SUMMARY Fungal pathogens infect humans, animals, and plants and cause severe consequences on global human health and crop production. Communication between hosts and pathogens is essential for host defense and pathogen virulence, but the underlying mechanisms are not well understood. Previous studies in my lab discovered that some non-coding regulatory small RNAs (sRNAs) from fungal pathogens, such as Botrytis cinerea, which causes grey mold disease on more than 1000 plant species, can be transported into host plant cells and suppress host immunity genes, a mechanism called “Cross-Kingdom RNAi”. Recently, we discovered that such sRNA communication is bi-directional. Plant hosts have also developed the ability to deliver sRNAs, mainly using extracellular vesicles, to fungal cells and induce cross-kingdom RNAi of fungal virulence-related genes. Such sRNA communication was also observed between mammals and parasites. Although more and more studies across diverse systems demonstrate that mobile sRNAs are key regulatory molecules in host and pathogen interactions, the field of cross-kingdom/cross-species RNA communication is still in its infancy. This proposal is designed to use plant Arabidopsis and fungal pathogen Botrytis as a model system to address the outstanding questions in this field, including, how host cells control sRNA transport upon infection, how specific classes of small RNAs are sorted into extracellular vesicles, how fungal cells deliver sRNAs into host cells, what are the mechanisms of RNA and vesicle uptake in the host cells and fungal cells, and whether other classes of RNAs, such as mRNAs and long non-coding RNAs, move between host and fungal cells, how they function in the counter party, etc.. A combination of genetics, genomics, biochemical and molecular biology approaches will be used. This project is expected to provide unprecedented insight into the underlying mechanisms of cross-kingdom/cross-species RNA communications, which will ultimately help develop innovative and eco-friendly disease control strategies and RNA-based fungicides or antifungal drugs.