Double-Stranded RNA-Degrading Enzymes Reduce the Efficiency of RNA Interference in Plutella xylostella.

Jin-Zhi Chen,Ying-Xia Jiang,Jian-Wen Li,Guang Yang,Miao-Wen Li,Ben-Hu Zha

doi:10.3390/insects12080712

Abstract

Simple SummaryThe efficiency of Lepidoptera RNA interference (RNAi) is highly varied among different species, different periods, and different genes. The stability of dsRNA is one of the important factors. DsRNA-degrading enzymes (dsRNases) are the key factors affecting the stability of dsRNA in insects. The efficiency of RNAi in diamondback moths was low and unstable. Furthermore, in vitro experiments, we found that dsRNA was completely degraded when incubated with the hemolymph or gut fluid of diamondback moths. Therefore, we hypothesized that the efficiency of RNAi in diamondback moths was decreased predominantly due to degradation of dsRNA by dsRNase. In this study, we identified four dsRNases in diamondback moths: PxdsRNase1 was mainly expressed in the hemolymph; and PxdsRNase2 and PxdsRNase3 were mainly expressed in the intestinal tract. PxdsRNase1, PxdsRNase2, and PxdsRNase3 were verified to be involved in the RNAi process in diamondback moths. In vitro, the recombinant protein of PxdsRNase1 degraded dsRNA completely and PxdsRNase3 cleaved dsRNA without complete degradation. Overall, our findings provided a fundamental basis for understanding the mechanism of dsRNase involvement in the RNAi process and using RNAi to control diamondback moths in the future.DsRNA-degrading enzymes (dsRNases) have been recognized as important factors in reducing RNA interference (RNAi) efficiency in different insect species. However, dsRNases in Plutella xylostella are still unknown. We identified the full-length cDNAs of PxdsRNase1, PxdsRNase2, PxdsRNase3, and PxdsRNase4. Gene expression profile showed that PxdsRNase1 was mainly expressed in the hemolymph; and that PxdsRNase2 and PxdsRNase3 were mainly expressed in the intestinal tract. The expression of PxCht (Chitinase of P. xylostella) in P. xylostella larvae injected with the mixture of dsPxCht (dsRNA of PxCht) and dsPxdsRNase1 (dsRNA of PxdsRNase1), dsPxdsRNase2 (dsRNA of PxdsRNase2), or dsPxdsRNase3 (dsRNA of PxdsRNase3) was significantly higher than that in the larvae injected with the mixture of dsGFP (dsRNA of green fluorescent protein gene, GFP) and dsPxCht; the transcription level of PxCht in the larvae feeding on the mixture of dsPxCht and dsPxdsRNase1, dsPxdsRNase2, or dsPxdsRNase3 was significantly higher than that in the larvae feeding on the mixture of dsPxCht and dsGFP. The recombinant protein of PxdsRNase1 degraded dsRNA rapidly, PxdsRNase3 cleaved dsRNA without complete degradation, and PxdsRNase2 could not degrade dsRNA in vitro. These results suggested that PxdsRNases1, PxdsRNases2, and PxdsRNases3 were involved in the dsRNA degradation to reduce RNAi efficiency with different mechanisms.

Highlights

The efficiency of Lepidoptera RNA interference (RNAi) is highly varied among different species, different periods, and different genes [1]
The P. xylostella Fuzhou-sensitive strain (FZss) used in the experiment was maintained at the Institute of Applied Ecology, Fujian Agriculture and Forestry University, Fuzhou, China
The cDNA sequences were cloned by RT-PCR and sequenced

Summary

Introduction

The efficiency of Lepidoptera RNAi is highly varied among different species, different periods, and different genes [1]. The dsRNA degradation [2,3,4] or the inability of dsRNA to enter the cytoplasm [3,5,6] are the main factors affecting RNAi efficiency. Studies have shown that dsRNA needs to last long enough in the midgut or hemolymph to be absorbed into cells to produce an effective RNAi response [7]. DsRNases are the key factors affecting the stability of dsRNA in insects. The activity of dsRNase has been observed in several insects. In Bombyx mori, dsRNase is expressed in the digestive juice and midgut, secreted into the intestinal cavity for nucleic acid digestion [8,9]. DsRNases are found in more and more insects, such as Lygus lineolaris [10], Manduca sexta [11], Acyrthosiphon pisum [12], Schistocerca gregaria [13], and Spodoptera frugiperda [14]

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Discussion

Conclusion