Exogenous application of dsRNA for the control of viruses in cucurbits

Abstract

The recurrent emergence of virus diseases in intensive horticultural crops requires alternative control strategies. Topical application of dsRNA molecules homologous to pathogens has been proposed as a tool for plant virus control. These dsRNAs induce the silencing mechanism (RNAi) that degrades homologous dsRNAs. Cucumber green mottle mosaic virus (CGMMV) represents a serious threat to cucurbit crops. Since genetic resistance to the virus is not yet available in commercial varieties, we aimed to control this virus by RNAi. For this purpose, we obtained constructions both for expressing dsRNA in bacteria to treat cucumber plants by topical application and for agroinoculation in experiments done in the growth chamber. Besides, greenhouse tests were performed in spring and in summer when plants were challenged with the virus and differences in several parameters were investigated, including severity of symptoms, dry weight, total height, virus accumulation and virus-derived small interfering RNAs (vsiRNAs). Spraying of plants with dsRNA reduced significatively CGMMV symptoms in the plants in growth chamber tests. Agroinfiltration experiments done under identical conditions were also effective in limiting the progress of CGMMV disease. In the greenhouse assay performed in spring, symptoms were significatively reduced in dsRNA-sprayed plants and the development of the plants improved with respect to non-treated plants. Virus titters and vsiRNAs were clearly reduced in dsRNA-treated plants. The effect of protection of the dsRNA was less evident in the greenhouse assay carried out in summer. Besides, we investigated the mobility of long (ds)RNA derived from spraying or agroinfiltrated dsRNA and found that it could be detected in local, close distal and far distal points from the site of application. VsiRNAs were also detected in local and distal points and the differences in accumulation were compared. In parallel, we investigated the capacity of dsRNAs derived from genes of tomato leaf curl New Delhi virus (ToLCNDV), another economically important virus in cucurbits, to limit the disease in zucchini, both by agroinfiltration or direct spraying, but found no protection effect. In view of the results, topical application of dsRNAs is postulated as a promising strategy for CGMMV control in cucumber.

Carbon dots boost dsRNA delivery in plants and increase local and systemic siRNA production

“…Hence, the more effective local and systemic silencing of GFP expression observed in 16c plants when siRNA is delivered as CD-nanocomposites [14] can be explained in terms of higher amount of siRNA available and the possible improvement of systemic movement of the coated RNAs, as results from our research. Although systemic GFP silencing has been so far observed only in N. benthamiana 16c plants [62], we and others have shown that virus-derived dsRNAs, or at least long RNA molecules, moves systemically in the plant, and result subjected to the RNAi machinery producing siRNAs and lead to symptom reduction after virus inoculation [16,18,63]. Thus, improving delivery methods for siRNA/dsRNA in foliar application will either decrease the amount of dsRNA needed or improve its effectiveness.…”

“…Detection and quantitation by RT-qPCR of the small RNAs derived of the dsRNAs was performed as described previously (18). In this section we evaluated the siRNAs derived from the sprayed dsRNAs product of the RNAi machinery in the plant cell.…”

“…The cDNA was then used to detect by qPCR the 6125-vsiRNA using the primer CG-6125 (5'-GCTAGGGCTGAGATAGATAATT-3') and the universal reverse primer (URP) (5'-GCGAGCACAGAATTAATACGAC-3'). Reaction and cycling conditions were described previously [18]. For the reference with an endogenous plant siRNA, we used the primer CUC5.8S based on the 5.8S rRNA of C. sativus (5'-CTTGGTGTGAATTGCAGGATC-3') [18].…”

“…Reaction and cycling conditions were described previously [18]. For the reference with an endogenous plant siRNA, we used the primer CUC5.8S based on the 5.8S rRNA of C. sativus (5'-CTTGGTGTGAATTGCAGGATC-3') [18]. Six biological replicas were included in each condition and the experiment was repeated twice.…”

“…There are examples of their application by spraying at higher or lower pressure [13,14] or mechanically [rubbing] with or without abrasives [15,16]. In the case of plant viruses, several successful cases of spray-induced RNAi control have been described [17], in general performed under laboratory conditions and more recently in greenhouse conditions [18]. The application of dsRNA or siRNA molecules on plants has also been considered with the aim of being sucked by the harmful insects that feed on them and thus exert a control effect by RNAi [8].…”