拟南芥免疫反应负调节因子的基因克隆与功能鉴定

Plants respond in various ways to defend themselves against pathogen infections, in which resistance (R) protein-mediated defense is one of the most effective mechanisms. In Arabidopsis, snc1 encodes a TIR-NB-LRR R-like protein that carries a unique gain-of-function mutation, leading to constitutive activation of defense. Over a dozen MOS (Modifier of SNC1) proteins have been identified as positive regulators of plant immunity, indicating a complicated signaling network involved in NB-LRR R protein activation. However, constitutive activation or over- accumulation of NB-LRR R proteins can result in unwarranted autoimmune responses, their activity and stability must be tightly regulated. Despite the importance of protein level control of NB-LRR R proteins, the molecular mechanisms by which their turnover is regulated is poorly understood. To study negative regulation of snc1-mediated resistance, genetic screen was performed in mos4 snc1 background. Mutants restoring snc1-mediated autoimmunity phenotypes were isolated and named as muse (mutants, snc1-enhancing) mutants. In this program, we aim to finish the identification and characterization of a muse mutant, through positional cloning, Illumina whole genome sequencing, transgenic complementation, genetic analysis, RT-PCR, subcellular localization, Co-IP and so on. Cloning and detailed functional studies on MUSEs may contribute to uncovering the negative regulatory mechanisms in signal transduction pathway plants utilize to defend themselves against pathogen attack.

植物以不同方式抵御病原菌侵染来保护自己，抗性蛋白介导的免疫反应是最有效的机制之一。拟南芥中，功能获得性突变基因snc1编码一个TIR-NB-LRR抗性蛋白，导致突变体中免疫反应持续激活。以snc1为背景的遗传筛选，发现十几个植物免疫的正调控因子 (MOS蛋白)，它们的功能研究揭示NB-LRR抗性蛋白的激活涉及一个复杂的信号网络。NB-LRR抗性蛋白持续激活或过量积累会导致不必要的自身免疫反应，其活性和稳定性必须受到严格调控，然而对该过程的分子机制仍不清楚。为了研究snc1介导抗性的负调控机制，我们在mos4 snc1的背景下开展遗传筛选，获得恢复snc1抗病表型的muse突变体。本项目结合基因图位克隆和全基因组测序对muse (55-1)突变体进行基因克隆，同时利用转基因互补验证、遗传分析、基因定量表达、蛋白定位、免疫共沉淀等技术和手段进行功能验证，了解植物抗病信号转导途径中的负调控机制。

本项目以拟南芥muse (55-1) 突变体为研究材料，结合基因图位克隆和全基因组测序的手段，通过转基因互补验证克隆鉴定出一个从未报道过的泛素E3连接酶编码基因At2g37150，对其突变体的抗病表证分析发现该基因在抗病免疫途径中行使负调控作用。利用合成生物学手段进行体外泛素化活性鉴定表明该基因编码E3连接酶具有自我泛素化活性。结合其生物学功能，说明At2g37150编码E3可能通过泛素化途径降解抗病正调控因子而行使其抗病负调控作用。遗传关系及生化关系验证都发现抗性蛋白RPS2可能是At2g37150编码E3连接酶的目标蛋白。进一步的体外泛素化实验证实南芥基因At2g37150编码蛋白作为E3连接酶，通过识别抗病免疫途径中的正调控因子，如抗性蛋白RPS2使其发生泛素化修饰而被降解，从而在抗病免疫途径中行使负调控的功能。