Agrobacterium-mediated insertional mutagenesis (AIM) of the entomopathogenic fungus Beauveria bassiana

Agrobacterium tumefaciens was used to stably transform the entomopathogenic deuteromycete Beauveria bassiana to hygromycin B resistance by integration of the hph gene of Escherichia coli into the fungal genome. The transformation protocol was optimized to generate a library of insertion mutants of Beauveria. Transformation frequencies around 10^–4 and suppression of background growth were achieved. Over 90% of the AIM mutants investigated contained single-copy T-DNA integrations at different chromosomal locations. Integrated T-DNAs were re-isolated from ten transformants by a marker rescue approach. When the sequences flanking these T-DNAs were compared with the corresponding locations of the wild-type genome, truncations of T-DNA borders were found to be common, while none of the sites of integration had suffered deletion or rearrangement. Thus, AIM can be considered a promising tool for insertional mutagenesis studies of entomopathogenic filamentous fungi.Communicated by J. HeitmanA. Leclerque and H. Wan contributed equally to this work