Isolation and characterization of barley (Hordeum vulgare) extracellular vesicles towards assessing their role in RNA spray-based crop protection

The demonstration that spray-induced gene silencing (SIGS) can confer strong disease resistance bypassing the laborious and time-consuming transgenic expression of double-stranded (ds)RNA to induce gene silencing of pathogenic targets was ground-breaking. However, future field applications will require fundamental mechanistic knowledge on dsRNA uptake, processing, and its transfer. There is increasing evidence that extracellular vesicles (EVs) mediate the transfer of transgene-derived small interfering (si)RNAs in host-induced gene silencing (HIGS) applications. Here, we established a protocol for barley EV isolation and assessed the possibility of EVs regarding the translocation of sprayed dsRNA from barley (Hordeum vulgare) to its interacting fungal pathogens. We found barley EVs with 156 nm in size containing predominantly 21 and 19 nucleotide (nt) siRNAs starting with a 5’-terminal Adenine. Although direct comparison of RNA cargo between HIGS- and SIGS EV isolates is improper given their underlying mechanistic differences, we identified sequence-identical siRNAs in both systems. Overall, the number of siRNAs isolated from EVs of dsRNA sprayed barley plants with sequence complementarity to the sprayed dsRNA precursor was low. However, whether these few siRNAs are sufficient to induce SIGS of pathogenic target genes needs further research. Together our results raise the possibility that EVs may not be mandatory for spray delivered siRNA uptake and induction of SIGS.