RetS-Mediated Environmental Sensing Coordinates TetR-Dependent Regulation of Type III Secretion System and Virulence in Pseudomonas syringae pv. actinidiae
Kiwifruit bacterial canker, caused by Pseudomonas syringae pv. actinidiae (Psa), is an emerging global concern in kiwifruit production. Successful Psa infection in kiwifruit relies on the type III secretion system (T3SS), which is governed by the HrpR/S-HrpL regulatory pathway. However, the mechanisms by which certain transcriptional factors regulate this pathway remain poorly understood. In this study, we identified the TetR/AcrR family transcription factors C_22735 (tetR1) and C_04700 (hybrid histidine kinase; retS) in the P. syringae pv. actinidiae (biovar 3) strain S26 using transposon libraries and a DNA pull-down strategy. Both the mutant strains tetR1 and retS exhibited significantly reduced pathogenicity in kiwifruit leaf discs and a diminished ability to induce hrpRS, hrpL, and hrpA expression in vitro and showed a reduced hypersensitive response in Nicotiana benthamiana. Moreover, transcriptomic studies showed a considerable overlap in down-regulated genes between the tetR1 and two-component system (TCS) retS mutant, highlighting the intertwined regulatory roles of both genes. This overlap suggests a tightly coordinated regulatory system, where tetR1 serves as the response regulator of the hybrid histidine kinase retS to regulate T3SS and virulence. The electrophoretic mobility shift assays, and luciferase-based promoter activity detection further revealed that TetR1 positively regulates the T3SS by directly binding to the promoter regions of hrpR/S and hrpL. This study identifies the tetR1 gene as the first TetR-family transcription factor directly linking the hybrid TCS retS to HrpR/S-HrpL -mediated T3SS regulatory pathway activation in Psa for regulation. Overall design: RNA-seq profiling of wild type strain S26 and its knockout strains (C_22735, C_4700) induced by HDM (hrp-depress) for 8h. Grant awarded to Zhao Zhibo: National Key Research and Development Program of China (Grant No. 2022YFD1400200) Grant awarded to Zhao Zhibo: National Natural Science Foundation of China (Grant No. 31860486, 32001864) Grant awarded to Zhao Zhibo: Fund for the China Agriculture Research System (Grant No. CARS-26-04A)