植物先天免疫是植物与病原微生物长期共同进化产生的对抗病原微生物侵害的防御机制。植物的先天免疫系统主要由病原微生物模式分子引起的免疫途径（Pathogen-associated molecular pattern triggered immunity，PTI）和效应蛋白引起的免疫反应（Effector ETI）两大类组成。其中ETI主要由位于胞内的NB-LRR（NLR）抗病蛋白介导，根据其N端结构域的不同，可进一步分成TIR-NB-LRR（TNL）类抗病蛋白和CC-NB-LRR（CNL）类抗病蛋白。关于CNL类抗病蛋白的配体识别和活化机制，已经有研究阐明。但是不同于CNL，TNL类抗病蛋白的作用机制尚未阐明。之前有文献报道植物的TIR结构域具有NAD+水解酶活性，对于TNL类抗病蛋白的功能至关重要。但是，截止到本文发表之前，TNL类抗病蛋白如何识别配体和活化的机制、TNL是如何发挥NAD+水解酶活性的以及下游信号传递的具体机制尚缺乏深入研究。RPP1作为TNL类抗病蛋白的典型代表，主要介导拟南芥对Hyaloperonospora arabidopsis (Hpa)病原菌产生抗性。Hpa在侵染拟南芥的过程中，会分泌效应蛋白ATR1，ATR1能引起表达相应RPP1抗病蛋白的拟南芥植株产生抗病反应。根据现有的模型，RPP1在未感知ATR1的情况下，通过自身各结构域之间的相互作用维持自抑制状态。当RPP1识别ATR1后，会激活并通过下游的免疫信号通路诱导宿主植物产生抗病反应。 本文将RPP1和ATR1在昆虫细胞里进行共表达，通过串联纯化得到了高纯度活化状态的RPP1-ATR1复合物，命名为RPP1抗病小体。通过冷冻电镜单颗粒重构技术，解析了RPP1抗病小体3.16 ?的高分辨率同源四聚体结构。该结构还揭示了一个C末端集成的Jelly roll/ IgG样结构域(C-JID)，负责特异性识别ATR1。功能分析表明，ATR1与C-JID和LRR结构域的结合能够激活RPP1组装形成四聚体并诱导产生NADase全酶活性。RPP1四聚化产生两个活性位点，每个位点由非对称的同源二聚体介导形成。 总之，本文通过结构生物学、生物化学和遗传学手段，成功揭示了植物TNL类抗病蛋RPP1直接识别结合效应蛋白ATR1并组装形成四聚体，诱导产生NADase全酶活性的分子机制。
Plant innate immunity is a defense mechanism against the invasion of pathogenic micro-organisms which coevolved by plants and pathogens. Plant innate immune system consists of two types of immunity: Pathogen-associated molecular pattern triggered immunity (PTI) and Effector triggered immunity (ETI). ETI is mainly mediated by intracellular NB-LRR (NLR) disease resistant proteins, which can be further divided into TIR-NB-LRR (TNL) disease resistant proteins and CC-NB-LRR (CNL) disease resistant proteins according to the different N-terminal domains. The ligand recognition and activation mechanisms of CNL disease resistant proteins have been elucidated. However, unlike CNLs, the mechanism of TNL resistance proteins has not yet been clarified. It has been previously reported that the TIR domain of plant TNL resistant proteins has NAD+ hydrolase activity, which is crucial for the function of TNL disease resistant proteins. However, until the publication of this paper, the mechanism of how TNL disease resistant proteins recognize ligand and activate, how TNL exerts the activity of NAD+ hydrolase and the specific mechanism of downstream signal transmission have not been thoroughly studied.RPP1, as a typical representative of TNL disease resistant protein, mainly mediates resistance of Arabidopsis thaliana to Hyaloperonospora arabidopsis (Hpa) pathogen. During the infection of Arabidopsis thaliana, Hpa secretes effector protein ATR1, which can induce Arabidopsis thaliana ecotypes expressing corresponding RPP1 resistant proteins to produce disease resistance response. In the absence of ATR1, RPP1 maintains self-inhibition state through intermolecular interactions among its own domains. When the pathogen infects the plant, it will secrete the effector protein ATR1 into the host cell. Upon recognition of ATR1, RPP1 will release the auto-inhibition and become activated, then catalyze the hydrolysis of NAD+ molecules, generating a second messenger called v-cADPR, which will transmit and activate the downstream EDS1-SAG101-NRG1 pathway, and finally induce plant disease resistance response.In this study, RPP1 and ATR1 were co-expressed in insect cells, and high purity activated RPP1-ATR1 complex was obtained by tandem purification, which was named as RPP1 resistosome. The 3.16 ? homo-tetramer structure of RPP1 resistosome was analyzed by Cryo-EM single particle reconstruction technique. The structure also reveals a C-terminal integrated Jelly Roll/IgG-like domain (C-JID) responsible for the specific recognition of ATR1. Functional analysis showed that ATR1 binding to C-JID and LRR domains could activate tetrameric RPP1 assembly and induce NADase holoenzyme activity. Tetramerization of RPP1 yields two active sites, each mediated by an asymmetric homodimer.In conclusion, we demonstrate the molecular mechanism by which TNL disease resistant protein RPP1 can directly recognize binding effector protein ATR1 and assemble into tetramer to induce NADase holoenzyme activity through structural biology, biochemistry and genetic methods.
