Double-stranded RNA uptake through topical application, mediates silencing of five CYP4 genes and suppresses insecticide resistance in Diaphorina citri.

Nabil Killiny,Siddarame Gowda,Siddharth Tiwari,Lukasz L Stelinski,Subhas Hajeri,Immo A Hansen

doi:10.1371/journal.pone.0110536

Nabil Killiny, Siddarame Gowda + Show 4 more

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https://doi.org/10.1371/journal.pone.0110536

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Journal: PloS one	Publication Date: Oct 20, 2014
Citations: 161	License type: CC BY 4.0

Affiliation: Florida Department of Citrus, University of Florida

Abstract

Silencing of genes through RNA interference (RNAi) in insects has gained momentum during the past few years. RNAi has been used to cause insect mortality, inhibit insect growth, increase insecticide susceptibility, and prevent the development of insecticide resistance. We investigated the efficacy of topically applied dsRNA to induce RNAi for five Cytochrome P450 genes family 4 (CYP4) in Diaphorina citri. We previously reported that these CYP4 genes are associated with the development of insecticide resistance in D. citri. We targeted five CYP4 genes that share a consensus sequence with one dsRNA construct. Quantitative PCR confirmed suppressed expression of the five CYP4 genes as a result of dsRNA topically applied to the thoracic region of D. citri when compared to the expression levels in a control group. Western blot analysis indicated a reduced signal of cytochrome P450 proteins (45 kDa) in adult D. citri treated with the dsRNA. In addition, oxidase activity and insecticide resistance were reduced for D. citri treated with dsRNA that targeted specific CYP4 genes. Mortality was significantly higher in adults treated with dsRNA than in adults treated with water. Our results indicate that topically applied dsRNA can penetrate the cuticle of D. citri and induce RNAi. These results broaden the scope of RNAi as a mechanism to manage pests by targeting a broad range of genes. The results also support the application of RNAi as a viable tool to overcome insecticide resistance development in D. citri populations. However, further research is needed to develop grower-friendly delivery systems for the application of dsRNA under field conditions. Considering the high specificity of dsRNA, this tool can also be used for management of D. citri by targeting physiologically critical genes involved in growth and development.

Highlights

RNA interference (RNAi) is a promising tool for studying functional genomics in eukaryotes and insects in particular [1,2]
Relative expression levels for the five Cytochrome P450 genes family 4 (CYP4) genes were compared between double-stranded RNA (dsRNA)- P450, dsRNA-gfp-treated and control psyllids (Figure 1)
The expression level of a-tubulin remained constant among all treatments indicating the specificity of dsRNA- P450 to CYP4 genes

Summary

Introduction

RNA interference (RNAi) is a promising tool for studying functional genomics in eukaryotes and insects in particular [1,2]. Anti-sense (nonsense) RNA strand transcription has been used for over three decades to inhibit gene activity [3]. Since the discovery of double-stranded RNA (dsRNA) mediated gene-specific silencing in the nematode, Caenorhabditis elegans (Maupas) [4], dsRNAmediated RNAi has been employed with various insects to silence specific genes [2]. The systemic nature of dsRNA-mediated RNAi has allowed this tool to be used in the management of various insect pests [8,9,10,11,12,13]

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