RNA interference as a gene silencing tool to controlTuta absolutain tomato (Solanum lycopersicum)

Abstract

RNA interference (RNAi), a gene-silencing mechanism that involves providing double-stranded RNA molecules that match a specific target gene sequence, is now widely used in functional genetic studies. The potential application of RNAi-mediated control of agricultural insect pests has rapidly become evident. The production of transgenic plants expressing dsRNA molecules that target essential insect genes could provide a means of specific gene silencing in larvae that feed on these plants, resulting in larval phenotypes that range from loss of appetite to death. In this report, we show that the tomato leafminer (Tuta absoluta), a major threat to commercial tomato production, can be targeted by RNAi. We selected two target genes (Vacuolar ATPase-A and Arginine kinase) based on the RNAi response reported for these genes in other pest species. In view of the lack of an artificial diet for T. absoluta, we used two approaches to deliver dsRNA into tomato leaflets. The first approach was based on the uptake of dsRNA by leaflets and the second was based on “in planta-induced transient gene silencing” (PITGS), a well-established method for silencing plant genes, used here for the first time to deliver in planta-transcribed dsRNA to target insect genes. Tuta absoluta larvae that fed on leaves containing dsRNA of the target genes showed an ∼60% reduction in target gene transcript accumulation, an increase in larval mortality and less leaf damage. We then generated transgenic ‘Micro-Tom’ tomato plants that expressed hairpin sequences for both genes and observed a reduction in foliar damage by T. absoluta in these plants. Our results demonstrate the feasibility of RNAi as an alternative method for controlling this critical tomato pest.

A gene silencing of V‐ATPase subunit A interferes with survival and development of the tomato leafminer, Tuta absoluta

“…Similarly, Camargo et al (2016) used oral assay to show the transcript level of vacuolar ATPase in the treated tomato leafminers after silencing. They performed two approaches.…”

“…Vacuolar-type ATPase (v-ATPase) is one of the most promising genes involved in pest management using RNAi technology (Camargo et al, 2016). This evolutionarily conserved enzyme found in the membranes of intracellular compartments of eukaryotic cells is a multisubunit protein consisting of two domains, V 1 and V 0 .…”

“…Subunit A, belonging to the V 1 domain is the catalytic site and responsible for ATP hydrolysis (Upadhyay et al, 2011). Thus, suppression of v-ATPase subunit A expression lead to mortality and reduction in weight and reproduction of various insects (Camargo et al, 2016;Mao et al, 2015).…”

Identification and validation of potential reference gene for effective dsRNA knockdown analysis in Chilo partellus

“…The larvae were sorted according to their growth stages and maintained on fresh maize stem for approximately 3 days to adapt to the new artificial environment (26 °C ± 2 and 65% humidity). dsRNA were targeted against vital genes that have been reported to induce silencing in order Lepidoptera: Chitinase , Vacuolar ATPase , Arginine kinase 47 , acetylcholinesterase 45 , and cytochrome P450 48 . dsRNAs were induced in bacteria as described by 14 and purified according to 49 .…”

Epigenetic Modifications: An Unexplored Facet of Exogenous RNA Application in Plants

“…Unsurprisingly, plant researchers have lately resorted to induction of RNAi by exogenous application of RNA molecules having the potential to trigger RNAi (exogenous RNAi, exo-RNAi), as an effective and transgene-free alternative to GM crops [31][32][33][34][35]. Indeed, dsRNAs/siRNAs were applied in plants by methods such as spraying, petiole uptake, trunk injection and root absorption in order to modify plant gene expression [36][37][38][39] and to confer resistance against viruses [40][41][42][43][44], fungi [45][46][47][48][49] and insects [50][51][52][53][54]. In the near future, one may envisage that exo-RNAi could potentially replace conventional herbicides, fungicides and insecticides, and a great amount of effort is invested in this direction by most major crop industries [55].…”