Journal
FUNGAL GENETICS AND BIOLOGY
Volume 42, Issue 11, Pages 904-913Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.fgb.2005.07.003
Keywords
Cryptococcus neoformans; C. gattii; Agrobacterium tumefaciens; ATMT; transformation; insertional mutagenesis
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Funding
- Intramural NIH HHS Funding Source: Medline
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Cryptococcus neoformans and Cryptococcus gattii are the causative agents of cryptococcal meningoencephalitis and are amenable to genetic manipulations, making them important models of pathogenic fungi. To improve the efficiency of Agrobacterium tumefaciens mediated transformation (ATMT) in C. neoformans, we optimized various co-cultivation conditions including incubation time and temperature, and bacteria to yeast ratio. ATMT was also applied to both serotypes (B and C) of C gattii. Transformation efficiency by ATMT in C neoformans was comparable to either electroporation or biolistic transformation and gave superior efficiencies in serotypes B and C, but unlike Saccharomyces cerevisiae, adenine auxotrophy did not increase ATMT efficiency in C neoformans or C. gattii. All transformants tested were stable, with a majority containing only a single T-DNA insertion; however, homologous recombination was not observed. Additionally, we isolated adenine auxotrophs containing a single T-DNA insertion in the ADE2 gene for representative serotype B and C strains. Published by Elsevier Inc.
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