期刊
JOURNAL OF INVERTEBRATE PATHOLOGY
卷 106, 期 2, 页码 274-279出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.jip.2010.11.003
关键词
Entomopathogenic fungi; Beauveria bassiana; Random-insertion mutagenesis; Hydrocarbon utilization
类别
The entomopathogenic fungus Beauveria bassiana displays a broad insect host range and serves as a model for examining host-pathogen interactions. Rapid construction and screening of random-insertion mutants of B. bassiana provides a powerful tool to dissect the molecular mechanisms of fungal virulence. LiAc/DMSO treated B. bassiana blastospores were found to be highly competent to transformation using linear DNA and a polyethylene glycol-based method. Selection on cellophane-layered Czapek-Dox agar at a lowered pH (from 7.5 to 6.3) greatly decreased background growth of non-transformed cells and improved screening of transformants. Optimization of the protocol using integration of the bar phosphinothricin resistance gene resulted in high transformation rates (200-250 transformants/mu g DNA/10(8) cells). A collection of similar to 4000 insertion mutants was examined via high-throughput screens for hydrocarbon utilization. One mutant was isolated that grew poorly on both n-hexadecane and tributyrin. The random insertion site was mapped to a gene that displayed homology to vitamin H (biotin)/tartrate transporters. Insect bioassays using Galleria mellonella as the target host revealed decreased virulence in the mutant. This system provides a simple and rapid method for the generation and screening of insertion mutants and should expand our ability to genetically analyze the B. bassiana lifestyle. (C) 2010 Elsevier Inc. All rights reserved.
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