RetS-mediated environmental sensing coordinates TetR-dependent regulation of type III secretion system and virulence in pv. .

Unlabelled: Kiwifruit bacterial canker, caused by pv. (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 () and (hybrid histidine kinase; ) in the pv. (biovar 3) strain S26 using transposon libraries and a DNA pull-down strategy. Both the mutant strains and exhibited significantly reduced pathogenicity in kiwifruit leaf discs and a diminished ability to induce , , and expression and showed a reduced hypersensitive response in . Moreover, transcriptomic studies showed considerable overlap in downregulated genes between the and two-component system (TCS) mutant, highlighting the intertwined regulatory roles of both genes. This overlap suggests a tightly coordinated regulatory system, where serves as the response regulator of the hybrid histidine kinase 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 / and . This study identifies the gene as the first TetR-family transcription factor directly linking the hybrid TCS to HrpR/S-HrpL-mediated T3SS regulatory pathway activation in Psa for regulation.

Importance: This study uncovers the critical roles of two regulatory genes, () and (), in pv. (Psa) virulence via the type III secretion system (T3SS). Through genetic knockouts, complementation, and transcriptomic analyses, we establish , a TetR/AcrR family transcription factor, as a pivotal activator of T3SS-related genes essential for Psa's virulence in kiwifruit. We further demonstrate that , a hybrid histidine kinase two-component system regulator, acts upstream by enhancing expression, forming a hierarchical network that drives the T3SS cascade during infection. serves as the response regulator of the hybrid histidine kinase RetS to regulate T3SS and virulence in Psa. These insights reveal how Psa orchestrates virulence to colonize kiwifruit tissues, advancing our understanding of bacterial plant interactions. Moreover, this regulatory framework offers promising targets for developing precise strategies to combat bacterial canker, mitigating its devastating economic toll on global kiwifruit production.