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 putative ferricrocin synthetase gene ferS in the fungal entomopathogen Beauveria bassiana BCC 2660 was identified and characterized. The 14,445-bp ferS encodes a multimodular nonribosomal siderophore synthetase tightly clustered with Fusarium graminearum ferricrocin synthetase. Functional analysis of this gene was performed by disruption with the bar cassette. ΔferS mutants were verified by Southern and PCR analyses. HPLC and TLC analyses of crude extracts indicated that biosynthesis of ferricrocin was abolished in ΔferS. Insect bioassays surprisingly indicated that ΔferS killed the Spodoptera exigua larvae faster (LT50 59 h) than wild type (66 h). Growth and developmental assays of the mutant and wild type demonstrated that ΔferS had a significant increase in germination under iron depletion and radial growth and a decrease in conidiation. Mitotracker staining showed that the mitochondrial activity was enriched in ΔferS under both iron excess and iron depletion. Comparative transcriptomes between wild type and ΔferS indicated that the mutant was increased in the expression of eight cytochrome P450 genes and those in iron homeostasis, ferroptosis, oxidative stress response, ergosterol biosynthesis, and TCA cycle, compared to wild type. Our data suggested that ΔferS sensed the iron excess and the oxidative stress and, in turn, was up-regulated in the antioxidant-related genes and those in ergosterol biosynthesis and TCA cycle. These increased biological pathways help ΔferS grow and germinate faster than the wild type and caused higher insect mortality than the wild type in the early phase of infection.
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