核小体结合蛋白VdNBP调控大丽轮枝菌致病性的分子机制研究

Nucleasome binding protein (NBP) is a kind of high mobility group protein which mediate the DNA-binding of chromatin to modulate gene expression of eukaryotic organism. In previous study, we found knockout of VdNBP, a gene encoding the nucleasome binding protein in Verticillium dahliae, reduced conidia production, the pathogenicity in cotton and ethylene biosynthesis. In this project, based on the RNA-Seq data between VdNBP knockout mutant and wild-type V592 strain, we will determine target genes regulated by VdNBP by combining chromatin immunoprecipitation sequencing (ChIP-Seq) and qRT-PCR. Knockout mutant strains of target genes and their complement strains will be generated and used to determine target genes involved in conidia production, pathogenicity and ethylene biosynthesis, respectively. The responses of cotton, Arabidopsis thaliana and A. thaliana mutant plant etr1-1 (ethylene receptor mutant) inoculated with knockout mutant strains of VdNBP and target genes will be used to explore the components in defense against V.dahliae and to elucidate the roles of ethylene biosynthesis regulated by VdNBP and target genes in V. dahliae. We will confirm the subcellular localization of VdNBP, and tissue location of knockout mutant strains of VdNBP and target genes during infection process. Based on these research results, it will be helpful for us to elucidate the molecular mechanism of VdNBP regulating the pathogencity in V. dahliae, and to explore a new type of method to control cotton wilt disease.

核小体结合蛋白（NBP）是真核生物体内通过与核小体结合调控基因表达的一类高迁移率蛋白。本项目前期研究发现，大丽轮枝菌的一个核小体结合蛋白基因（VdNBP）敲除严重影响其产孢和对棉花的致病力，并导致真菌乙烯合成能力明显下降。本项目拟通过ChIP-Seq技术筛选与VdNBP结合的DNA序列，结合转录组测序结果及qRT-PCR验证明确VdNBP调控的靶基因。获得靶基因敲除突变体及互补体，通过产孢量、乙烯含量及致病性测定，明确分别影响大丽轮枝菌产孢、乙烯合成及致病力的靶基因。将VdNBP敲除体及与乙烯合成相关的靶基因敲除体分别接种棉花、拟南芥及拟南芥乙烯受体突变体etr1-1，分析VdNBP及靶基因调控的乙烯合成与致病力及寄主抗病之间的关系。结合VdNBP亚细胞定位、VdNBP及其靶基因在侵染过程中的定位研究，以期阐明VdNBP调控大丽轮枝菌致病的分子机制，为开展新的黄萎病防治策略提供理论依据。

核小体结合蛋白（NBP）是真核生物体内通过与核小体结合调控基因表达的一类高迁移率蛋白。本研究发现，棉花黄萎病菌V592菌株用棉花根系诱导培养的10 h，VdNBP基因在菌丝中的表达量最高，表明VdNBP基因在大丽轮枝菌中的表达具有组织和时间的差异性，并受寄主诱导表达，可能与寄主互作有关。VdNBP基因敲除导致大丽轮枝菌产孢量和致病力显著低于野生型菌株V592，表明VdNBP基因是大丽轮枝菌产孢和致病所需的基因。用棉花根系分别诱导培养VdNBP敲除突变体和野生型菌株V592 10 h，再分别对2个菌株进行RNA-seq和ATAC-Seq高通量测序，通过联合分析，筛选出ABC转运体基因（VDAG_07300）、内质网蛋白加工信号通路中的基因（VdSiL1、VdDnaJ和VdBip）和TPR结合蛋白基因（VdSET）为VdNBP调控的下游靶基因。对靶基因进行功能分析，结果显示，与V592菌株相比，VdSET、VdSiL1和VdDnaJ单个基因敲除均导致大丽轮枝菌产孢量显著下降；但是VdSET、VdSiL1和VdDnaJ单个基因敲除或VdSET和VdDnaJ双基因敲除均导致大丽轮枝菌对棉花的毒力显著增强，表明VdSET、VdSiL1和VdDnaJ基因可能与植物免疫调控有关。为了进一步明确VdNBP基因与靶基因的相关性，将靶基因VdSET和VdDnaJ分别与VdNBP基因进行双敲除，结果显示，双敲突变体均导致大丽轮枝菌不形成微菌核、产孢量比各基因单独敲除的产孢量下降更显著。然而当靶基因VdSET和VdDnaJ基因分别与VdNBP基因双敲时，均导致大丽轮枝菌对棉花的致病力比VdNBP基因单独敲除时的致病力明显减轻。上述结果表明表明VdNBP基因通过调控VdSET、VdSiL1和VdDnaJ基因影响大丽轮枝菌微菌核形成、产孢和致病过程。