Genetic transformation of Coccidioides immitis facilitated by Agrobacterium tumefaciens

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

Research output: Contribution to journalArticle

86 Citations (Scopus)

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 - 2000

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hygromycin-B kinase
Coccidioides
Genetic Transformation
Agrobacterium tumefaciens
Coculture Techniques
Fungi
Genes
Mycelium
Germination
Southern Blotting
Mutagenesis
Cell Wall
Digestion
Plasmids
Technology
Bacteria
Equipment and Supplies
Polymerase Chain Reaction

ASJC Scopus subject areas

  • Public Health, Environmental and Occupational Health
  • Immunology

Cite this

Genetic transformation of Coccidioides immitis facilitated by Agrobacterium tumefaciens. / Abuodeh, Raed O.; Orbach, Marc Joel; Mandel, M. Alejandra; Das, Anath; Galgiani, John N.

In: Journal of Infectious Diseases, Vol. 181, No. 6, 2000, p. 2106-2110.

Research output: Contribution to journalArticle

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