Abstract: Gram-negative bacteria are known to release extracellular vesicles (EVs) into their surrounding environment. However, the biological functions of the proteins contained within these vesicles remain largely unknown. Here, we used tandem mass tag (TMT) proteomic analysis to characterize protein cargoes within EVs of the phytopathogen Pseudomonas syringae pv. tomato DC3000 (Pto DC3000). Our investigation revealed that one catalase, KatB, is enriched in bacterial EVs. This enzyme confers EVs with the capacity to detoxify both exogenous and plant-produced H₂O₂, thereby contributing to the pathogen's proliferation within the plants. Interestingly, reactive oxygen species (ROS) stress stimulates bacterial EV secretion and enhances the package of KatB into these vesicles. This regulatory process depends on a periplasmic ankyrin-like protein, AnkB. Both AnkB and KatB are encoded within a small operon, and their mutant strains exhibit impaired growth in plant hosts. Furthermore, the treatment of EVs pelleted from bacterial culture supernatants activates the immune responses of plants, and the absence of KatB in EVs further enhances this protective activity. Collectively, our findings indicate that bacteria secreted KatB via EVs to interact with and reduce the host's oxidative environment, thereby promoting their proliferation within plants.

Key words: bacteria, extracellular vesicles, plant–microbe interaction, reactive oxygen species