RT Journal Article SR Electronic T1 Host-induced gene silencing involves transfer of dsRNA-derived siRNA via extracellular vesicles JF bioRxiv FD Cold Spring Harbor Laboratory SP 2020.02.12.945154 DO 10.1101/2020.02.12.945154 A1 A Koch A1 T Schlemmer A1 L Höfle A1 BT Werner A1 C Preußer A1 M Hardt A1 A Möbus A1 D Biedenkopf A1 M Claar A1 C Perlet A1 L Jelonek A1 A Goesmann A1 V Garikapati A1 B Spengler A1 T Busche A1 J Kalinowski A1 KH Kogel YR 2020 UL http://biorxiv.org/content/early/2020/02/13/2020.02.12.945154.abstract AB Small (s)RNA molecules are crucial factors in the communication between hosts and their interacting pathogens, where they function as effectors that can modulate both host defense and microbial virulence/pathogenicity through a mechanism termed cross-kingdom RNA interference (ckRNAi). Consistent with this recent knowledge, sRNAs and their double-stranded (ds)RNA precursors have been adopted to control diseases in crop plants through transgenic expression (host-induced gene silencing, HIGS) or exogenous application (spray-induced gene silencing, SIGS). While these strategies proved to be effective, the mechanism of RNA transfer at the plant - pathogen interface is widely unresolved. Here we show that extracellular vesicles (EVs) purified from Arabidopsis (Arabidopsis thaliana) leaf extracts and apoplastic fluids contain transgene-derived sRNAs. EVs from plants expressing CYP3RNA, a 791 nt long dsRNA, which was originally designed to target the three CYP51 genes of the fungal pathogen Fusarium graminearum, contain CYP3RNA-derived small interfering (si)RNAs as shown by RNA sequencing (RNA-seq) analysis. Notably, the EVs cargo retained the same CYP3RNA-derived siRNA profile as the respective leaf extracts, suggesting that there was no selective uptake of specific artificial sRNAs into EVs. In addition, mutants of the ESCRT-III complex were impaired in HIGS further indicating that endosomal vesicle trafficking supports transfer of transgene-derived siRNAs between donor host cells and recipient fungal cells. Further supporting the relevance of EV-mediated transport of sRNA, we demonstrate that HIGS plants, expressing a 100 nt dsRNA-target-sequence identified via EV-sRNA-seq of CYP3RNA Arabidopsis, confers strong resistance to F. graminearum. Together, these findings support the view that EVs are key mediators in the transport of HIGS-related sRNAs to reduce the virulence of interacting fungal pathogens during host-pathogen interaction.