Tomato (Solanum lycopersicum) is one of the most important vegetable plants in the world, with global production estimated as 180 million tons (FAOSTAT 2019, http://www.fao.org/faostat/); due to its nutritional importance as food and source of health-promoting compounds, and to the balanced mixture of minerals and antioxidants (Dorais et al. 2008). However, its production is affected by a diversity of insect pests and diseases. Among them, whiteflies (Bemisia tabaci) and whitefly-transmitted viruses present some of the most important constraints worldwide.
Whitefly is recognized as a complex of cryptic species. It is a polyphagous insect that causes huge damage in hundreds of host species, including horticultural crops, such as tomato, lettuce, eggplant, cauliflower, and cucumber (Greathead 1986; Oliveira et al. 2001; Shah and Liu 2013). Besides sucking nutrients from the phloem, which results in accumulation of toxic molecules leading to plant breakdown, B. tabaci transmits several plant viruses, such as begomoviruses (Geminiviridae), criniviruses (Closteroviridae), and torradoviruses (Secoviridae) (Jones 2003; Oliveira et al. 2001; Navas-Castillo et al. 2011) and these then cause yield losses, even with a low insect population. In addition, whitefly excretes honeydew on the leaf surface, which promotes its colonization by Ascomycete fungi, generally called “sooty mold”, which reduce photosynthesis efficiency (Byrne and Bellows Junior 1991; Perring et al. 2018). Whiteflies are particularly difficult to manage, due to their diversity, adaptability, rapid reproduction lifecycle, extensive host range, and the ability to quickly select populations resistant to insecticides, and also to transmit several viruses. Numerous management methods have been employed, such as biological control and agricultural practice strategies. However, the use of chemicals remains the common practice, which causes the development of insecticidal resistance. Consequently, there is considerable interest in the introduction of whitefly resistance into plants by classical and molecular breeding.
RNA interference (RNAi) has evolved as an important defense mechanism in eukaryotes against viruses and transposable elements (Obbard et al. 2009). In insects, RNAi have been reported in species from Lepidoptera, Coleoptera, Diptera, Hemiptera, Hymenoptera, Dictyoptera, Isoptera and Orthoptera orders (Swevers and Smagghe 2012). Three RNAi pathways have been identified mediated by different small RNA molecules playing a role in defense against viruses and transposons (via siRNA), regulating gene expression (via miRNA) and suppression of germ-line transposon expression (via piRNA) (Yan et al. 2020).
Although some insect species are insensitive to RNAi through feeding, gene silencing is usually triggered by supplying exogenous dsRNA by topical applications, such as foliar spray, micro-injection, root dipping and seed treatment, and expression of dsRNA in transgenic plants (for a review see Yan et al. 2020). The effectiveness of RNAi as a tool to generate transgenic crops resistant to insect pests has been demonstrated (Baum et al. 2007; Mao et al. 2007; Gordon and Waterhouse 2007) and is now a reality with the development of some commercial products (Head et al. 2017).
RNAi-mediated gene silencing has been demonstrated in whiteflies. For example, silencing of genes (Cyp315a1, Cyp18a1, EcR and E75) from the ecdysone pathway through leaf-mediated dsRNA feeding assays had a limited effect on the survival and fecundity of adult whiteflies, while in nymphs, gene silencing reduced survival and delayed development Luan et al. (2013). Raza et al. (2016) reported 70% insect mortality in transgenic tobacco (Nicotiana tabacum) engineered to express dsRNAs for silencing insect aquaporin and alpha glucosidase genes. Similarly, acetylcholinesterase (AChE) and ecdysone receptor (EcR) genes from whitefly were used to induce mortality of up to 90% in tobacco (Malik et al. 2016). Thakur et al. (2014) showed mortality of 34 % to 85% among insects feeding on transgenic tobacco leaf discs expressing a v-ATPase gene target. Recently, Xia et al. (2021) reported resistance in transgenic tomato plants modified to silence the BtPMaT1 gene by impairing whitefly ability to detoxicate plant phenolic glucosides.
We have previously shown mortality of 83–98% in adult whiteflies and 95% reduction in the number of eggs of whiteflies feeding on transgenic lettuce expressing dsRNA corresponding to a vATPase gene (Ibrahim et al. 2017). Since transmembrane ATPases are crucial for several functions in the cell metabolism, intracellular membrane transport, and their processing, and transport of neurotransmitters, they seem to be promising candidates for engineering RNAi-based resistance to whiteflies in transgenic plants (Beyenbach and Wieczorek 2006; Upadhyay et al. 2011; Thakur et al. 2014; Ibrahim et al. 2017). However, since vATPase genes present some similarities among insect species, the effect of gene silencing on non-target organisms might be a constraint that should be investigated. Here we report the generation of transgenic tomato lines engineered to express dsRNA molecules targeting a B. tabaci vATPase gene. We hypothesized that the expression of this vATPase intron-hairpin construction would interfere in insect survival, generating tolerant tomato plants.