Xinjunan Binds to Pseudomonas syringae Pv.Actinidiae FleQ to Inhibit Flagella and Biofilm Formation and Reduce Pathogenicity

Abstract Pseudomonas syringae pv. actinidiae (Psa) is a devastating kiwifruit pathogen causing severe economic losses. In this study, Xinjunan (dioctyldiethylenetriamine) exerts potent antibacterial activity against Psa (EC50 = 4.29 mg/L), outperforming thiodiazole-copper (34.8 mg/L). Transcriptomic analysis showed that Xinjunan affected pathways associated with flagellar assembly, biofilm formation, membrane biogenesis, and chemotaxis. Xinjunan treatment also induced reactive oxygen species (ROS) accumulation, leading to bacterial death. Deletion of FleQ enhances Psa resistance to Xinjunan, reduces virulence, and inhibits flagellar formation. Under Xinjunan treatment, the mutant strain showed better growth than the wild-type strain, with no significant differences in motility capacity or ROS levels; the complemented strain restored sensitivity to Xinjunan. Microscale thermophoresis and isothermal titration calorimetry determined dissociation constants of Xinjunan binding to the FleQ protein as 1.64 and 6.41 μM, respectively. Molecular docking and molecular dynamics simulations identified FleQ Glu264 as a critical binding residue. These findings provide promising targets for the management of bacterial phytopathogen Psa.