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

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/μg DNA/108 cells). A collection of ∼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.

Graphical abstractA robust transformation protocol based upon the bar gene marker conferring resistance to phosphinothricin for selection of recombinants in the entomopathogenic fungus, B. bassiana, was developed. Selection on Czapek-Dox agar adjusted to pH 6.3, containing 200 mg/ml phosphinothricin and 0.01% bromocresol purple allowed for high transformation efficiency with low background. As proof of principle regarding the utility of the method, a collection of random insertion mutants was generated and screened for reduced/loss of hydrocarbon substrate utilization, e.g. loss of halo formation on tributyrin/Victoria blue agar. The insertion site of one such mutant was mapped and insect bioassays revealed reduced virulence in the mutant.Figure optionsDownload full-size imageDownload as PowerPoint slideResearch highlights► Method for high throughput transformation of B. bassiana developed. ► Protocol was validated for construction of random-insertion mutant library. ► Screening of transformants using hydrocarbon utilization assays were performed. ► Putative mutants were isolated and the marker insertion site mapped. ► One such characterized mutant displayed decreased virulence in insect bioassays.