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.

A 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 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.