Genetic transformation of Coccidioides immitis facilitated by Agrobacterium tumefaciens

Raed O. Abuodeh, Marc J. Orbach, M. Alejandra Mandel, Anath Das, John N. Galgiani

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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 105 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.

Original languageEnglish (US)
Pages (from-to)2106-2110
Number of pages5
JournalJournal of Infectious Diseases
Volume181
Issue number6
DOIs
StatePublished - Jul 10 2000

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ASJC Scopus subject areas

  • Immunology and Allergy
  • Infectious Diseases

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