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Entomopathogenic fungi utilize specific secondary metabolites to defend against insect immunity, thereby enabling colonization of their specific hosts. We are particularly interested in the polyketide synthesis gene  pks15 , which is involved in metabolite production, and its role in fungal virulence. Targeted disruption of  pks15  followed by genetic complementation with a functional copy of the gene would allow for functional characterization of this secondary metabolite biosynthesis gene. Using a  Beauveria bassiana  ∆ pks15  mutant previously disrupted by a bialophos-resistance ( bar ) cassette, we report here an in- cis  complementation at  bar  cassette using CRISPR/Cas9 gene editing. A  bar -specific short guide RNA was used to target and cause a double-strand break in  bar , and a donor DNA carrying a wild-type copy of  pks15  was co-transformed with the guide RNA. Isolate G6 of  ∆pks15  complemented with  pks15  was obtained and verified by PCR, Southern analyses and DNA sequencing. Compared to ∆ pks15  which showed a marked reduction in sporulation and insect virulence, the complementation in G6 restored with insect virulence, sporulation and conidial germination to wild-type levels. Atomic force and scanning electron microscopy revealed that G6 and wild-type conidial wall surfaces possessed the characteristic rodlet bundles and rough surface while ∆ pks15  walls lacked the bundles and were relatively smoother. Conidia of ∆ pks15  were larger and more elongated than that of G6 and the wild type, indicating changes in their cell wall organization. Our data indicate that PKS15 and its metabolite are likely not only important for fungal virulence and asexual reproduction, but also cell wall formation.

The polyketide synthase PKS15 has a crucial role in cell wall formation in Beauveria bassiana