Abstract
BackgroundRNA silencing is an important mechanism for regulation of endogenous gene expression and defense against genomic intruders in plants. This natural defense system was adopted to generate virus-resistant plants even before the mechanism of RNA silencing was unveiled. With the clarification of that mechanism, transgenic antiviral plants were developed that expressed artificial virus-specific hairpin RNAs (hpRNAs) or microRNAs (amiRNAs) in host plants. Previous works also showed that plant-mediated RNA silencing technology could be a practical method for constructing insect-resistant plants by expressing hpRNAs targeting essential genes of insects.Methodology/Principal findingsIn this study, we chose aphid Myzus persicae of order Hemiptera as a target insect. To screen for aphid genes vulnerable to attack by plant-mediated RNA silencing to establish plant aphid resistance, we selected nine genes of M. persicae as silencing targets, and constructed their hpRNA-expressing vectors. For the acetylcholinesterase 2 coding gene (MpAChE2), two amiRNA-expressing vectors were also constructed. The vectors were transformed into tobacco plants (Nicotiana tabacum cv. Xanti). Insect challenge assays showed that most of the transgenic plants gained aphid resistance, among which those expressing hpRNAs targeting V-type proton ATPase subunit E-like (V-ATPaseE) or tubulin folding cofactor D (TBCD) genes displayed stronger aphicidal activity. The transgenic plants expressing amiRNAs targeting two different sites in the MpAChE2 gene exhibited better aphid resistance than the plants expressing MpAChE2-specific hpRNA.Conclusions/SignificanceOur results indicated that plant-mediated insect-RNA silencing might be an effective way to develop plants resistant to insects with piercing-sucking mouthparts, and both the selection of vulnerable target genes and the biogenetic type of the small RNAs were crucial for the effectiveness of aphid control. The expression of insect-specific amiRNA is a promising and preferable approach to engineer plants resistant to aphids and, possibly, to other plant-infesting insects.
Highlights
The phenomenon of RNA silencing was first discovered during plant transgenic studies where it was termed co-suppression [1,2]
Selection and Cloning of M. persicae Target Genes Based on the results of a dsRNA-feeding assay in western corn rootworm conducted by Baum et al [18], eight aphid genes encoding proteins with essential functions were selected as our RNA interference (RNAi) targets (Table 1)
Three independent transgenic lines of each of the hairpin RNAs (hpRNAs)- and artificial microRNA (amiRNA)-expressing plants were chosen for insect-resistance assay, none of which displayed obvious phenotypic aberrations
Summary
The phenomenon of RNA silencing was first discovered during plant transgenic studies where it was termed co-suppression [1,2] It has various terms, post-transcriptional gene silencing (PTGS) in plants, RNA interference (RNAi) in animals and quelling in fungi, RNA silencing is based on a highly conserved mechanistic frame [1,2,3], and is a natural regulatory mechanism acting against genomic intruders and modulating endogenous gene expression in eukaryotes [4,5,6,7]. Because RNA silencing can be exploited to regulate gene expression through knock-down of the nucleotide sequence-matched target transcripts, it has become an effective reverse genetics approach in functional genomics and a powerful tool to develop transgenic plants that have enhanced resistance against diseases caused by e.g. virus pathogens or insect pests [8,9,10,11,12,13]. Previous works showed that plant-mediated RNA silencing technology could be a practical method for constructing insectresistant plants by expressing hpRNAs targeting essential genes of insects
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