蛋白酶体参与大麦白粉病抗病蛋白MLA介导专化性抗病反应的机理研究

The plant immune system against microbial pathogens is consisted of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI). NLR-type R protein can reprogram defense-related gene expression upon effector recognition and activate and mediate ETI, however, its mechanism yet remains unclear. Ubiquitin-proteasome system (UPS) is the major protein degradation machinery in eukaryotic cells and is pivotal to plant ETI in various aspects, however, its mechanism remains unclear, too. We recently found in barley that NLR-type R protein MLA directly and specifically interacts with RPTX subunit of proteasome during the MLA-mediated ETI responses against powdery mildew. In this study, first, we plan to profile in depth the characteristics of the MLA-RPTX interaction, with an emphasis on structural analysis of the interaction interface. Next, we will perform structure-function analysis and mutagenesis on RPTX peptide sequence, and identify mutated RPTX (mRPTX) that no longer interacts with MLA but retains fundamental functions in proteasome. Then, we will replace the native RPTX gene in barley genome with mRPTX gene through stable transformation and CRISPR-Cas-based DNA modification technology. Meanwhile, we will establish in vitro experimental systems to investigate the effects of MLA on the activities and functions of proteasome. Finally, in mRPTX barley, we will examine the changes in PTI and ETI responses upon powdery mildew challenge, the changes in defense-related protein expression and turnover, and changes in proteasome activities in vivo. To summarize, in this study we plan to elucidate the function of MLA-RPTX interaction during barley-powdery mildew interaction, and explore new mechanisms in plant immunity mediated by ubiquitin-proteasome system through its interaction with NLR-type R proteins.

植物免疫系统包括识别与病原微生物相关的分子模式后引发的基础性免疫反应和识别病原微生物效应蛋白后引发的专化性抗病反应；后者由NLR类免疫受体蛋白介导，但其作用机理尚不十分清楚。泛素-蛋白酶体系统是细胞内蛋白降解的主要途径，参与植物免疫抗病反应，但其作用机制尚有待深入研究。最新研究发现，在大麦NLR类抗病蛋白MLA介导的白粉病专化性抗病反应中，MLA特异性地与蛋白酶体相互作用，并伴随蛋白酶体活性上升。本项目拟深入分析模式抗病蛋白MLA与蛋白酶体间相互作用的生物学特征，建立体外实验系统解析MLA与蛋白酶体对抗病相关蛋白的调控作用、及MLA对蛋白酶体活性和功能的影响，并结合蛋白结构-功能分析和遗传学等手段，了解两者间相互作用对建立专化性抗病反应的影响，阐明蛋白酶体在参与MLA激发下游抗病相关基因表达及建立超敏反应过程中的作用，进而揭示蛋白酶体参与大麦抗病蛋白MLA介导白粉病专化性抗病反应的新机理。

本项目旨在研究大麦蛋白酶体与NB-LRR类抗病蛋白MLA互作并影响其稳定性来调节大麦对大麦白粉菌小种专化抗性的机理，包括大麦蛋白酶体与MLA之间相互作用的生物化学，细胞生物学和结构生物学特征、蛋白酶体对MLA活性和功能的影响、以及蛋白酶体参与MLA介导专化性抗病反应的机制研究。本项目所取得的主要研究成果可归纳为以下三方面：（1）揭示了大麦蛋白酶体与NB-LRR类抗病蛋白MLA相互作用的关键位点、时序及亚细胞定位；（2）阐明了蛋白酶体对NB-LRR类抗病蛋白MLA活性的影响；（3）发现蛋白酶体通过调节NB-LRR类抗病蛋白MLA活性来参与大麦对大麦白粉菌专化性抗病反应。