芸薹根肿菌分泌型E3泛素连接酶PbRING1的生物学功能研究

Clubroot disease, caused by Plasmodiophora brassicae, is an economically important disease of crucifer crops. Clubroot management remains a challenging task due to limited knowledge of molecular basis of P.brassicae pathogenesis. In order to further clarify the molecular mechanism underlying pathogenesis of P. brassicae, putative effectors secreted by P. brassicae were chosen for further study. Real-time PCR revealed that expression of PbRING1 was significantly increased in clubroot, indicative of an important role in pathogenicity; in silico analysis showed that PbRING1 harbors a signal peptide and a RING domain which shows similarity with reported E3 ligase in both plants and pathogens. Therefore, the main objectives of this project were: (1) to investigate the biochemical function of PbRING1 by employing site-directed mutagenesis, heterologous expression, in vitro enzyme assay; (2) to identify interacting proteins by affinity capture combined with yeast two hybrid; (3) to explore the effect of PbRING1-host protein interaction by domain deletion, overexpression, omic apporaches. The results from this project are expected to be helpful in elucidating the role of PbRING1 in P.brassicae pathogenesis, which definitely will advance our understanding of molecular basis of pathogenesis by P. brassicae and facilitate the development of resistant cultivar and clubroot management.

芸薹根肿菌引起的根肿病是十字花科生产上的重要病害，由于对根肿病的致病分子机理所知有限，目前缺乏科学有效的防治方法。为了进一步揭示其致病分子机制，我们对芸薹根肿菌效应子进行研究。通过荧光定量PCR发现芸薹根肿菌PbRING1在侵染植物组织中表达量显著上升，说明该基因很可能在芸薹根肿菌致病过程中发挥重要功能；通过生物信息学分析发现PbRING1具有信号肽，并且与E3泛素连接酶存在同源。在此基础上，本研究拟采用定点突变、原核表达、离体酶活检测研究PbRING1的生化功能；通过免疫亲和捕获、酵母双杂交鉴定PbRING1的互作蛋白；通过结构域缺失、过表达技术、组学分析研究芸薹根肿菌PbRING1与寄主蛋白互作的生物学效应，借此探明芸薹根肿菌PbRING1的作用机理，并为通过基因编辑手段定向修饰PbRING1的分子靶标改良植物对根肿病的抗性做铺垫，最终为抗病育种和芸薹根肿病的防治策略制定提供重要基础。

本项目解析了芸薹根肿菌分泌型E3泛素连接酶PbRING1的生物学功能。通过生物信息学分析发现，芸薹根肿菌中存在139个E3泛素连接酶。通过信号肽和跨膜结构域预测发现，12个E3泛素连接酶具有分泌蛋白特征。通过转录组学分析发现分泌型E3泛素连接酶PbRING1表达量在原生质团时期特异上调，表明其参与芸薹根肿菌的侵染过程。PbRING1的氨基端（第1-30个氨基酸）编码一个信号肽，通过酵母蔗糖酶分泌实验发现该信号肽具有功能，可以引导蛋白分泌。PbRING1在羧基端还存在一个RING结构域，通过点突变、原核表达和体外泛素化实验表明PbRING1具有E3泛素连接酶活性，且第187和191位组氨酸是维持其活性所必需的。通过植物过表达和免疫亲和捕获，鉴定了一些PbRING1的互作蛋白。瞬时表达实验表明PbRING1能够抑制Bax诱导的细胞凋亡。综上所述，我们鉴定了12个芸薹根肿菌分泌型E3泛素连接酶，解析了分泌型E3泛素连接酶PbRING1的酶活，探索了PbRING1参与芸薹根肿菌致病的机制，为推进芸薹根肿菌致病机理研究提供理论依据。