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Agrobacterium tumefaciens -Mediated Transformation of Aspergillus fumigatus : an Efficient Tool for Insertional Mutagenesis and Targeted Gene Disruption

2005; American Society for Microbiology; Volume: 71; Issue: 4 Linguagem: Inglês

10.1128/aem.71.4.1798-1802.2005

1098-5336

Janyce A. Sugui, Yun C. Chang, Kyung J. Kwon‐Chung,

Chromosomal and Genetic Variations

ABSTRACT Agrobacterium tumefaciens was used to transform Aspergillus fumigatus by either random or site-directed integration of transforming DNA (T-DNA). Random mutagenesis via Agrobacterium tumefaciens -mediated transformation (ATMT) was accomplished with T-DNA containing a hygromycin resistance cassette. Cocultivation of A. fumigatus conidia and Agrobacterium (1:10 ratio) for 48 h at 24°C resulted in high frequencies of transformation (>100 transformants/10 7 conidia). The majority of transformants harbored a randomly integrated single copy of T-DNA and were mitotically stable. We chose alb1 , a polyketide synthase gene, as the target gene for homologous integration because of the clear phenotype difference between the white colonies of Δ alb1 mutant strains and the bluish-green colonies of wild-type strains. ATMT with a T-DNA-containing alb1 disruption construct resulted in 66% albino transformants. Southern analysis revealed that 19 of the 20 randomly chosen albino transformants (95%) were disrupted by homologous recombination. These results suggest that ATMT is an efficient tool for transformation, random insertional mutagenesis, and gene disruption in A. fumigatus .