紫云英类受体激酶AsNIP43在根瘤菌侵染中的共生互作机制研究

It is imperative to elucidate the symbiotic receptor and signaling pathway in legume host plant in response to the rhizobial effector protein Nops (nodulation outer proteins). In our latest research progress, a B_lectin-S_locus plant receptor-like kinase in Astragalus sinicus, namely AsNIP43, was isolated. It was identified that AsNIP43 interacts with a type III effector protein NopP of Mesorhizobium huakuii. It is speculated that this interaction can modulate the signaling pathway of host plant cells and plays an important role in rhizobial infection. This project aims to reveal the symbiotic interacting mechanism and pathway of AsNIP43 during the processes of rhizobia invasion into the host plant. The project will be carried out through the following three approaches. Firstly, with the aspect of spatial functional localization of AsNIP43 protein, the studies of protein-protein interactions will be conducted, and the symbiotic interactions in vivo, (sub) cellular and (co)-localization of AsNIP43-NopP will be confirmed and characterized; Secondly, with the aspect of functional mechanism of AsNIP43, the interactive relationships between AsNIP43 protein kinase activity and NopP, as well as the potential competitive interacting between NopP and LPS (lipopolisaccaride) with AsNIP43, will be examined and determined; Thirdly, with the aspect of functional pathway mediated by AsNIP43, downstream proteins of the host plant interacting with AsNIP43 will be screened and identified by yeast two-hybrid technique, the symbiotic phenotypes of the target proteins in the rhizobial infection will be corroborated. Meanwhile, a global overview analysis will be performed in regard to the candidate key genes and pathways modulated by AsNIP43 in terms of symbiotic defense. The results of this study will, for the first time, reveal the functional mechanism and signaling pathway of a novel legume host plant RLK to recognize a rhizobial effector protein Nop, by which to control the successful infection of rhizobia. It will produce achievement and advances with indigenous innovation and intellectual property in this research field. In addition, this study is able to provide a new perspective and pointcut to profoundly explore the inter-relationship of signaling pathway between symbiosis and defense during the legume-rhizobium interactions.

我们前期发现，紫云英的一个B_lectin-S_locus类受体激酶（RLK，receptor-like kinase）AsNIP43与华癸根瘤菌效应蛋白NopP相互作用，可能调制宿主细胞内信号通路，在介导根瘤菌侵染中发挥重要作用。为揭示其功能作用机制，拟从3个层面开展研究：1）在功能定位层面，确证AsNIP43在共生条件下与NopP的原位互作与定位；2）在功能机制层面，阐明AsNIP43激酶活性与NopP的互作关系，以及AsNIP43与NopP／LPS之间潜在的竞争互作；3）在作用途径层面，筛选和鉴定与AsNIP43互作的下游宿主蛋白，全局分析AsNIP43调制共生防御中的关键基因与途径。预期研究结果将首次阐明宿主豆科植物RLKs与根瘤菌效应蛋白发生相互作用、控制根瘤菌入侵的信号途径，为该领域提供自主创新研究成果和进展。还可提供新视角深入探究根瘤菌-宿主互作中共生与防御的相互关系。

我们前期发现紫云英的一个B_lectin-S_locus类受体激酶AsNIP43与华癸根瘤菌效应蛋白NopP相互作用，可能调制宿主细胞内信号通路，在介导根瘤菌侵染中发挥重要作用。为揭示其功能机制，本项目主要从以下3个方面开展了研究：1）在功能定位层面，确证AsNIP43在共生条件下与NopP的原位互作与定位；2）在功能机制层面，阐明AsNIP43与NopP／LPS之间潜在的竞争互作；3）在作用途径层面，筛选和鉴定与AsNIP43互作的下游宿主蛋白，全局分析AsNIP43调制共生防御中的关键基因与途径。.通过项目实施，我们发现AsNIP43定位于细胞膜和内质网，NopP和AsNIP43共定位于细胞质膜及细胞质内膜系统；AsNIP43含有B-lectin结构域，是介导其与NopP互作的关键结构域，且与根瘤菌脂多糖LPS 具有结合能力，AsNIP43与共生受体NFR5具有相互作用；筛选获得AsNIP43互作宿主蛋白AsEPN，确定AsEPN亚细胞定位在细胞质和细胞膜，共生表型鉴定表明AsEPN影响根瘤菌入侵及共生体发育；这些功能机制在模式豆科植物苜蓿中获得了系统的验证和鉴定；还发现华癸根瘤菌NopP突变体在不同紫云英品种上具有不同的共生表型，在信紫一号等品种上促进结瘤固氮。基于上述发现，我们提出一个工作模型：NopP与AsNIP43的相互作用可能阻止了根瘤菌脂多糖（LPS）与AsNIP43中B_lectin结构域的结合，弱化或阻断了宿主植物防御信号通路；并通过NFR5互作引发共生反应；进一步通过互作蛋白AsEPN的协同作用，调控根瘤菌侵染和早期结瘤过程。.本项目取得的成果揭示了豆科宿主植物S型类受体激酶与根瘤菌效应蛋白互作、从而控制根瘤菌侵染的功能机制，为该领域提供了自主创新的研究成果。也为深入研究豆科植物响应根瘤菌效应蛋白、LPS、NFs信号，以及R蛋白之间的信号整合调制与互作网络，提供了切入点。