Functional diversification of oxalate decarboxylases in terms of enzymatic activity, morphosporogenesis, stress regulation and virulence in Colletotrichum siamense

Oxalate decarboxylase (OxdC) is an enzyme that degrades oxalic acid and may affect the virulence of necrotrophic fungal pathogens that rely on oxalic acid as a pathogenicity factor. However, the biological function of OxdCs in hemibiotropic fungi is still unknown. Our previous studies revealed four OxdC-encoding genes in the whole genome, with <i>CsOxdC3</i> playing important roles in morphosporogenesis, fungicide resistance and virulence in <i>Colletotrichum siamense</i>. Here, we systematically analyzed the biological functions of four oxalate decarboxylase genes in <i>C. siamense</i> via a loss-of-function method. The results revealed <i>CsOxdC1</i>, <i>CsOxdC2</i>, and <i>CsOxdC4</i> played major roles in degrading oxalic acid in <i>C. siamense</i>, whereas <i>CsOxdC3</i> did not. All four <i>CsOxdCs</i> positively modulated morphosporogenesis, including vegetative growth, conidial size, conidial germination rate and the appressorium formation rate, to different extents. In particular, the <i>CsOxdC3</i> deletion mutant failed to form appressoria. The four <i>OxdC</i> gene deletion mutants had different responses to Mn²⁺, Cu²⁺, and multiple fungicides. Among them, <i>CsOxdC2</i> and <i>CsOxdC4</i> exhibited positive roles in resistance to Mn²⁺ and Cu²⁺ stresses; <i>CsOxdC1</i> played a slightly positive role in <i>C. siamense</i> resistance to azole fungicides; and <i>CsOxdC3</i> had a significantly positive role in regulating the sensitivity of <i>C. siamense</i> to multiple fungicides, including pyrrole and azole, but not <i>CsOxdC2</i> and <i>CsOxdC4</i>. Furthermore, compared with the wild-type strain, Δ<i>CsOxdC2</i> and Δ<i>CsOxdC3</i>, but not Δ<i>CsOxdC1</i> and Δ<i>CsOxdC4</i>, displayed significantly reduced virulence. In conclusion, our data indicated that <i>CsOxdCs</i> exerted diverse functions in morphogenesis, stress homeostasis, fungicide resistance, and virulence in <i>C. siamense</i>. This study provides insights into the biological function of <i>OxdCs</i> in the hemibiotrophic fungus <i>C. siamense</i>.