Genetic transformation of Coccidioides immitis facilitated by Agrobacterium tumefaciens

Agrobacterium tumefaciens was used to facilitate genetic transformation of Coccidioides immitis. A gene cassette containing the gene encoding hygromycin phosphotransferase (hph) was cloned into a T-DNA vector plasmid and introduced into A. tumefaciens, and the resultant strain was used for cocultivation with germinated arthroconidia, This procedure produced numerous colonies 60- to >500-fold more resistant to hygromycin than untransformed mycelia, Both polymerase chain reaction and Southern blot analysis of the transformants indicated that all contained hph, usually as a single genomic copy. A transformation frequency of 1 per 10(5) arthroconidia was obtained by varying the germination time prior to cocultivation and altering the bacterium : fungus ratio. This approach requires no special equipment that might complicate biocontainment, Furthermore, transformation does not require digestion of fungal cell walls, further simplifying this procedure, A. tumefaciens-facilitated transformation should make possible the development of tagged mutagenesis and targeted gene disruption technology for C, immitis and perhaps other fungi of medical importance.