Table_1_β-Glucosidase VmGlu2 Contributes to the Virulence of Valsa mali in Apple Tree.docx

The apple tree canker is caused by Valsa mali, which produces major pathogenic factors involving multiple cell wall-degrading enzymes (CWDEs) and toxins. The β-glucosidases are among the main CWDEs, and thus, they play important roles in the virulence of necrotrophic pathogens. However, the specific roles of β-glucosidases in the virulence of V. mlai remain largely unknown. In this study, we identified a β-glucosidase gene, VmGlu2, which was upregulated during the V. mali infection. We found that VmGlu2 protein had high enzyme activity of β-glucosidase using p-nitrophenyl-β-D-glucopyranoside (pNPG) as a substrate, while the VmGlu2 could convert phloridzin to phloretin with the release of glucose. The deletion and overexpression of VmGlu2 showed no effect on vegetative growth, but gene deletion mutants of V. mlai showed significantly reduced pycnidia formation. The gene deletion mutants had lower β-glucosidase activities and toxin levels as compared to the wild-type strain. Therefore, these mutants showed a reduced virulence. Moreover, the overexpression of VmGlu2 did not affect toxin levels, but it significantly enhanced β-glucosidase activities, which resulted in an increased pathogenicity. Thus, we conclude that VmGlu2 is required for the full virulence of V. mali. These results provide valuable evidence to the complex role of CWDEs in the fungal pathogenicity.

苹果树溃疡病由 Valsa mali 引起，该病原体产生多种致病因子，包括多种细胞壁降解酶（CWDEs）和毒素。β-葡萄糖苷酶是主要的 CWDEs 之一，因此，它们在坏死性病原体的致病力中发挥着至关重要的作用。然而，β-葡萄糖苷酶在 V. mali 病原体致病力中的具体作用尚不清楚。在本研究中，我们鉴定了一个 β-葡萄糖苷酶基因，命名为 VmGlu2，该基因在 V. mali 感染期间上调表达。我们发现，VmGlu2 蛋白以对硝基苯基-β-D-吡喃葡萄糖苷（pNPG）为底物，表现出高酶活性，同时 VmGlu2 可以将花青素转化为儿茶素，并释放葡萄糖。VmGlu2 的基因敲除和过表达对营养生长没有影响，但 V. mali 的基因敲除突变体显示出显著减少的子囊壳形成。与野生型菌株相比，基因敲除突变体具有较低的 β-葡萄糖苷酶活性和毒素水平。因此，这些突变体表现出降低的致病力。此外，VmGlu2 的过表达没有影响毒素水平，但它显著增强了 β-葡萄糖苷酶活性，从而增加了致病性。因此，我们得出结论，VmGlu2 对于 V. mali 的完全致病力是必需的。这些结果为 CWDEs 在真菌致病性中的复杂作用提供了宝贵的证据。