Pathogenic bacteria deploy biofilm as a key virulence factor to cause plant vascular diseases, which are devastating to global agricultural practices. Extracellular DNA (eDNA) constitutes the backbone of bacterial biofilm and is key to biofilm stability, thereby representing as an attractive therapeutic target. Here, we engineered the plant chloroplast-localized Holliday junction (HJ) resolvase MOC1 by replacing its native chloroplast transit peptide with a secretory signal, successfully relocating it to the apoplast. Transgenic tomato and rice expressing secreted MOC1 exhibited robust resistance to bacterial wilt and bacterial blight, respectively, without growth or yield penalties. Additionally, we implemented bacterial pathogen-inducible promoters to achieve precisely spatial and temporal control over the resistance trait. Secreted MOC1 degrades eDNA in situ, disrupts biofilm architecture, and markedly reduces bacterial colonization and systemic spread. Our work presents a novel strategy for controlling vascular diseases by engineering plant HJ resolvases to disrupt biofilms. This approach provides a new blueprint for molecular resistance breeding and disease resistance gene exploration.

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