Engineered Yarrowia lipolytica inducing gene silencing confers control ability against gray mold in plants

Plant gray mold disease, caused by the necrotrophic fungus Botrytis cinerea, threatens global food security by infecting over 1,400 species. The resistance of B. cinerea to fungicides poses a major challenge for its control. RNA interference (RNAi) shows promise for pathogen management, but high production costs, poor stability, and inefficient delivery of double-stranded RNA (dsRNA) restrict its practical use. In this study, we developed Yarrowia lipolytica strain MP181-2 as a novel RNAi platform, leveraging its ability to colonize the phyllosphere of crop plants (tomato and alfalfa) to enable simultaneous dsRNA production and pathogen targeting via microbe-induced gene silencing (MIGS). Through target screening, we identified three essential fungal genes, BcRpd3, BcNat1, and BcArd1, whose silencing significantly reduced the pathogenicity of B. cinerea. We further constructed a chimeric dsRNA molecule (BcANR-dsRNA) targeting these three genes, which demonstrated superior disease suppression compared to single-gene targeting. The engineered Y. lipolytica strains that produce dsRNA could effectively interfere with the expression of target genes in B. cinerea, and the strain expressing BcANR-dsRNA (designated Yl-dsANR) acquired the ability to suppress the growth of B. cinerea. In planta tests confirmed that Yl-dsANR successfully interfered with the expression of three target genes, leading to decreased fungal biomass and reduced lesion development on different hosts. Notably, foliar application of Yl-dsANR provided durable protection (over 70% efficacy for 5 d) in potted plants. Our integrated approach combines the advantages of microbial biocontrol with RNAi precision, establishing Y. lipolytica as a versatile chassis for sustainable crop protection. This study also provides an effective solution to current limitations in RNA pesticide development and offers a scalable, eco-friendly alternative to chemical fungicides for gray mold management.