The orchid Gastrodia-derived extracellular vesicles deliver miR535 to suppress a fungal effector and facilitate symbiosis with Armillaria.

Plant-derived extracellular vesicles (PDEVs) are nanoscale lipid bilayer structures that facilitate cross-kingdom communication by transporting bioactive molecules, including proteins and small RNAs. While their roles in plant immunity against fungal pathogens are well-established, their functions in beneficial or symbiotic interactions, particularly with mycorrhizal fungi, remain largely unexplored. Here, we investigated the function of PDEVs in the symbiosis between the fully mycoheterotrophic orchid Gastrodia elata and its fungal partner Armillaria gallica. We isolated and characterized G. elata-derived EVs (GeEVs), and confirmed their typical morphology and the presence of the EV marker PEN1. We demonstrated that GeEVs significantly inhibit A. gallica growth and downregulate fungal genes associated with pathogenicity. Small RNA sequencing revealed that GeEVs are rich in specific miRNAs, including gel-miR535, which is undetectable in EVs from model autotrophic plants. Overexpression of gel-miR535 in A. gallica phenocopied the inhibitory effect of GeEV treatment on fungal growth. We further identified that gel-miR535 directly targets the fungal effector gene Ag_07049. Functional assays showed that Ag_07049 is a secreted protein localized to the plant cytoplasm and nucleus. Upon expression in plant cells, it suppresses BAX- and INF1-triggered programmed cell death (PCD), reactive oxygen species (ROS) accumulation, and callose deposition, suggesting that Ag_07049 functions as an immunosuppressive effector. Our findings indicate a novel regulatory mechanism in which a mycoheterotrophic plant utilizes EVs to deliver a miRNA targeting a fungal effector gene, potentially limiting the pathogenic capacity of its symbiotic partner and support balanced mutualism. This work extends the paradigm of cross-kingdom RNAi to beneficial symbiosis and provides critical insights into the molecular dialogue underpinning orchid mycoheterotrophy.