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 barspecific 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 wildtype 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. Beauveria bassiana, an entomopathogenic fungus, has a broad host spectrum and is considered to have high potential for insect biocontrol in agriculture. While B. bassiana can cause mycosis in several insect species 1,2 , insect killing is fairly slow due to several limiting factors, particularly in the field. The fungus is also vulnerable to environmental stress factors such as UV radiation, high temperature and drought 3. A better understanding of the biological and physiological characteristics of this entomopathogen should allow us to improve its virulence and stress tolerance. Secondary metabolites are abundant in entomopathogenic fungi and include polyketides, nonribosomal peptides, terpenes and alkaloids that play important roles in various aspects of the fungal life cycle. B. bassiana BCC2660, a widely used biocontrol fungus in Thailand, has 12 polyketide synthase (PKS) genes in its genome 4. Two PKS genes, pks15 and pks14, have crucial roles in virulence against insects, as previously demonstrated by targeted gene deletion 4,5. The pks15 mutant exhibits loss in phagocytic survival ability, a phenotype likely associated with changes in the cell wall, the outermost layer of fungal conidia. Unfortunately, little is known regarding the relationship between polyketides and the fungal wall. In a few reports, melanin, the metabolite of a n...
The reducing clade IIb polyketide synthase gene, pks14, is preserved throughout the evolution of entomopathogenic fungi. We examined the functions of pks14 in Beauveria bassiana using targeted gene disruption, and pks14 disruption was verified by Southern blot and PCR analyses. The radial growth, cell dry weight and conidial germination of Δpks14 were comparable to that of the wild type. Our sequence and gene expression analyses of the pks14 biosynthetic cluster demonstrated: (i) cotranscription and constitutive expression of nearly all the genes of the aforementioned cluster including the C2H2 zinc finger transcription regulator gene, but not pks14 and the cytochrome P450 gene; (ii) expression of the pks14 gene in the insect-containing culture condition only; and (iii) a KAR9-like gene in direct proximity with pks14 is the only gene showing co-regulation. The Δpks14-infected Spodoptera exigua larvae survived significantly longer than those infected by the wild type, indicating a marked reduction in the virulence of Δpks14 against the insect. LT50 of Δpks14 was increased by 1.55 days. Hyphal body formation was decreased in the hemolymph of insects infected by Δpks14 as compared with those inoculated by the wild type. Our results suggest that PKS14-catalyzed polyketide enhances virulence and pathogenicity of B. bassiana on insects.
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