PT  - JOURNAL ARTICLE
AU  - A Koch
AU  - T Schlemmer
AU  - L Höfle
AU  - BT Werner
AU  - C Preußer
AU  - M Hardt
AU  - A Möbus
AU  - D Biedenkopf
AU  - M Claar
AU  - C Perlet
AU  - L Jelonek
AU  - A Goesmann
AU  - V Garikapati
AU  - B Spengler
AU  - T Busche
AU  - J Kalinowski
AU  - KH Kogel
TI  - Host-induced gene silencing involves transfer of dsRNA-derived siRNA via extracellular vesicles
AID  - 10.1101/2020.02.12.945154
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.02.12.945154
4099  - http://biorxiv.org/content/early/2020/02/13/2020.02.12.945154.short
4100  - http://biorxiv.org/content/early/2020/02/13/2020.02.12.945154.full
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.