Engineered Flock House Virus for Targeted Gene Suppression Through RNAi in Fruit Flies (Drosophila melanogaster) in Vitro and in Vivo.

Clauvis N T Taning,Olivier Christiaens,Xiuxia Li,Martine Maes,Hans Casteels,Luc Swevers,Guy Smagghe

doi:10.3389/fphys.2018.00805

Abstract

RNA interference (RNAi) is a powerful tool to study functional genomics in insects and the potential of using RNAi to suppress crop pests has made outstanding progress. However, the delivery of dsRNA is a challenging step in the development of RNAi bioassays. In this study, we investigated the ability of engineered Flock House virus (FHV) to induce targeted gene suppression through RNAi under in vitro and in vivo condition. As proxy for fruit flies of agricultural importance, we worked with S2 cells as derived from Drosophila melanogaster embryos, and with adult stages of D. melanogaster. We found that the expression level for all of the targeted genes were reduced by more than 70% in both the in vitro and in vivo bioassays. Furthermore, the cell viability and median survival time bioassays demonstrated that the recombinant FHV expressing target gene sequences caused a significantly higher mortality (60–73% and 100%) than the wild type virus (24 and 71%), in both S2 cells and adult insects, respectively. This is the first report showing that a single stranded RNA insect virus such as FHV, can be engineered as an effective in vitro and in vivo RNAi delivery system. Since FHV infects many insect species, the described method could be exploited to improve the efficiency of dsRNA delivery for RNAi-related studies in both FHV susceptible insect cell lines and live insects that are recalcitrant to the uptake of naked dsRNA.

Highlights

RNA interference (RNAi), the process that is triggered by double stranded RNA molecules and results in specific gene silencing through hybridization of processed small RNAs to target mRNAs, has evolved rapidly to a standard technique to evaluate gene function in eukaryotes (Fire et al, 1998; Huvenne and Smagghe, 2010; Zotti et al, 2017)
We hypothesized that engineering Flock House virus (FHV) to express D. melanogaster target gene sequences could lead to Virus-induced gene silencing (VIGS) when D. melanogaster cells are infected by recombinant clones of the virus
We show that a single stranded RNA insect virus can be engineered as a virus-based delivery system, for both in vitro and in vivo RNA silencing in an insect

Summary

Introduction

RNA interference (RNAi), the process that is triggered by double stranded (ds) RNA molecules and results in specific gene silencing through hybridization of processed small RNAs to target mRNAs, has evolved rapidly to a standard technique to evaluate gene function in eukaryotes (Fire et al, 1998; Huvenne and Smagghe, 2010; Zotti et al, 2017). Efficient cellular uptake of dsRNA is crucial, amongst other factors A much higher stability and tissue penetrance of dsRNA was found for the RNAi-sensitive coleopteran insects than for the RNAi-recalcitrant lepidopterans (Ivashuta et al, 2015). These all imply that the stability of dsRNA in the insect body could be affected by both enzymatic hydrolysis and other elements of the gut environment (Price and Gatehouse, 2008). Viruses have several attractive properties that make them excellent delivery vehicles for nucleic acids such as, efficiency and specificity of infection and the evolved avoidance of the immune response

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