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June 2000

Volume 181, Number 6
The Journal of Infectious Diseases 2000;181:2106–2110
© 2000 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2000/18106-0041$02.00
DOI: 10.1086/315525
CONCISE COMMUNICATION

Genetic Transformation of Coccidioides immitis Facilitated by Agrobacterium tumefaciens

Raed O. Abuodeh,1,2,4,a

Marc J. Orbach,1,3

M. Alejandra Mandel,1,3

Anath Das,5 and

John N. Galgiani1,2,4

1Valley Fever Center for Excellence; 2Medical and Research Services, Southern Arizona Veterans Affairs Health Care System; and 3Department of Plant Pathology, College of Agriculture, and 4Department of Internal Medicine, College of Medicine, University of Arizona, Tucson; 5Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul

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.

Received 13 December 1999; revised 14 February 2000; electronically published 5 June 2000.

Reprints or correspondence: Dr. John N. Galgiani, Valley Fever Center for Excellence (1‐111), 3601 S. Sixth Ave., Tucson, AZ 85723 ().

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Caroline B. Michielse, Paul J. J. Hooykaas, Cees A. M. J. J. Hondel, Arthur F. J. Ram. (2005) Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Current Genetics 48:1, 1-17
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  • Presented in part: 100th annual meeting of the American Society for Microbiology, Los Angeles, 24 May 2000.

    Financial support: US Department of Veterans Affairs; California Health Care Foundation; Purdue University (grant U.S.D.A. Prime 96‐34340‐2711).

  • Present affiliation: College of Health Sciences, University of Sharjah, Sharjah, United Arab Emirates.

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