The polyketide synthase PKS15 has a crucial role in cell wall formation in Beauveria bassiana
| Autor: | Rudsamee Wasuwan, Chettida Srisuksam, Nuchnudda Wichienchote, Nur Amalina Shamsun Nahar, Udom Sae-Ueng, Wachiraporn Toopaang, Somsiri Udompaisarn, Alongkorn Amnuaykanjanasin, Morakot Tanticharoen |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
DNA End-Joining Repair Insecta DNA recombination 030106 microbiology Mutant Virulence lcsh:Medicine Biology Article Fluorescence Fungal Proteins 03 medical and health sciences chemistry.chemical_compound Cell growth Fungal genes Phagocytosis Cell Wall Polyketide synthase Animals Beauveria lcsh:Science Gene Gene Editing Multidisciplinary Microbial Viability Base Sequence lcsh:R Genetic Complementation Test Wild type Spores Fungal Cell biology Complementation 030104 developmental biology chemistry Cell wall organization Genetic Loci Mutagenesis Mutation biology.protein lcsh:Q CRISPR-Cas Systems Polyketide Synthases DNA |
| Zdroj: | Scientific Reports Scientific Reports, Vol 10, Iss 1, Pp 1-16 (2020) |
| ISSN: | 2045-2322 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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