Interfering Transposable Elements: IS Xoo 15 Transposase as a First‐in‐Class Antibacterial Target Against Xanthomonas oryzae pv. oryzae

ABSTRACT Current challenges in controlling phytopathogenic bacteria lie in widespread chemical resistance, biosafety concerns, and the scarcity of novel biomacromolecule targets. While transposable elements have emerged as critical drivers of genetic variability and virulence in plant pathogens, their potential as druggable targets remains unexplored. Here, we report the first discovery of IS Xoo 15 transposase in Xanthomonas oryzae pv. oryzae (Xoo) as the bactericidal receptor for J9, a pyrimidine‐substituted pleuromutilin derivative. In vitro assays demonstrate J9's superior anti‐Xoo activity, with an EC 50 of 0.12 mg/L—significantly lower than commercial agents thiodiazole copper (86.39 mg/L) and zinc thiazole (26.15 mg/L). In vivo pot trials reveal enhanced curative and protective efficacy of J9 against rice bacterial leaf blight compared to these metal‐based controls. A photoaffinity probe, P‐J9, is synthesised and coupled with activity‐based protein profiling to unequivocally identify IS Xoo 15 transposase (encoded by PXO_03433 ) as J9's specific target. Reverse transcription‐quantitative PCR confirmed significant downregulation of PXO_03433 expression in J9‐treated Xoo. Physiological and virulence‐related functional analyses of a homologous recombination‐mediated PXO_03433 ‐knockout strain (ΔPXO_03433) showed markedly attenuated virulence and impaired pathogenicity. Conversely, PXO_03433 ‐complemented strain CΔPXO_03433 possessed substantial restoration of pathogenicity‐related traits. Proteomic profiling revealed significant downregulation of pathways associated with DNA repair, recombination and binding proteins in both J9‐treated and mutant strains. IS Xoo 15 transposase may serve as a key regulator in enabling the homeostasis of the DNA metabolic network in the bacteria. This study provides pioneering evidence for targeting bacterial transposases as a novel antibacterial strategy, establishing a foundation for effective management of phytopathogenic bacteria.