Engineered Rhizobacteria Exploit Bacterial Vesicles for Cross-Kingdom RNAi and Soilborne Disease Control.

The escalating severity of antifungal resistance necessitates novel crop protection strategies. RNAi is promising but faces challenges including environmental instability of dsRNA and poor cellular delivery efficiency. In this study, we engineered the cotton rhizobacterium Pantoea agglomerans YC4 to deliver dsRNA into Verticillium dahliae. Small RNA-seq revealed that bacterial outer membrane vesicles (OMVs) selectively enrich specific endogenous sRNAs compared with intracellular compartments, with a congruent sRNA enrichment pattern observed in fungal cells, thus confirming OMVs as the primary carriers for cross-kingdom RNA delivery. Synthetic biology strategies, including promoter engineering and OmpA-DRB4 fusion, further boosted dsRNA production and OMVs loading capacity. Greenhouse trials demonstrated the persistent colonization of engineered strain in the cotton rhizosphere, and alleviated Verticillium wilt symptoms and reduced fungal burden. This work establishes an engineered rhizosphere symbiont platform for in situ RNAi delivery, offering a practical and sustainable strategy for controlling soil-borne fungal diseases.