Miao Pei, Zhou Jian-Min, Wang Wei
State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China.
College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, 100049, China.
Stress Biol. 2024 Feb 16;4(1):14. doi: 10.1007/s44154-024-00152-2.
Many Gram-negative pathogens employ the type III secretion system (T3SS) to deliver effector proteins into host cells, thereby modulating host cellular processes and suppressing host immunity to facilitate pathogenesis and colonization. In this study, we developed a straightforward, rapid, and quantitative method for detecting T3SS-mediated translocation of Pseudomonas syringae effectors using a self-assembling split Nano luciferase (Nluc)-based reporter system. It was demonstrated that this system can detect effector secretion in vitro with an exceptionally high signal-to-noise ratio and sensitivity, attributed to the strong affinity between the split domains of Nluc and the intense luminescence generated by functional Nluc. During natural infections, effectors fused to a small C-terminal fragment of Nluc were successfully translocated into plant cells and retained their virulence functions. Furthermore, upon infection of plants expressing the N-terminal fragment of Nluc with these P. syringae strains, functional Nluc proteins were spontaneously assembled and produced bright luminescence, demonstrating that this system enables the straightforward and rapid assessment of P. syringae T3SS-mediated effector translocation during natural infections. In conclusion, the self-assembling split Nluc-based reporting system developed in this study is suitable for efficient in vitro and in planta detection of effectors secreted via T3SS.
许多革兰氏阴性病原体利用Ⅲ型分泌系统（T3SS）将效应蛋白输送到宿主细胞中，从而调节宿主细胞过程并抑制宿主免疫，以促进发病机制和定植。在本研究中，我们开发了一种直接、快速且定量的方法，用于使用基于自组装分裂纳米荧光素酶（Nluc）的报告系统检测丁香假单胞菌效应蛋白的T3SS介导的转运。结果表明，该系统能够在体外以极高的信噪比和灵敏度检测效应蛋白的分泌，这归因于Nluc分裂结构域之间的强亲和力以及功能性Nluc产生的强烈发光。在自然感染期间，与Nluc的小C末端片段融合的效应蛋白成功转运到植物细胞中并保留其毒力功能。此外，用这些丁香假单胞菌菌株感染表达Nluc N末端片段的植物后，功能性Nluc蛋白自发组装并产生明亮的发光，表明该系统能够在自然感染期间直接、快速地评估丁香假单胞菌T3SS介导的效应蛋白转运。总之，本研究中开发的基于自组装分裂Nluc的报告系统适用于高效地体外和体内检测通过T3SS分泌的效应蛋白。
