Fungal extracellular vesicles mediate cross-kingdom trafficking of virulence effectors into plant cells to promote infection.

Extracellular vesicles (EVs) facilitate cross-kingdom communication by delivering bioactive molecules between cells. Although the role of fungal EVs in cross-kingdom RNA trafficking is well documented, whether and how they deliver pathogen-derived virulence effectors into host plants to facilitate infection remains largely unknown. Here, we report that the fungal pathogen Rhizoctonia solani secretes vesicles enriched with the EV marker R. solani tetraspanin 2 (RsTsp2) and the effectors R. solani necrosis-promoting protein 8 (RsNP8) and R. solani serine protease (RsSerp). These proteins are upregulated during infection and are critical for fungal virulence. Notably, clathrin-coated vesicles accumulate at the fungal infection sites, and RsTsp2, RsSerp, and RsNP8 are detectable within these vesicles, indicating their entry into plant cells via clathrin-mediated endocytosis. RsNP8 is translocated into the chloroplast, where it interacts with NP8-interacting chloroplast protein 1 (NICP1) in Arabidopsis. NICP1 contributes to plant immunity by regulating the reactive oxygen species burst during infection, whereas RsNP8 suppresses this immune response. Silencing of RsTsp2, RsSerp, and RsNP8 in R. solani attenuates sheath blight disease progression in rice. Taken together, these findings demonstrate that fungal EVs enable cross-kingdom delivery of effectors into plant cells, revealing a previously unrecognized mechanism by which eukaryotic pathogens invade host plants.