Isolation and Characterization of Barley (Hordeum vulgare) Extracellular Vesicles to Assess Their Role in RNA Spray-Based Crop Protection

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.

Extracellular vesicles isolated from dsRNA-sprayed barley plants exhibit no growth inhibition or gene silencing in Fusarium graminearum

“…Notably, plant-derived EVs were shown to contain stress response-related proteins and lipids [8,12,32,45,47] and exhibit antifungal activity [12,48]. It is therefore surprising that we did not observe any inhibitory effects of barley EVs on F. graminearum.…”

“…To test the possibility of plant EV uptake by F. graminearum in vitro, we isolated EVs from control [tris-EDTA (TE) buffer] and CYP3RNA-sprayed barley leaves using a protocol modified from those described by Rutter and Innes [45] and Schlemmer et al [48]. In our recent studies, we observed that state-of-the-art EV purification from apoplastic fluids leads to impure EV isolates containing additional co-purified apoplastic substances [47]. This finding aligns with recent debates discussing the pitfalls of current plant EV research methods and the need for standardization, with different contamination risks reported for different plant EV separation and characterization methods [33,40,46].…”

“…We previously found that EVs isolated from HIGS-A. thaliana and SIGS-barley plants principally contain transgene-and dsRNA-spray-derived siR-NAs [47,49]. However, since the amount of HIGS and SIGS-related siRNA inside EVs was low, we assessed here whether these siRNAs could induce the silencing of F. graminearum CYP51 genes and thus fungal growth inhibition, despite their low abundance.…”

“…These examples intensify the assumption that plant-or pathogen-derived EVs contribute bidirectionally to this highly specialized interspecies communication through the release of lipids, proteins, and small RNAs (sRNAs) that regulate and deregulate defensive and offensive responses [5,51]. In particular, the identification of plant EV-derived sRNAs stimulated a debate about whether EVs function as shuttles in interspecies communication, directing plant antifungal defence responses [2,7,44,[47][48][49]. Conversely, fungal pathogens secrete sRNAs to dampen plant immunity [13,29,57,58].…”

“…Studying the role or requirement of EVs in transferring HIGS-and SIGS-associated RNAs, we recently showed that EVs isolated from CYP3RNA-expressing A. thaliana plants contain CYP3RNA-derived siRNAs [47]. Notably, subsequent differential digestive treatments of EVs with RNase, protease, and a detergent revealed that the amount of intravesicular siRNA was low [47], more than 70% of the CYP3RNA-derived siR-NAs were found to be extravesicular. EVs isolated from CYP3RNA-sprayed barley plants revealed CYP3RNAderived siRNAs, too; however, their abundance was even lower compared with EVs isolated from HIGS A. thaliana plants [49].…”