Host-induced gene silencing (HIGS) involves Arabidopsis extracellular vesicles and the endosomal sorting complex required for transport III (ESCRT-III).
Small interfering (si)RNAs and their double-stranded (ds)RNA precursors have been adopted to control diseases in crop plants through expression in transgenic plants and targeted gene silencing (host-induced gene silencing, HIGS). While HIGS strategies have proven effective against several pathogens, the mechanism of RNA transfer at the plant-pathogen interface is widely unknown. Here, we show that extracellular vesicles (EVs) purified from Arabidopsis thaliana plants expressing CYP3RNA, a dsRNA originally designed to target the three CYP51 genes of the fungal pathogen Fusarium graminearum, co-purify with CYP3RNA-derived siRNAs as shown by RNA sequencing (RNA-seq) analysis. These transgene-derived siRNAs showed a bias towards 5'-uracil (U) and 5'-adenine (A). Digestive treatment of EVs led to shift in RNA population and nucleotide identity. Notably, stringent protease and RNase treated EV fractions contained >70% less CYP3RNA-derived siRNAs, suggesting the presence of co-purified extravesicular nucleoprotein complexes stabilizing siRNAs outside of EVs. In addition, mutants of the ESCRT-III complex showed a loss of HIGS-mediated F. graminearum disease resistance and EVs isolated from these mutants were free of CYP3RNA-derived siRNAs. Together, these findings indicate that ESCRT-mediated vesicle trafficking is required for HIGS, while the precise contribution of EVs to siRNA transfer remains elusive.