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.
Abuodeh
R O
RO
Valley Fever Center for Excellence, Southern Arizona Veterans Affairs Health Care System, Tucson, AZ, USA.
Orbach
M J
MJ
Mandel
M A
MA
Das
A
A
Galgiani
J N
JN
D016960:Agrobacterium tumefaciens / Q000235:genetics* / Q000502:physiology; D003045:Coccidioides / Q000235:genetics*; D018014:Gene Transfer Techniques; D014170:Transformation, Genetic