Isolation and Characterization of Barley (Hordeum vulgare) Extracellular Vesicles to Assess 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 pathogenic targets, was ground-breaking. However, future field applications will require fundamental mechanistic knowledge dsRNA uptake, processing, transfer. There is increasing evidence extracellular vesicles (EVs) mediate transfer transgene-derived small interfering (si)RNAs in host-induced (HIGS) applications. In this study, we establish a protocol for barley EV isolation assess possibilities EVs regarding translocation sprayed from (Hordeum vulgare) its interacting fungal pathogens. We found were 156 nm size, containing predominantly 21 19 nucleotide (nts) siRNAs, starting with 5?-terminal Adenine. Although direct comparison RNA cargo between HIGS SIGS isolates improper given their underlying differences, identified sequence-identical siRNAs both systems. Overall, number isolated dsRNA-sprayed plants sequence complementarity precursor low. whether these few are sufficient target genes requires further research. Taken together, our results raise possibility may not be mandatory spray-delivered siRNA uptake induction SIGS.