Abstract
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 the gene silencing of pathogenic targets, was ground-breaking. However, future field applications will require fundamental mechanistic knowledge of dsRNA uptake, processing, and 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. In this study, we establish a protocol for barley EV isolation and assess the possibilities for EVs regarding the translocation of sprayed dsRNA from barley (Hordeum vulgare) to its interacting fungal pathogens. We found barley EVs that were 156 nm in size, containing predominantly 21 and 19 nucleotide (nts) siRNAs, starting with a 5′-terminal Adenine. Although a direct comparison of the 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 the 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 the SIGS of pathogenic target genes requires further research. Taken together, our results raise the possibility that EVs may not be mandatory for the spray-delivered siRNA uptake and induction of SIGS.
Highlights
To study whether barley (Hordeum vulgare) extracellular vesicles (EVs) contain double-stranded RNA (dsRNA) spray-derived small interfering RNAs (siRNAs), we established a protocol for EV isolation from barley leaves by adjusting the EV isolation protocol we previously adopted for Arabidopsis preparation [5]
To assess the involvement of EVs in mediating the transport and uptake of SIGSderived siRNA, barley leaves were sprayed with CYP3RNA, as previously described [6]
We found less siRNA in barley EVs than in Arabidopsis EVs, which led to a low read coverage compared to Arabidopsis EVs (Figure 1e) [5]
Summary
To study whether barley (Hordeum vulgare) EVs contain dsRNA spray-derived siRNAs, we established a protocol for EV isolation from barley leaves by adjusting the EV isolation protocol we previously adopted for Arabidopsis preparation [5]. We analyzed the relative abundance of siRNAs of each length in comparison to all identified siRNAs, which we mapped to the precursor to compare the siRNA amounts between both species, and found that barley EVs revealed a second peak for 19 nts siRNAs, which we did not observe in EVs from Arabidopsis (Figure 1h,i). This finding—along with previously discovered differences in efficiencies between dsRNA originating from endogenous expression (HIGS). Further research is required to unravel the routes of dsRNAs and siRNAs necessary to determine the strengths and limitations of the SIGS strategy in a pathosystem-specific manner
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