Knockdown of Midgut Genes by dsRNA-Transgenic Plant-Mediated RNA Interference in the Hemipteran Insect Nilaparvata lugens

Abstract

BackgroundRNA interference (RNAi) is a powerful technique for functional genomics research in insects. Transgenic plants producing double-stranded RNA (dsRNA) directed against insect genes have been reported for lepidopteran and coleopteran insects, showing potential for field-level control of insect pests, but this has not been reported for other insect orders.Methodology/Principal FindingsThe Hemipteran insect brown planthopper (Nilaparvata lugens Stål) is a typical phloem sap feeder specific to rice (Oryza sativa L.). To analyze the potential of exploiting RNAi-mediated effects in this insect, we identified genes (Nlsid-1 and Nlaub) encoding proteins that might be involved in the RNAi pathway in N. lugens. Both genes are expressed ubiquitously in nymphs and adult insects. Three genes (the hexose transporter gene NlHT1, the carboxypeptidase gene Nlcar and the trypsin-like serine protease gene Nltry) that are highly expressed in the N. lugens midgut were isolated and used to develop dsRNA constructs for transforming rice. RNA blot analysis showed that the dsRNAs were transcribed and some of them were processed to siRNAs in the transgenic lines. When nymphs were fed on rice plants expressing dsRNA, levels of transcripts of the targeted genes in the midgut were reduced; however, lethal phenotypic effects after dsRNA feeding were not observed.ConclusionsOur study shows that genes for the RNAi pathway (Nlsid-1 and Nlaub) are present in N. lugens. When insects were fed on rice plant materials expressing dsRNAs, RNA interference was triggered and the target genes transcript levels were suppressed. The gene knockdown technique described here may prove to be a valuable tool for further investigations in N. lugens. The results demonstrate the potential of dsRNA-mediated RNAi for field-level control of planthoppers, but appropriate target genes must be selected when designing the dsRNA-transgenic plants.

Diet-delivered RNAi in Helicoverpa armigera – Progresses and challenges

“…In the case of H. armigera, ingested dsRNA is presumably exposed and imported into epithelial cells in the midgut (Price and Gatehouse, 2008;Zha et al, 2011). The presence of at least 2 sid-1 homologs in H. armigera has been reported (unpublished data, Xue et al (2012)), but analysis of published insect genomes thus far has not produced clear correlation between the presence of sid-1 homologs and systemic RNAi response in insects (Tomoyasu et al, 2008).…”

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 generation of Agrobacterium-mediated transgenic plants encoding dsRNA involves i) subcloning of the sense and antisense regions in vectors such as pDONR 207 (Pitino et al, 2011) or pHANNIBAL (Huang et al, 2006;Fairbairn et al, 2007;Zha et al, 2011;Zhang et al, 2011), ii) transfer of the silencing cassette into a plant binary vector, iii) transformation of Agrobacterium with the desired plasmids, and iv) plant transformation by Agrobacterium. The procedure is relatively complex and time consuming, and problems with plasmid instability related to antibiotic selection have been reported.…”

DNA Sequencing and Crop Protection

“…In fact the current expression of Bt insecticidal proteins in plant offers a better degree of protection in field condition against Lepidoptera and Coleoptera, while none of Cry toxins of B. thuringiensis is effective on sup-sucking pests (Virla et al, 2010). The good new results obtained with the whitefly Bemisia tabaci (Gennadius) (Upadhyay et al, 2011), the aphid M. persicae (Pitino et al, 2011) and the brown planthopper Nilaparvata lugens Stål (Zha et al, 2011) are promising. Anyhow, recent reports of resistance to Bt toxins expressed in the plants (Tabashnik et al, 2008) will provide more interest for alternative strategies also for Lepidoptera and Coleoptera.…”