The increasing resistance displayed by plant phytopathogenic bacteria to conventional pesticides has heightened the urgency for the exploration of novel antibacterial agents possessing distinct modes of action (MOAs). In this study, a series of novel phenylalanine derivatives with the unique structure of acylhydrazone dithioether have been designed and synthesized. Bioassay results demonstrated that most target compounds exhibited excellent in vitro antibacterial activity against Xanthomonas oryzae pv oryzae (Xoo) and Xanthomonas axonopodis pv citri (Xac). Among them, the EC₅₀ values of L₃, L₄, L₆, L₂₁, and L₂₂ against Xoo were 7.4, 9.3, 6.7, 8.9, and 5.1 μg/mL, respectively, superior to that of bismerthiazol (BT) and thiodiazole copper (TC) (41.5 and >100 μg/mL); the EC₅₀ values of L₃, L₄, L₅, L₆, L₇, L₈, L₂₀, L₂₁, and L₂₂ against Xac were 5.6, 2.5, 6.2, 4.1, 4.2, 6.4, 6.3, 3.6, and 5.2 μg/mL, respectively, superior to that of BT and TC (43.3 and >100 μg/mL). An unmodified drug affinity responsive target stability (DARTS) technology was used to investigate the antibacterial MOAs of active compound L₂₂, and the 50S ribosomal protein L2 (RL2) as an unprecedented target protein in Xoo cells was first discovered. The target protein RL2 was then expressed and purified. Furthermore, the in vitro interactions by microscale thermophoresis (K_d = 0.050 μM) and fluorescence titration (K_a = 1.4 × 10⁵ M^-1) experiments also demonstrated a strong binding force between compound L₂₂ and RL2. Overall, these results not only facilitate the development of novel antibacterial agents but also establish a reliable method for exploring the targets of bactericides.

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