水稻PPR蛋白OsNBL3通过参与线粒体RNA代谢调控植物防卫反应的分子机制

Pentatricopepetide repeat (PPR) proteins comprise one of the largest protein family involved in RNA metabolism in plant organelles. Although functions of many PPR proteins have been studied, the knowledge about PPR proteins is still limited concerning this large protein family andthe relationship between PPR proteins and plant defense is largely unknown. In previous studies, a rice mutant nbl3 from T-DNA insertion lines was identified. This mutant with slow growth, early leaf blight companied with reactive oxygen species burst enhanced resistance to the blast fungus. The DNA sequences flanking the T-DNA insertion site were identified by SiteFinding-PCR method, and the full length cDNA of OsNBL3 was cloned. Complementation of this mutant with OsNBL3 proved that disruption of OsNBL3 is responsible for the phenotypes of nbl3 mutant. OsNBL3 encodes a novel P-subclass PPR protein harboring a mitochondrial signal peptide. In this study, in addition to determine the sub-cellular location of OsNBL3, we will elucidate whether and how OsNBL3 involves in mitochondrial RNA metabolism and which gene is the target of OsNBL3. To meet this aim, C-to-U changes of mitochondrial RNAs and splicing efficiency will determined by RT-PCR and subsequently sequencing. 5’- and 3’- termini of mitochondrial RNAs will be determined by CR-RT-PCR. The differential of Respiratory complexes between wild type and the mutant will be compared by blue native polyacrylamide gel electrophoresis, western blot analysis and in-gel activity assays. Also, the mechanism of OsNBL3 regulating ROS releasing and hypersensitive cell death will be investigated. Furthermore, transcriptional analysis of nbl3, wild type and OsNBL3 overexpression line will be carried out to elucidate the defense pathway regulated by OsNBL3, and the key gene(s) regulated by OsNBL3 in rice defense will be identified. Overall, our findings will shed light on revealing molecular mechanism of mitochondria-targeted P-subclass PPR proteins which are involved in RNA metabolism and defense regulation.

PPR蛋白是植物中最大的蛋白家族之一，参与调控细胞器基因的RNA代谢。尽管很多PPR蛋白的功能已被报道，但相对于庞大的PPR蛋白家族而言，人们对PPR蛋白的认识还十分有限，尚无有关PPR基因参与植物抗病性的报道。本项目前期获得了一个水稻T-DNA插入突变体nbl3。该突变体生长缓慢、叶片早枯并伴随活性氧突发，对稻瘟菌的抗性增强。基因克隆和互补试验证明nbl3的表型突变系OsNBL3基因被破坏引起。OsNBL3是一个新的P亚家族PPR蛋白，预测定位在线粒体。在本项目中，将研究其如何参与调控线粒体基因RNA修饰，进而研究其突变如何影响线粒体呼吸链蛋白的含量及其活性，以及其调控线粒体活性氧产生和细胞过敏性坏死的机制。在此基础上，解析其参与植物抗病反应的调控路径并鉴定其调控的关键防卫基因。研究结果将有助于揭示位于线粒体的P亚家族PPR蛋白在调控线粒体基因转录后修饰及在调控植物防卫反应中的分子机制。

Pentatricopeptide repeat（PPR）蛋白是一类由核基因编码且多在线粒体或叶绿体发挥功能的蛋白质，其主要参与线粒体或叶绿体RNA的内含子剪接、碱基编辑、转录后的修饰和翻译以及非翻译区的稳定等。线粒体基因编码的蛋白质大部分属于线粒体电子传递呼吸链 (mitochondrial electron transport chain, mtETC) 复合体的亚基。mtETC的功能紊乱会激活呼吸替代酶（alternative oxidases，AOX）途径，并产生线粒体活性氧（mitochondrial reactive oxygen species, mtROS）。mtROS的累积可以破坏线粒体的结构和功能，与植物PCD密切相关。已有研究表明，一些PPR蛋白的功能缺失会影响线粒体电子传递链复合体的完整性及质子传递功能，导致植物表现种子和胚发育缺陷、生长迟滞、对非生物逆境和ABA敏感等表型缺陷，但其与植物类病斑的形成及防卫反应的关系还所知甚少。本研究从T-DNA插入突变体库中鉴定了一个自然叶枯突变体nbl3，其表现出自发性细胞死亡、ROS积累、抗病性和耐盐性增强以及过早衰老，并且激活多个抗病和耐盐相关基因的表达。OsNBL3编码一个线粒体定位的五肽重复序列 (PPR) 蛋白，其主要参与线粒体基因nad5 内含子4的剪接。OsNBL3的缺失导致nbl3中线粒体呼吸链复合物I的活性降低、替代呼吸途径被激活和线粒体形态被破坏。同时，OsNBL3的RNA干扰株系表现出与nbl3相似的增强的抗病性和耐盐性，而过表达株系的抗病性和耐盐性与野生型没有差异。综上所述，该研究结果解析了一个定位在线粒体的PPR蛋白的作用机制，同时，为揭示程序性细胞死亡和植物对生物和非生物胁迫的反应机制提供了新的见解。