VdNUC-2, the Key Regulator of Phosphate Responsive Signaling Pathway, Is Required for Verticillium dahliae Infection

In fungal cells, a phosphate (Pi) responsive signaling and metabolism (PHO) pathway regulates Pi-homeostasis. NUC-2/PHO81 and its homologs are one of the most important components in the regulation pathway. In soil-borne phytopathogenic fungus Verticillium dahliae, we identified a Neurospora crassa nuc-2 homolog gene VdNUC-2. VdNUC-2 is composed of 1,018 amino acids, and is highly conserved in tested filamentous fungi. Under conditions of Pi-starvation, compared with the wild-type strain and ectopic complementation strains, the VdNUC-2 knocked out mutants exhibited reduced radial growth, decreased production of conidia and microsclerotia, and were more sensitive to hydrogen peroxide stress. The virulence of VdNUC-2 defective mutants was significantly compromised, and that was unable to be restored by exogenous application of extra Pi. Additionally, the deletion mutants of VdNUC-1, a key transcription factor gene positively controlled by VdNUC-2 in the PHO pathway, showed the similar cultural phenotypes as VdNUC-2 mutants when both of them grew in Pi-limited conditions. However, the virulence of VdNUC-1 mutants was comparable to the wild-type strain. These evidences indicated that the virulence reduction in VdNUC-2 mutants is not due to the interruptions in the PHO pathway or the disturbance of Pi-homeostasis in V. dahliae cytoplasm. VdNUC-2 is not only a crucial gene in the PHO pathway in V. dahliae, but also is required for the full virulence during host-infection.

在真菌细胞中，无机磷酸盐（Pi）响应性信号转导与代谢（PHO）通路负责调控Pi稳态。NUC-2/PHO81及其同源物是该调控通路中最为关键的组分之一。在土传植物病原真菌大丽轮枝菌（Verticillium dahliae）中，我们鉴定得到了粗糙脉孢菌（Neurospora crassa）nuc-2基因的同源基因VdNUC-2。VdNUC-2由1018个氨基酸残基组成，在已检测的丝状真菌中保守性极高。在Pi饥饿培养条件下，与野生型菌株及异位互补菌株相比，VdNUC-2基因敲除突变体的径向生长速率显著降低，分生孢子与微菌核的产生量下降，且对过氧化氢胁迫更为敏感。VdNUC-2功能缺失突变体的致病力显著减弱，且该表型无法通过外源施加额外Pi来恢复。此外，作为PHO通路中受VdNUC-2正向调控的关键转录因子基因，VdNUC-1的敲除突变体在Pi限制培养条件下，表现出与VdNUC-2突变体相似的培养表型。但VdNUC-1突变体的致病力与野生型菌株并无显著差异。上述实验结果表明，VdNUC-2突变体的致病力下降并非源于PHO通路的阻断或大丽轮枝菌胞内Pi稳态的紊乱。VdNUC-2不仅是大丽轮枝菌PHO通路中的关键基因，同时也是其宿主侵染过程中实现完全致病力所必需的基因。