluorescence microscopy showed a high number of transformed AM hyphae expressing GFP fluorescence. This is the first report of a mycorrhizal fungus where GFP fluorenscence has been observed and it shows that the Agrobacterium-mediated transformation could be further developed for functional analysis of fungal genes in the AM symbiosis.
Full conference title:
23rd Fungal Genetics Conference
- Fungal Genetics Conference 23rd (2002)