Functional characterization of three trehalase genes regulating the chitin metabolism pathway in rice brown planthopper using RNA interference.

Lina Zhao,Xiaojun Liu,Shigui Wang,Qida Shen,Bin Tang,Mengmeng Yang,Zuokun Shi

doi:10.1038/srep27841

Abstract

RNA interference (RNAi) is an effective gene-silencing tool, and double stranded RNA (dsRNA) is considered a powerful strategy for gene function studies in insects. In the present study, we aimed to investigate the function of trehalase (TRE) genes (TRE 1-1, TRE 1-2, and TRE-2) isolated from the brown planthopper Nilaparvata lugens, a typical piercing-sucking insect in rice, and investigate their regulating roles in chitin synthesis by injecting larvae with dsRNA. The results showed that TRE1 and TRE2 had compensatory function, and the expression of each increased when the other was silenced. The total rate of insects with phenotypic deformities ranged from 19.83 to 24.36% after dsTRE injection, whereas the mortality rate ranged from 14.16 to 31.78%. The mRNA levels of genes involved in the chitin metabolism pathway in RNA-Seq and DGEP, namely hexokinase (HK), glucose-6-phosphate isomerase (G6PI) and chitinase (Cht), decreased significantly at 72 h after single dsTREs injection, whereas two transcripts of chitin synthase (CHS) genes decreased at 72 h after dsTRE1-1 and dsTREs injection. These results demonstrated that TRE silencing could affect the regulation of chitin biosynthesis and degradation, causing moulting deformities. Therefore, expression inhibitors of TREs might be effective tools for the control of planthoppers in rice.

Highlights

Changes in TRE1-1, TRE1-2, and TRE2 expression at 48 h and 72 h after dsTRE1-1, dsTRE1-1, dsTRE2, dsTREs, and dsGFP injection. (D,E) Changes in TRE1 and TRE2 activity at 48 h and 72 h after dsTRE1-1, dsTRE1-1, dsTRE2, dsTREs, and dsGFP injection. (F) Changes in trehalose content at 48 h and 72 h after dsTRE1-1, dsTRE1-1, dsTRE2, dsTREs, and dsGFP injection
We hypothesised that TRE controls the process of moulting by regulating the genes involved in the chitin biosynthesis pathway and we aimed to study the functions and relationships of three TRE genes (TRE 1-1, TRE 1-2, and TRE-2) which were found from the genome of N. lugens[42], as well as their regulating roles in chitin synthesis using RNA interference (RNAi) and RNA-Sequencing (RNA-Seq)
We investigated the relative expression of TRE1 and TRE2 after dsTRE and dsTREs injection and found that they have selective, discriminate, and compensatory functions; TRE1-1 and TRE1-2 probably have similar functions[3,8]

Summary

Introduction

Changes in TRE1-1, TRE1-2, and TRE2 expression at 48 h and 72 h after dsTRE1-1, dsTRE1-1, dsTRE2, dsTREs, and dsGFP injection. (D,E) Changes in TRE1 and TRE2 activity at 48 h and 72 h after dsTRE1-1, dsTRE1-1, dsTRE2, dsTREs, and dsGFP injection. (F) Changes in trehalose content at 48 h and 72 h after dsTRE1-1, dsTRE1-1, dsTRE2, dsTREs, and dsGFP injection. TRE regulates the chitin biosynthesis pathway by decreasing the expression of some of the involved genes, whereas chitinase (Cht) or chitinase-like genes are responsible for the degradation of chitin and the completion of moulting[13,36]. It has been reported that TRE regulates the expression of CHS in the cuticle and midgut of planthoppers, and the inhibition of chitin synthesis by suppressing or knocking down TRE leads to abnormal moulting and mortality. We hypothesised that TRE controls the process of moulting by regulating the genes involved in the chitin biosynthesis pathway and we aimed to study the functions and relationships of three TRE genes (TRE 1-1, TRE 1-2, and TRE-2) which were found from the genome of N. lugens[42], as well as their regulating roles in chitin synthesis using RNAi and RNA-Sequencing (RNA-Seq)

Objectives

Methods

Results

Conclusion