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J Infect Dis. 2000 Jun;181(6):2106-10. Epub 2000 Jun 5.

Genetic transformation of Coccidioides immitis facilitated by Agrobacterium tumefaciens.

Author information

1
Valley Fever Center for Excellence, Southern Arizona Veterans Affairs Health Care System, Tucson, AZ, USA.

Abstract

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.

PMID:
10837205
DOI:
10.1086/315525
[Indexed for MEDLINE]

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