Efficient insertional mutagenesis system for the dimorphic pathogenic fungus Sporothrix schenckii using Agrobacterium tumefaciens.
Sporothrix schenckii is a dimorphic pathogenic fungus that causes human and animal sporotrichosis globally. Here we developed and optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) system of S. schenckii for insertional mutagenesis. The transformation efficiency reached more than 600 transformants per 10(6) conidia. Using this protocol enabled us to obtain a large number of T-DNA insertional mutants within a short experimental period. Several mutants with altered phenotypes were obtained during the transformation experiments. The mutants displayed mitotic stability. Transferred DNA (T-DNA) flanking sequences were cloned by thermal asymmetric interlaced PCR (TAIL-PCR). Our results demonstrated that the ATMT system can be an effective tool for insertional mutagenesis in S. schenckii. This is the first report of a suitable mutagenesis system which may provide valuable mutants and information for both forward and reverse genetics research in the future for this medically important fungus.
Department of Pathogenobiology, Norman Bethune College of Medicine, Jilin University Mycology Research Center, Jilin University, Changchun 130021, China.
Yanhua
Y
Zhang
Guangquan
G
Li
Dan
D
He
Baodong
B
Yu
Koji
K
Yokoyama
Li
L
Wang
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D000818:Animals; D005827:Genetics, Microbial / Q000379:methods*; D006801:Humans; D016254:Mutagenesis, Insertional / Q000379:methods*; D013175:Sporothrix / Q000235:genetics*; D014170:Transformation, Genetic; D014481:United States