Exogenously applied dsRNA molecules deriving from the Zucchini yellow mosaic virus (ZYMV) genome move systemically and protect cucurbits against ZYMV

Abstract

Zucchini yellow mosaic virus (ZYMV) causes serious damage in a large number of cucurbits, and control measures are necessary. Transgenic cucurbits expressing parts of the ZYMV genome have been shown to be resistant to the cognate virus. A non-transgenic approach involving the exogenous application of double-stranded RNA (dsRNA) has also been shown to induce resistance in tobacco against Cucumber mosaic virus (CMV) and Tobacco mosaic virus (TMV). In the present study, dsRNA molecules derived from the helper component-proteinase (HC-Pro) and coat protein (CP) genes of the ZYMV_DE_2014 isolate were produced in vitro. On exogenous dsRNA application in cucumber, watermelon and squash plants, dsRNA HC-Pro conferred resistance of 82%, 50% and 18%, and dsRNA CP molecules of 70%, 43% and 16%, respectively. On deep sequencing analysis of ZYMV-infected watermelon, hot-spot regions for viral small interfering RNAs (vsiRNAs) in the genome of ZYMV were identified. Stem-loop reverse transcription-polymerase chain reaction (RT-PCR) detection of selected 21-nucleotide-long vsiRNAs in plants that received only dsRNA molecules suggested that the dsRNAs exogenously applied onto plants were successfully diced, thus initiating RNA silencing. dsRNA molecules were found to be progressively degraded in planta, and strongly detected by semi-quantitative RT-PCR for at least 9 days after exogenous application. Moreover, dsRNA molecules were detected in systemic tissue of watermelon and squash, showing that dsRNA is transported long distances in these plants.

RNA‐based biocontrol compounds: current status and perspectives to reach the market

“…7,8 Some studies have also shown that topically applied dsRNAs can act on both the treated plant area and distal non-treated parts by systemic translocation of dsRNAs via the plant vascular system, following application via mechanical wounding. [9][10][11] These results demonstrate the possibility of active uptake of exogenously applied dsRNAs by plant cells and their processing into siRNAs, which should extend the time of control/prevention against some plant diseases. However, specific features of the target organism (i.e.…”

“…Examples include the induction of plant resistance to fungi, insects and viruses following external application of bacterially produced or in vitro synthesized long dsRNAs, hpRNA, or small interfering RNAs (siRNAs) designed to selectively target essential genes of pests and pathogens . Some studies have also shown that topically applied dsRNAs can act on both the treated plant area and distal non‐treated parts by systemic translocation of dsRNAs via the plant vascular system, following application via mechanical wounding . These results demonstrate the possibility of active uptake of exogenously applied dsRNAs by plant cells and their processing into siRNAs, which should extend the time of control/prevention against some plant diseases.…”

Phloem-mediated spreading of SIGS-derived non-coding RNAs inHordeum vulgare

“…Moreover, they showed that dsRNA levels continuously decreased in the local (treated) tissue from 3 dpi to 9 dpi where dsRNA was no longer detectable (Mitter et al, 2017). More recently, Kaldis et al (2018) showed that exogenously applied dsRNA derived from the silencing suppressor HC-Pro and the coat protein genes of zucchini yellow mosaic virus (ZYMV) protect watermelon and cucumber against ZYMV and spread systemically over long distances in cucurbits (Kaldis et al, 2018).…”

A Plant SMALL RNA-BINDING PROTEIN 1 Family Mediates Cell-to-Cell Trafficking of RNAi Signals

“…To further confirm that AtSRBP1 and AtSRBP4 bind vsiRNA, we next synthesized two ZYMV vsiRNAs, termed vsiRNA21 (21 nt) and vsiRNA22 (22 nt). These specific vsiRNAs were chosen based on their high read counts from an earlier ZYMV study of watermelon plants (Kaldis et al, 2018). Electrophoretic mobility-shift assays (EMSAs) indeed established that both AtSRBP1 and AtSRBP4 bound vsiRNAs ( Figure 6E and Supplemental Figure 5A).…”