Silencing of Aphid Genes by dsRNA Feeding from Plants

Abstract

BackgroundRNA interference (RNAi) is a valuable reverse genetics tool to study gene function in various organisms, including hemipteran insects such as aphids. Previous work has shown that RNAi-mediated knockdown of pea aphid (Acyrthosiphon pisum) genes can be achieved through direct injection of double-stranded RNA (dsRNA) or small-interfering RNAs (siRNA) into the pea aphid hemolymph or by feeding these insects on artificial diets containing the small RNAs.Methodology/Principal FindingsIn this study, we have developed the plant-mediated RNAi technology for aphids to allow for gene silencing in the aphid natural environment and minimize handling of these insects during experiments. The green peach aphid M. persicae was selected because it has a broad plant host range that includes the model plants Nicotiana benthamiana and Arabidopsis thaliana for which transgenic materials can relatively quickly be generated. We targeted M. persicae Rack1, which is predominantly expressed in the gut, and M. persicae C002 (MpC002), which is predominantly expressed in the salivary glands. The aphids were fed on N. benthamiana leaf disks transiently producing dsRNA corresponding to these genes and on A. thaliana plants stably producing the dsRNAs. MpC002 and Rack-1 expression were knocked down by up to 60% on transgenic N. benthamiana and A. thaliana. Moreover, silenced M. persicae produced less progeny consistent with these genes having essential functions.Conclusions/SignificanceSimilar levels of gene silencing were achieved in our plant-mediated RNAi approach and published silencing methods for aphids. Furthermore, the N. benthamiana leaf disk assay can be developed into a screen to assess which genes are essential for aphid survival on plants. Our results also demonstrate the feasibility of the plant-mediated RNAi approach for aphid control.

Interplay of Plasma Membrane and Vacuolar Ion Channels, Together with BAK1, Elicits Rapid Cytosolic Calcium Elevations in Arabidopsis during Aphid Feeding

“…Aphid Fecundity Assay M. persicae fecundity was assessed as previously described (Pitino et al, 2011). Briefly, five adult aphids from the stock colony were added to each plant at the beginning of the experiment, and after 48 h all adults were removed.…”

Use of chromatin remodeling ATPases as RNAi targets for parental control of western corn rootworm (Diabrotica virgifera virgifera) and Neotropical brown stink bug (Euschistus heros)

“…Successes in inducing RNAi in aphids (Hemiptera: Aphididae) by introducing dsRNA in artificial diet and/or via in planta expression of dsRNA hairpins to control green peach aphid, Myzus persicae (Coleman et al, 2015;Pitino et al, 2011), the pea aphid Acyrthosiphon pisum (Mao et al, 2013;Mao and Zeng, 2012), and the grain aphid, Sitobion avenae , bolster the potential for achieving RNAi in stink bugs. Since stink bugs can have multiple generations per year (Pilkay, 2013), parental RNAi-based approaches could be particularly effective in controlling this pest complex.…”

Development of an improved RNA interference vector system for Agrobacterium-mediated plant transformation

“…As a second improvement, the constructs allow in vitro synthesis of dsRNA through the addition of the T7 RNA polymerase promoter so that the intervening DNA templates can be used for in vitro synthesis of dsRNAs. Plant-mediated RNAi possesses great potential as an insect control strategy in the field (Gordon and Waterhouse, 2007;Huvenne and Smagghe, 2010 (Baum et al, 2007), cytochrome P450 monooxygenase, and glutathione-Stransferase (Mao et al, 2007); receptor of activated kinase C (Pitino et al, 2011); hexose transporter; carboxypeptidase; and trypsin-like serine protease (Zha et al, 2011). Targeting midgut genes through plant-mediated RNAi could have great potential as an insect control strategy because of its essential role in insect digestive physiology and the indirect contact with the external environment, which allows for a per os application.…”

“…The procedure is relatively complex and time consuming, and problems with plasmid instability related to antibiotic selection have been reported. For example, many Agrobacterium strains, such as C58 (Huang et al, 2006), GV3101 (Pitino et al, 2011;Zhang et al, 2011), and A136 and BG53 (Rogers et al, 2002) that are routinely used for Arabidopsis thaliana transformation possess an inducible resistance mechanism against chloramphenicol (Tennigkeit and Matzura, 1991), suggesting that effective selection with this antibiotic is not possible with many Agrobacterium strains (Rogers et al, 2002;Bent, 2006;Szakasits et al, 2007). This response also leads to promiscuous recombination and plasmid instability (Ballester et al, 1986(Ballester et al, , 1989.…”