Host‐induced gene silencing in wild apple germplasm Malus hupehensis confers resistance to the fungal pathogen Botryosphaeria dothidea

SUMMARY Host‐induced gene silencing (HIGS) is an inherent mechanism of plant resistance to fungal pathogens, resulting from cross‐kingdom RNA interference (RNAi) mediated by small RNAs (sRNAs) delivered from plants into invading fungi. Introducing artificial sRNA precursors into crops can trigger HIGS of selected fungal genes, and thus has potential applications in agricultural disease control. To investigate the HIGS of apple (Malus sp.) during the interaction with Botryosphaeria dothidea, the pathogenic fungus causing apple ring rot disease, we evaluated whether apple miRNAs can be transported into and target genes in B. dothidea. Indeed, miR159a from Malus hupehensis, a wild apple germplasm with B. dothidea resistance, silenced the fungal sugar transporter gene BdSTP. The accumulation of miR159a in extracellular vesicles (EVs) of both infected M. hupehensis and invading B. dothidea suggests that this miRNA of the host is transported into the fungus via the EV pathway. Knockout of BdSTP caused defects in fungal growth and proliferation, whereas knockin of a miR159a‐insensitive version of BdSTP resulted in increased pathogenicity. Inhibition of miR159a in M. hupehensis substantially enhanced plant sensitivity to B. dothidea, indicating miR159a‐mediated HIGS against BdSTP being integral to apple immunity. Introducing artificial sRNA precursors targeting BdSTP and BdALS, an acetolactate synthase gene, into M. hupehensis revealed that double‐stranded RNAs were more potent than engineered MIRNAs in triggering HIGS alternative to those natural of apple and inhibiting infection. These results provide preliminary evidence for cross‐kingdom RNAi in the apple–B. dothidea interaction and establish HIGS as a potential disease control strategy in apple. Significance Statement To investigate host‐induced gene silencing (HIGS) in apple immunity against Botryosphaeria dothidea, we evaluated the translocation of Malus hupehensis miRNAs during infection and their potential in targeting fungal genes, demonstrating miR159a delivered into fungal cells silenced BdSTP to restrict B. dothidea pathogenicity, which conferred resistance to M. hupehensis. We further explored artificial sRNA precursors as more efficient HIGS triggers targeting selected fungal genes, providing a basis for applying HIGS‐based strategies in controlling B. dothidea in apple.