Fgk3, a Glycogen\nSynthase Kinase, Regulates Chitin\nSynthesis through the Carbon Catabolite Repressor FgCreA in Fusarium graminearum

The glycogen synthase kinase-3 (GSK3) orthologs are well-conserved\nin eukaryotic organisms. However, their functions remain poorly characterized\nin filamentous fungi. In our previous study, we unveiled the function\nof Fgk3, the GSK3 ortholog, in glycogen metabolism in Fusarium graminearum, the causal agent of Fusarium\nhead blight. Interestingly, the <i>fgk3</i> mutant was unstable\nand tended to produce fast-growing suppressors, including secondary\nsuppressors. Using whole-genome sequencing, we identified suppressor\nmutations in <i>FgCHS5</i>, <i>FgFKS1</i>, <i>FgCREA</i>, <i>FgSSN6</i>, <i>FgRGR1</i>, and <i>FgPP2A</i> in nine primary and four secondary\nsuppressors. Subsequently, we validated that deletion of <i>FgCHS5</i> or <i>FgCREA</i><sup>ΔH253</sup> mutation partially\nsuppressed the defects of <i>fgk3</i> in vegetative growth\nand cell wall integrity, suggesting that Fgk3 may regulate the chitin\nsynthesis through FgCreA-mediated transcriptional regulation in F. graminearum. Accordingly, the <i>FGK3</i> deletion led to hyphal swelling with abnormal chitin deposition,\nand deletion of <i>FGK3</i> or <i>FgCREA</i> caused\nthe upregulation of the expression of chitin synthases <i>FgCHS5</i> and <i>FgCHS6</i>. The interaction between Fgk3 and FgCreA\nwas verified by Yeast two-hybrid and Co-Immunoprecipitation assays.\nMore importantly, we verified that the nuclear localization and protein\nstability of FgCreA relies on the Fgk3 kinase, while the H253 deletion\nfacilitated the re-localization of FgCreA to the nucleus in the <i>fgk3</i> mutant background, potentially contributing to the\nsuppression of the <i>fgk3</i> mutant’s defects.\nIntriguingly, the ΔH253 mutation of FgCreA, identified in suppressor\nmutant S3, is adjacent to a conserved phosphorylation site, S254,\nsuggesting that this mutation may inhibit the S254 phosphorylation\nand promote the nuclear localization of FgCreA. Collectively, our\nfindings indicate that the glycogen synthase kinase Fgk3 regulates\nthe chitin synthesis through the carbon catabolite repressor FgCreA\nin F. graminearum.