Direct Foliar Application of dsRNA Derived From the Full-Length Gene of NSs of Groundnut Bud Necrosis Virus Limits Virus Accumulation and Symptom Expression

Abstract

Groundnut bud necrosis virus (GBNV) is the most significant member of the genus Orthotospovirus occurring in the Indian subcontinent. There is hardly any effective measure to prevent GBNV in crop plants. In order to develop GBNV infection prevention procedure, we examined the effect of the direct foliar application of double-stranded RNA (dsRNA) derived from the full-length NSs gene (1,320 nucleotides) of GBNV. The bacterially expressed dsRNA to the non-structural (dsNSs) gene of GBNV was purified and delivered to plants as an aqueous suspension containing 0.01% Celite for evaluating its efficacy in preventing GBNV infection in systemic host, Nicotiana benthamiana as well as in local lesion and systemic host, cowpea cv. Pusa Komal (Vigna unguiculata). The dsNSs application and challenge-inoculation were conducted in three different combinations, where plants were challenge-inoculated with GBNV a day after, immediately, and a day before the application of dsNSs. N. benthamiana plants, which were not treated with dsRNA showed severe systemic wilting and death by 9–16 days post-inoculation (dpi). The non-treated cowpea plants exhibited many chlorotic and necrotic lesions on the cotyledonary leaves followed by systemic necrosis and death of the plants by 14–16 dpi. The dsNSs treated plants in all the combinations showed significant reduction of disease severity index in both N. benthamiana and cowpea. The treatment combination where the GBNV inoculation was conducted immediately after the dsNSs treatment was found to be the most effective treatment in preventing symptom expression. The viral RNA analysis by real time PCR also showed 20 and 12.5 fold reduction of GBNV in cowpea and N. benthamiana, respectively. Our results suggest that the foliar application of dsRNA derived from the full-length NSs gene of GBNV through Celite is successful in delivering long dsRNA leading to effective prevention of GBNV infection.

Exogenous application of dsRNA for the control of viruses in cucurbits

“…Nevertheless, a reduction in virus accumulation in the plants will help limiting the damages and will probably reduce the transmission between plants during in routine crop management practices in the greenhouse such as planting, pruning, harvesting, and others. The reduction in virus accumulation in greenhouse conditions of 48-and 5-fold depending on the season was comparable to the results obtained in groundnut bud necrosis virus (GBNV), where it has been reported 20-and 12.5-fold reduction after rubbing dsRNA on plant leaves in cowpea and N. benthamiana, respectively (Gupta et al, 2021). The correlation between symptom severity and virus accumulation has been previously reported for many plant pathosystems, specifically for CGMMV in N. benthamiana by ELISA (Ali et al, 2016), in cucumber by RT-qPCR (Crespo et al, 2018), and for CVYV in cucumber, also by RT-qPCR (Galipienso et al, 2013).…”

“…Plant cuticles are barriers to dsRNA entry that has also to reach the cell wall and cross the plasma membrane. High pressure spraying allows the entry of the dsRNA, as well as do lesions made by abrasives such as carborundum, like in our experiments when applied the dsRNA by rubbing or celite, and as reported for groundnut bud necrosis virus (GBNV) control using dsRNA (Gupta et al, 2021). Other authors have proposed surfactants to facilitate the entry of the dsRNA and the movement of the silencing signals (Schwartz et al, 2020;Bennet et al, 2021).…”

“…The introduction of resistance in transgenic plants expressing virus genes leads to reduced symptom expression that is correlated with a decrease in virus accumulation (Bagewadi et al, 2014). Similarly, induced resistance with exogenous delivery of dsRNA or siRNAs leads to reduction in virus accumulation in plants as shown by serology or qPCR (Tenllado et al, 2003;Rego-Machado et al, 2020;Holeva et al, 2021;Gupta et al, 2021;Necira et al, 2021).…”

“…A PMMoV-derived dsRNA was detected by Northern-blot in pepper at 7 dpt (Tenllado and Díaz-Ruiz, 2001). More recently, a GBNV-derived dsRNA was locally and systemically detected by semiquantitative RT-qPCR at 7 dpt in N. benthamiana and at 5 dpt in cowpea (Gupta et al, 2021). Delivery of insect-derived dsRNA into barley plants showed systemic dsRNA movement and distal siRNAs were present as shown by HTS (Biedenkopf et al, 2020).…”

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

“…In most cases these nucleic acids have been delivered naked, in aqueous phase or buffered [7]. 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].…”