Exogenous Application of dsRNA for the Control of Viruses in Cucurbits

Abstract

The recurrent emergence of viral diseases in intensive horticultural crops requires alternative control strategies. The topical application of double-stranded RNA (dsRNA) molecules homologous to pathogens has been proposed as a tool for virus control in plants. These dsRNAs induce the silencing mechanism, the RNA interference (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 the 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 significantly reduced in dsRNA-sprayed plants, and the development of the plants improved with respect to non-treated plants. Virus titers 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 the 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 by direct spraying, but found no protective effect. In view of the results, the topical application of dsRNAs is postulated as a promising strategy for CGMMV control in the cucumber.

RNA interference‐based exogenous double‐stranded RNAs confer resistance to Rhizoctonia solaniAG‐3 on Nicotiana tabacum

“…The successful entry of dsRNA into plants is a prerequisite for the application of dsRNA spraying on the surface of plants to suppress pathogen infection. The plant cuticle is an entry barrier for dsRNA that has to reach the cell wall and cross the plasma membrane, 60 and the possibility of dsRNA stomatal uptake also exists in plants 61 . Exogenous dsRNA sprayed on the surface of the plant can be absorbed by the plant and induce the RNAi response in plant 47,62 .…”

Hairpin-RNA Spray Confers Resistance to Mungbean Yellow Mosaic India Virus in Mungbean

“…Though, exogenously applied dsRNA presents a promising tool for virus control, its efficacy may vary across different viruses. For DNA viruses like the begomoviruses Tomato Severe Rugose Virus (ToSRV) in tomatoes (73) and Tomato Leaf Curl New Delhi Virus (ToLCNDV) in zucchini squash (50), the application of exogenous dsRNA has shown limited effectiveness. Considering the existing research gap, this study aimed to assess the effectiveness of exogenously applied hpRNA for the control of a bipartite begomovirus, MYMIV, in economically significant mungbean plants.…”

“…Here, we employed a E. coli HT115 expression system to generate hpRNA (hpTR-1+2) in large quantities. The dose of exogenous dsRNA varied across studies: 40 to 60 μg against Zucchini Yellow Mosaic Virus (ZYMV) (51), almost 200 μg/plant against Tobacco Mosaic Virus (TMV) (49), 2.5 μg/plant against MYMV (55), 250 μg/plant against Tomato Spotted Wilt Virus (TSWV) (72), 60 μg/plant against Cucumber Green Mottle Mosaic Virus (CGMMV) (50), 15 μg/plant against ChiLCV (57), 100 μg/plant against Pepper Mild Mottle Virus (PepMoV) (48), 12 to 16 μg/plant against Cucumber Mosaic Virus (CMV) (53), 10 μg/plant against TSWV (70), and 20 to 30 μg/plant against ToLCV (54). Machado et al (2020) found a threshold, with concentrations below 16 μg/plant providing no protection in Tobacco Mosaic Virus (ToMV)-dsRNA tests, and a dose-dependent response ranging from 50 to 400 μg/plant (52).…”

“…The use of externally applied naked dsRNA to prevent infection by various plant viruses was first reported by Tennellado and Diaz Ruiz in 2001 (46). Subsequent studies confirmed the efficacy of dsRNA vaccination against several plant RNA viruses (47)(48)(49)(50)(51)(52)(53). Although limited, there are reports of dsRNA vaccination against DNA viruses like begomoviruses.…”

Exogenous Application of dsRNA for Protection against Tomato Leaf Curl New Delhi Virus

“…Partial symptom remission and reduction in the viral titer was observed in blackgram plants treated with dsRNAs targeting the CP and Rep coding regions of mungbean yellow mosaic virus [ 22 ]. However, no detectable antiviral effect mediated by topical application of dsRNAs has been observed until now against tomato leaf curl New Delhi virus in cucurbits [ 23 ] or tomato severe rugose virus in tomato [ 23 , 24 ].…”