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

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 the gene silencing of pathogenic targets, was ground-breaking. However, future field applications wil...

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Bibliographic Details
Published in:International journal of molecular sciences Vol. 22; no. 13; p. 7212
Main Authors: Schlemmer, Timo, Barth, Patrick, Weipert, Lisa, Preußer, Christian, Hardt, Martin, Möbus, Anna, Busche, Tobias, Koch, Aline
Format: Journal Article
Language:English
Published: Basel MDPI AG 01-07-2021
MDPI
Subjects:
Adenine
Barley
Biosynthesis
Communication
Complementarity
Disease resistance
Double-stranded RNA
dsRNA
Extracellular vesicles
Flowers & plants
Fungi
Gene expression
Gene silencing
Genes
Hordeum vulgare
Nucleotides
Pesticides
Plant diseases
plant EV
Plant protection
Risk assessment
RNA interference
RNAi
siRNA
Vesicles
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Summary: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.
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ISSN:1422-0067
1661-6596
1422-0067
DOI:10.3390/ijms22137212