水稻E3-EIP蛋白分子模块调控水稻自身免疫的分子机理研究

Bacterial blight and Rice blast diseases, caused by Xanthomonas oryzae pv. oryzae and Magnaporthe oryzae, respectively, are two of the most devastating diseases in rice worldwide. Previously, we screened a mutant ebr1 (enhanced blight and blast resistance 1) showed enhanced resistance to multiple strains of Xoo as well as M. oryzae. Plants of ebr1 developed a spontaneous cell death phenotype on leaves in the absence of pathogens, similar to hypersensitive response (HR) cell death in during plant immune activation. Together with Map-based cloning and RNA-seq analysis, we successfully identified the EBR1 gene. Biochemical analysis revealed that EBR1 encodes a previously unknown RING-type protein with E3 ubiquitin ligase activity. RNA interference transgenic lines with reduced EBR1 transcripts exhibited the lesion mimic and enhanced resistance to diseases phenotype similar to that of ebr1 plants, while expression of EBR1 in the ebr1 mutant background complemented the ebr1 phenotype, confirming that the mutation identified in EBR1 is responsible for the ebr1 mutant phenotype. Meanwhile, the EBR1 overexpression transgenic plants show more susceptible to Xoo as well as M. oryzae in comparison with the wild-type. All these together revealed EBR1 is a negative regulator of cell death and defense in rice. To elucidate the mechanism of EBR1-mediated immunity and to identify its interaction partners, we performed an extensive yeast two-hybrid screening, and successful identified 11 possible EBR1-interacting proteins (EIPs). Among them, at least EIP1 could interactive with EBR1 in vivo, which was confirmed by BiFC. However, it is still unclear about the biochemical role of EIPs, and how EIPs involved in rice innate immune activation. Therefore, in this project, We will validate the in vivo interaction between EBR1 and EIPs. Furthermore, by investigating the biological function of EIPs, we will trying to illustrate how EBR1 affects plant immunity through EIPs, especially EIP1. These results will provide clues for our understanding of the broad resistance of plant against pathogens, and finally benefit the molecular breeding in agriculture.

水稻白叶枯病和稻瘟病是水稻的两大主要病害，每年都对其产量造成严重损失。我们前期从水稻资源库中筛选到一株广谱抗病植株ebr1。ebr1表现出全生育期自发免疫类病斑表型，且对白叶枯及稻瘟病具有广谱抗性。研究发现，ebr1表型由隐性基因EBR1控制，EBR1是参与细胞死亡和免疫防御的一个负调控因子。EBR1编码一个未知E3泛素连接酶。利用酵母双杂技术，我们已成功筛选出11个与EBR1互作的蛋白EIPs, 并验证了EIP1与EBR1存在体内互作。然而，EBR1互作蛋白EIPs的明确生物学功能及其如何参与到ebr1广谱抗病的机理尚不明确。本项目将在已有研究基础上着重对EBR1与EIPs在植物体内互作进行验证，重点对E3-EIP1模块在植物抗病信号传导中的作用及机理进行深入研究，并利用这些基因进行水稻抗病育种研究。本项目对深入理解水稻抗病机制及发掘新的广谱抗源有重要的理论和实践意义。

在前期工作中，我们筛选到广谱抗病植株ebr1,对水稻两大病害白叶枯病及稻瘟病均有较强抗性，且具有自发免疫类病斑、植株矮小等表型。随后研究结果表明，EBR1利用其泛素连接酶活性，负调控植物免疫因子而调控植物免疫。为进一步研究EBR1调控免疫分子机理，我们利用酵母双杂技术筛选出11个与其互作的蛋白(EBR1 interaction proteins， EIPs)。以此为基础，本项目主要研究内容为：验证EIPs与EBR1的体内互作，并揭示EBR1-EIP参与植物免疫调控的机理。结果表明，EIP1与EBR1在植物体内能够直接互作。同时，EBR1能够泛素化EIP1,并控制EIP1在植物体内的累积。过表达EIP1的植株能产生与ebr1类似的类病斑、抗白叶枯与抗稻瘟病表型。在ebr1背景下，利用RNAi技术knock-down EIP1基因也能够回补ebr1表型。可见，EIP1正调控植物细胞程序性死亡与免疫，负调控植物生长发育；且EIP1在植物体内的含量，受到EBR1介导的26S蛋白酶体的直接调控。因此，我们在ebr1材料中观察到的对白叶枯病与稻瘟病的广谱抗病表型、类病斑表型及生长发育受抑制等表型，均与EIP1在植物体内的精细调控紊乱有关。至此，我们成功解析了EBR1-EIP1这一蛋白模块在水稻自身免疫与广谱抗病的分子机理。我们也同时验证了EBR1能与EIP2、EIP5及EIP8在体内互作。但过表达相关EIP后，植物抗病性并未改变。说明相关EIP并不参EBR1的抗病过程。过表达EIP10也能增强植物对白叶枯病的抗病性，但对稻瘟菌抗病性及植物生长并无影响，提示EIP10可能为潜在的植物免疫正调控因子。然而，我们并没有检测到EIP10与EBR1在体内的互作，也未检测到EIP10蛋白被泛素化修饰。表明虽然EIP10也参与植物抗细菌过程，但显然与EIP1参与植物免疫分子机理是有所不同的。生物信息分析表明，EIP10含有磷酸化激酶结构域。通过对EIP10在体内的自磷酸化的初步探索，我们发现：EIP10在酪氨酸、苏氨酸/丝氨酸磷酸化位点与对照组带型无明显差异,但酪氨酸磷酸化的EIP10含量有所增加。因此，我们仍需寻找新的思路，研究EIP10参与植物抗病的分子机理。