Polymyxin B inhibits T3SS gene expression via WspR in Pseudomonas syringae pv. actinidiae

• Polymyxin B suppresses the expression of T3SS genes through WspR in Pseudomonas syringae pv. actinidiae . • The TetR family transcription factor PsrA directly binds to the hrpL promoter and positively regulates hrpL transcription and Psa virulence. • WspR interacts with PsrA, inhibiting its binding to the hrpL promoter. Kiwifruit bacterial canker (KBC), caused by Pseudomonas syringae pv. actinidiae ( Psa ), severely threatens the kiwifruit industry. The type III secretion system (T3SS) is a key virulence factor in Psa , but the regulatory mechanisms remain poorly understood. Polymyxin B1, the main component of polymyxin B, inhibits T3SS gene expression in Psa , yet its underlying mechanism is unclear. Cyclic diguanosine monophosphate (c-di-GMP), a crucial bacterial second messenger, is synthesized by diguanylate cyclases (DGCs) containing a GGDEF domain. In this study, we identified and characterized PSA_1379 (WspR), a GGDEF domain-containing protein in Psa . Biochemical assays demonstrated that WspR exhibits DGC activity. Virulence assays showed that WspR negatively regulates Psa virulence. RT-qPCR analyses revealed that polymyxin B induces wspR expression. Additionally, polymyxin B upregulates intracellular c-di-GMP levels and inhibits the expression of T3SS genes through WspR. Bacterial two-hybrid and GST pull-down assays confirmed that WspR interacts with the transcription factor PsrA. Both WspR and c-di-GMP inhibit the binding of PsrA to the promoter of the T3SS master regulator hrpL , thereby suppressing PsrA-mediated transcriptional activation of hrpL and ultimately repressing T3SS gene expression. This study provides new insights into Psa virulence regulation and suggests potential targets for KBC control through the WspR-c-di-GMP pathway.