Data Sheet 2_Functional diversification of oxalate decarboxylases in terms of enzymatic activity, morphosporogenesis, stress regulation and virulence in Colletotrichum siamense.pdf

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 CsOxdC3 playing important roles in morphosporogenesis, fungicide resistance and virulence in Colletotrichum siamense. Here, we systematically analyzed the biological functions of four oxalate decarboxylase genes in C. siamense via a loss-of-function method. The results revealed CsOxdC1, CsOxdC2, and CsOxdC4 played major roles in degrading oxalic acid in C. siamense, whereas CsOxdC3 did not. All four CsOxdCs positively modulated morphosporogenesis, including vegetative growth, conidial size, conidial germination rate and the appressorium formation rate, to different extents. In particular, the CsOxdC3 deletion mutant failed to form appressoria. The four OxdC gene deletion mutants had different responses to Mn2+, Cu2+, and multiple fungicides. Among them, CsOxdC2 and CsOxdC4 exhibited positive roles in resistance to Mn2+ and Cu2+ stresses; CsOxdC1 played a slightly positive role in C. siamense resistance to azole fungicides; and CsOxdC3 had a significantly positive role in regulating the sensitivity of C. siamense to multiple fungicides, including pyrrole and azole, but not CsOxdC2 and CsOxdC4. Furthermore, compared with the wild-type strain, ΔCsOxdC2 and ΔCsOxdC3, but not ΔCsOxdC1 and ΔCsOxdC4, displayed significantly reduced virulence. In conclusion, our data indicated that CsOxdCs exerted diverse functions in morphogenesis, stress homeostasis, fungicide resistance, and virulence in C. siamense. This study provides insights into the biological function of OxdCs in the hemibiotrophic fungus C. siamense.

草酸脱羧酶（Oxalate decarboxylase, OxdC）是一类可降解草酸的酶，能够影响以草酸作为致病因子的死体营养型真菌病原菌的毒力。然而，草酸脱羧酶在半活体营养型真菌中的生物学功能仍未明确。我们前期的研究在暹罗炭疽菌（Colletotrichum siamense）的全基因组中鉴定出4个编码草酸脱羧酶的基因，其中CsOxdC3在该菌的形态发生、抗药性及毒力调控中发挥重要作用。本研究通过功能丧失（loss-of-function）手段，系统分析了暹罗炭疽菌中4个草酸脱羧酶基因的生物学功能。研究结果显示，CsOxdC1、CsOxdC2与CsOxdC4在暹罗炭疽菌的草酸降解过程中发挥主要作用，而CsOxdC3无此功能。4个CsOxdC基因均在不同程度上正向调控形态发生过程，包括营养生长、分生孢子大小、分生孢子萌发率及附着胞（appressorium）形成率；尤为关键的是，CsOxdC3的缺失突变体无法形成附着胞。4个草酸脱羧酶基因的缺失突变体对Mn²+、Cu²+及多种杀菌剂呈现不同的响应特征。其中，CsOxdC2与CsOxdC4在抵御Mn²+与Cu²+胁迫中发挥正向调控作用；CsOxdC1在暹罗炭疽菌对唑类（azole）杀菌剂的抗性中仅表现出微弱的正向调控效果；而CsOxdC3则显著正向调控暹罗炭疽菌对包括吡咯类（pyrrole）和唑类在内的多种杀菌剂的敏感性，但CsOxdC2与CsOxdC4无此功能。进一步研究发现，与野生型菌株（wild-type strain）相比，ΔCsOxdC2与ΔCsOxdC3的毒力显著降低，而ΔCsOxdC1与ΔCsOxdC4未表现出该表型。综上，本研究数据表明，CsOxdC家族基因在暹罗炭疽菌的形态发生、胁迫稳态、抗药性及毒力调控中发挥多样化的功能。本研究为阐明半活体营养型真菌暹罗炭疽菌中草酸脱羧酶的生物学功能提供了新的见解。