Host-induced gene silencing involves transfer of dsRNA-derived siRNA via extracellular vesicles

Koch, A.; Schlemmer, T.; Hoefle, L.; Werner, B.; Preusser, C.; Hardt, M.; Moebus, A.; Biedenkopf, D.; Claar, M.; Perlet, C.; Jelonek, L.; Goesmann, A.; Garikapati, V.; Spengler, B.; Busche, T.; Kalinowski, J.; Kogel, K.-H.

doi:10.1101/2020.02.12.945154

bioRxiv · 10.1101/2020.02.12.945154

Host-induced gene silencing involves transfer of dsRNA-derived siRNA via extracellular vesicles

Koch, A.·Schlemmer, T.·Hoefle, L.·Werner, B.·Preusser, C.·Hardt, M.·Moebus, A.·Biedenkopf, D.·Claar, M.·Perlet, C.·Jelonek, L.·Goesmann, A.·Garikapati, V.·Spengler, B.·Busche, T.·Kalinowski, J.·Kogel, K.-H.

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Abstract

Small (s)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 proved to be effective, 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, contain CYP3RNA-derived small interfering (si)RNAs as shown by RNA sequencing (RNA-seq) analysis. These transgene specific siRNAs had a length of 21 and 22 nucleotides with a bias towards 5-uracil (U) and 5-adenine (A). 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 disease resistance and EVs isolated from these mutants were free of CYP3RNA-derived siRNAs. Together, these findings support the view that endosomal vesicle trafficking is required for HIGS mediating the transfer of transgene-derived siRNAs between donor host cells and recipient fungal cells probably in an EV-independent manner.

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Koch, A., Schlemmer, T., Hoefle, L., Werner, B., Preusser, C., Hardt, M., Moebus, A., Biedenkopf, D., Claar, M., Perlet, C., Jelonek, L., Goesmann, A., Garikapati, V., Spengler, B., Busche, T., Kalinowski, J., Kogel, K.-H.. 2020-02-13. Host-induced gene silencing involves transfer of dsRNA-derived siRNA via extracellular vesicles. https://doi.org/10.1101/2020.02.12.945154

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