self-assembling split Nano luciferase-based assay for investigating Pseudomonas syringae effector secretion  

作　　者：Pei Miao Jian-Min Zhou Wei Wang 

机构地区：[1]State Key Laboratory of Plant Genomics,Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Beijing 100101,China [2]College of Advanced Agricultural Sciences,University of Chinese Academy of Sciences,Beijing 100049,China [3]CAS Center for Excellence in Biotic Interactions,University of Chinese Academy of Sciences,Beijing 100049,China [4]Yazhouwan National Laboratory,Sanya 572024,China

出　　处：《Stress Biology》2024年第1期594-599,共6页逆境生物学(英文)

基　　金：supported by grants from the Natural Science Foundation of China(32370322);the Hainan Excellent Talent Team,and the State Key Laboratory of Plant Genomics.

摘　　要：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.

关 键 词：Pseudomonas syringae T3SS effectors SECRETION Nano luciferase 

分 类 号：R73[医药卫生—肿瘤]