Construction of a versatile cloning vector for Agrobacterium tumefaciens-mediated transformation of Beauveria bassiana using benomyl for selection.

Abstract

Beauveria bassiana is a filamentous fungus that has considerable potential to combat insect pests. Therefore, an easy to use genetic transformation tool helps to investigate which fungal genes play a role in virulence and host specificity. However, B. bassiana is often very resistant to antibiotics used as selective agents for genetic transformation. We developed a binary cloning vector, pMBE1, for Agrobacterium tumefaciens-mediated transformation of B. bassiana. Selection of fungal transformants is based on the benomyl resistance gene that was introduced in this vector, as B. bassiana is very sensitive to this antibiotic. Genes of interest can be cloned into a multiple cloning site that was inserted downstream of a constitutive promoter. As proof of principle the gene for enhanced green fluorescence protein (egfp) was cloned in pMBE1 and successfully expressed in B. bassiana. Mitotic stability was demonstrated after growth of several generations on non-selective media. Interestingly, we also observed that homologous recombination can occur between the benomyl resistance gene and the beta-tubulin gene from B. bassiana resulting in transformants that are benomyl resistant but do not contain the egfp gene.