High-throughput insertion mutagenesis and functional screening in the entomopathogenic fungus Beauveria bassiana

作者：Fan Yanhua; Zhang Shizhu; Kruer Nathan; Keyhani Nemat O^*

来源：Journal of Invertebrate Pathology, 2011, 106(2): 274-279.

DOI：10.1016/j.jip.2010.11.003

• Entomopathogenic fungi
• Beauveria bassiana
• Random-insertion mutagenesis
• Hydrocarbon utilization

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.

• 出版日期2011-2