AAA ATP酶LRD6-6调控水稻细胞死亡和免疫反应的分子机制

The lesion resembling disease (lrd) mutants usually confer broad-spectrum resistance to diverse pathogens because they exhibit the cell death and enhanced immunity characterized with accumulation of ROS and increased expression of pathogenesis-related genes without pathogen attack. In rice, more than ten genes encoding different proteins have been cloned. However, only SPL11 has been well studied in molecular regulation of cell death and immunity. We previously characterized the rice mutant, lrd6-6, which was obtained from tissue-cultured rice Kittake. The mutant lrd6-6 showed enhanced immunity and thus conferred broad-spectrum resistance to blast caused by the fungal pathogen Magnaporthe oryzae and bacterial blight caused by the bacterial pathogen Xanthomonas oryzae pv oryzae, respectively. Map-based cloning revealed that Lrd6-6 encoded an AAA ATPase, and disruption of the AAA ATPase LRD6-6 led to autoimmunity and enhanced disease resistance in the lrd6-6 mutant. Here, we aim to uncover the molecular mechanism of the AAA ATPase LRD6-6-mediated regulation on cell death and immunity through revealing the subcellular localization and transcription expression pattern of LRD6-6, characterizing the ATPase activity, the key residues required for enzymatic activity and its regulation on immunity, and elucidating the molecular processes and metabolic regulations by using RNA-seq-mediated globally transcriptional analysis. Our study will broaden basic knowledge in understanding cell death and immunity, and applying broad-spectrum resistance to blast and bacterial blight.

植物类病斑突变体往往因活性氧（ROS）的积累、病程相关（PR）基因表达的上升而具有对病原菌的广谱抗病性。因此，类病斑突变体是研究细胞死亡和免疫反应的重要材料。水稻中目前已经克隆了约19个类病斑基因，仅Spl11 对细胞死亡和免疫反应的分子机制进行了深入的研究。在组培过程中，我们获得了一份类病斑突变体lrd6-6，该突变体中ROS明显富集、PR基因表达量明显上升，对白叶枯病和稻瘟病均表现出广谱抗病性。利用定位克隆策略我们成功分离了控制该性状的基因Lrd6-6，该基因编码一个AAA ATP酶。本项目拟分析蛋白LRD6-6的亚细胞定位和基因表达模式；分析LRD6-6的ATP酶活性、关键酶活性位点及其对生物学功能的影响；通过RNA-seq解析LRD6-6调控的分子网络，进而系统阐述AAA ATP酶LRD6-6调控水稻细胞死亡和免疫反应的分子机制，为水稻广谱抗病育种提供理论和应用依据。

水稻生产面临着包括稻瘟病、白叶枯病、稻曲病等多种病害的威胁，病害流行年份甚至会造成绝收。针对抗病育种目前抗性单一且容易丧失、隐性抗病基因不易利用、抗病基因聚合后无抗性等现状，研究者们提出通过研究类病斑突变体材料，来进一步解析植物抗病机制，解决抗病育种面临的问题。我们在粳稻材料Kitaake的转基因后代中分离得到一个水稻类病斑突变体，将其命名为lrd6-6。该突变体在苗期开始出现性状，表现为叶片从叶尖开始出现病斑并向叶基部延伸，老叶先出现病斑，病斑在整个生育期都有出现，但植物不会死亡。突变体对稻瘟病和白叶枯的抗性增强。通过图位克隆结合全基因组重测序，我们成功克隆了调控lrd6-6性状的基因。通过LRD6-6-GFP融合蛋白在烟草叶片和洋葱表皮细胞中的瞬时表达，结合渥曼青霉素的处理我们证明LRD6-6定位于植物多泡体MVBs上；通过分析LRD6-6的ATP酶活性、关键酶活性位点及其对生物学功能的影响，发现ATPase活性是LRD6-6行使生物学功能所必须的，过表达Lrd6-6E315Q点突变会引起野生型水稻出现类病斑表型；通过RNA-seq解析LRD6-6调控的分子网络，我们解析了AAA ATPase LRD6-6参与水稻细胞死亡及免疫反应调节的分子机制，并提出了将该基因应用于水稻抗病育种的方案。