棉花黄萎病菌效应子互作蛋白Nbnrp1调控植物免疫的分子机制

Nbnrp1 is a binding protein with Verticillium dahliae effector PevD1 and located in cytoplasm and nuclear. Bioassay with nbnrp1-overexpression and RNAi mutants showed that Nbnrp1 positively modulate PevD1-induced cell death and disease resistance. Bioinformatics analysis also indicate Nbnrp1 do not contain a typical architectures of receptor protein（NLRs）encode by central nucleotide-binding (NB) domain followed by a series of leucine-rich repeats (LRRs). whereas, Nbnrp1 contains binding site with transcription factor in its N-terminal sequence and belong to defense protein family. Whether Nbnrp1 act as a host factor to combine receptor protein to percept effector PevD1 and lead to initiate plant immunity remains unknown. In this project, we will use Nbnrp1 as a bait, fish and confirm Nbnrp1- association protein through more protein-protein interaction technology. The double genes mutants of both candidate receptor and Nbnrp1, and single gene mutants were generated through CRISPR-CAS9 technology to investigate the function of candidate receptor protein. We will analyzed the change of defense response between different mutants and wide type, including cell death, ROS, defense gene transcription, defense substance and hormone accumulation for investigating the role of the candidate receptor protein in PevD1 triggering plant immunity. Furthermore, we will analyze differential genes and differential protein expression between mutants and wild type plants post PevD1 treatment through transcriptomics and proteomics analysis, and illuminate the molecular mechanism of Nbnrp1 in modulation of plant immunity. This research will provide foundation for understanding the molecular mechanism of plant -Verticillium dahliae interaction and PevD1- triggering plant immunity.

Nbnrp1是申请人首次鉴定的棉花黄萎病菌效应子PevD1的互作蛋白，定位在植物细胞质和细胞核中。用nbnrp1基因过表达和RNAi沉默突变株验证了该蛋白能正调控效应子诱导的细胞坏死和抗病性。生物信息学分析表明，Nbnrp1蛋白无识别病原菌效应子的受体蛋白结构， 但其N端有转录因子结合位点，属于防御蛋白家族, 是否作为寄主因子参与免疫受体对效应子的间接识别而调控植物防御反应尚不清楚。本项目以Nbnrp1为诱饵蛋白，用多种蛋白互作技术钓取互作蛋白并进行体内体外验证；用CRISPR-CAS9基因组定点编辑技术，获得候选受体蛋白及Nbnrp1双基因和单基因的缺失突变株，通过候选受体基因定点剪切和修复技术明确受体蛋白在PevD1激发植物免疫中的功能，并结合转录组和蛋白组学分析与验证，阐述互作蛋白Nbnrp1调控PevD1激发植物免疫的分子机制，为进一步揭示大丽轮枝菌与寄主植物互作机制奠定理论基楚。

大丽轮枝菌(Verticillium dahliae)新效应蛋白PevD1的互作蛋白Nbnrp1属于植物NRP家族蛋白，是植物重要的防御蛋白。前期研究表明，Nbnrp1正调控PevD1诱导的植物免疫反应和抗病性，但是Nbnrp1如何参与PevD1激发植物免疫的分子机制尚不清楚。本研究利用转录组测序技术，明确了Nbnrp1-RNAi转基因植株响应PevD1诱导的差异表达基因显著富集在次生代谢产物合成通路中，如苯丙素生物合成、倍半萜和三萜等萜类物质合成等。这些次级代谢产物都是与植物抗性密切相关的化合物，其中木质素是苯丙素生物合成途径终产物，植保素是倍半萜类化合物的主要组份。因此，我们推测Nbnrp1介导PevD1诱导的抗病性主要与木质素积累和植保素合成有关。辣椒醇是茄科植物烟草重要的植保素之一，为了进一步明确Nbnrp1是否参与PevD1诱导的辣椒醇合成，我们检测了辣椒醇合成途径中的两个关键基因EAS和EAH的转录水平以及辣椒醇的积累。结果表明，PevD1处理后，WT和Nbnrp1-RNAi植物中EAS和EAH的转录水平均转录上调，但Nbnrp1-RNAi中的转录水平显著低于WT。与野生型相比，Nbnrp1-RNAi植物中辣椒醇含量也明显降低。在没有PevD1处理的WT和Nbnrp1- rnai植物中未能检测到辣椒醇，以上结果表明，Nbnrp1阻止了PevD1诱导的植物抗毒素辣椒醇积累。