锌簇转录因子VdZC1调控细胞壁降解功能参与大丽轮枝菌侵染寄主的致病机理研究

This project will provide the theoretical foundations for the pathogenicity mechanism of Zn2Cys6 transcription factor VdZC1 regulates expression of cell wall degradation-related genes in the Verticillium dahliae and other fungi pathogens. Zn2Cys6 proteins are one kind of fungal-specific transcription factors that can participate in the pathogenicity-related gene regulation during the plant infection. In Verticillium dahliae, Zn2Cys6 proteins belong to the transcription factors superfamily that encodes more than 200 Zn2Cys6 transcription factors in genome. However, few studies on the functional genes involved in the pathogenicity have been reported. In the previous research of this project, a Zn2Cys6 transcription factors VdZC1 involved in the pathogenicity were identified by a series of experiments, including identification of pathogenicity-related genes by T-DNA insertional mutagenesis, Zn2Cys6 gene family analysis, gene knockout and complementary. However, the mechanism of VdZC1 regulating the pathogenicity in Verticillium dahliae was unknown. Therefore, this project will identify the gene polymorphism and their morphological development to determine the characteristic of VdZC1. The VdZC1 regulatory genes and its regulation network will be identified by the integration analysis of RNA-seq and chromatin immunoprecipitation (ChIP) data. Gene knock-out experiments will be used to identify the virulence of target regulatory genes while the conserved cis-element in the promoters bind to VdZC1 will be identified by the regulatory genes. The binding activity will be confirmed by the yeast one-hybrid; and the localization characteristic of VdZC1 and its relationship with pathogenicity will be illustrated by analysis of subcellular localization and the mutation of nuclear localization signal and transcription activation domains.

Zn2Cys6锌簇蛋白是真菌特有的转录因子，可调控基因表达参与病原对植物的侵染过程。大丽轮枝菌编码超过200个锌簇转录因子，但参与致病性调控的功能基因少见报道。前期通过大丽轮枝菌T-DNA突变体筛选及基因敲除与回补，确定了1个参与细胞壁降解并影响致病性的锌簇转录因子VdZC1，但其调控基因表达参与侵染寄主的分子机理尚不清楚。因此，本项目拟通过VdZC1多态性、影响形态发育等分析明确基因的基本特性；结合表达谱和染色质免疫共沉淀分析，鉴定VdZC1调控降解细胞壁的靶标基因并验证功能；整合数据筛选VdZC1结合的顺式作用元件并通过酵母单杂交鉴定，明确调控靶标基因的作用方式；通过核定位信号/转录结构域突变基因回补明确VdZC1调控靶标基因的机制，最终构建VdZC1调控细胞壁降解功能参与致病性的基因调控网络。完成上述研究将为锌簇转录因子参与大丽轮枝菌以及其他真菌致病性调控的分子机理研究提供理论依据。

项目按照研究计划完成了既定研究内容。通过突变体形态发育、基因表达、多态性、亚细胞定位等分析，明确了Zn2Cys6转录因子VdFTF1调控大丽轮枝菌致病性但是不参与调控菌株生长的基本特性；结合基因表达谱分析，明确了VdZC1主要通过调控细胞壁降解功能基因参与致病的分子机制，并明确其中部分靶标基因具有调控毒力的功能；通过筛选VdZC1结合的潜在顺式作用元件并进一步利用酵母单杂交技术鉴定其结合特性，明确了VdZC1调控靶标基因的保守作用基序；最终构建了Zn2Cys6转录因子VdZC1通过调控细胞壁降解功能参与病原侵染寄主的基因调控网络。上述研究首次发现了大丽轮枝菌不含锌指结构域的Fungal_trans类转录因子具有致病性调控功能，揭示了其调控植物细胞壁降解酶基因表达参与病原侵染寄主的致病新机制，有助于阐明Zn2Cys6转录因子调控大丽轮枝菌致病性的功能特性、作用方式和调控机理，为转录因子参与病原菌侵染寄主的致病分子机理研究提供理论依据。