RNA exchange between plants and fungal pathogens initiates at the leaf surface during the epiphytic phase of infection

We have recently shown that plants secrete diverse RNA species onto leaf surfaces, a crucial interface for the initial interactions between plants and microbes.These extracellular RNAs (exRNAs) differ from cellular RNAs and exRNAs found inside leaves in both composition and size.Interestingly, leaf surface RNAs are neither protected by extracellular vesicles nor RNA-binding proteins.Moreover, the amount of leaf surface RNA increases rapidly following the perception of pathogen-derived molecules, such as flg22 from bacterial flagellin or hormones associated to biotic stress, including salicylic acid (SA) and jasmonic acid (JA).To assess whether leaf surface RNAs are enriched in silencing RNAs, we transiently expressed an artificial hairpin RNA in Nicotiana benthamiana targeting the SGE1/Gti1 transcript, which encodes a key transcription factor in the phytopathogenic fungus Colletotrichum higginsianum.Two days post-infiltration, plants showed an accumulation of long double-stranded RNA derived from the artificial hairpin on leaf surfaces.To test the efficiency of these leaf surface RNAs in inducing silencing in C. higginsianum during the epiphytic phase of infection, we applied these RNAs on Arabidopsis cotyledons followed by infection with C. higginsianum.Exogenous application of this RNA to the surface of Arabidopsis cotyledons blocked infection before penetration of host cell walls, indicating that C. higginsianum can take up RNA from leaf surfaces via a newly discovered mechanism of RNA transfer during plant-pathogen interaction.Step-wise guide to isolate leaf surface wash (LSW) and apoplastic wash fluid (AWF) from Arabidopsis plants