Distinct fitness costs associated with the knockdown of RNAi pathway genes in western corn rootworm adults.

Ke Wu,Elane Fishilevich,Xiuping Chen,Caitlin E Taylor,Blair D Siegfried,Carolina Camargo,Kenneth E Narva

doi:10.1371/journal.pone.0190208

Ke Wu, Elane Fishilevich + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0190208

Copy DOI

Abstract

RNA interference (RNAi) based approaches can potentially be used to control insect pests. These approaches may depend on the usage of microRNA (miRNA) or double stranded RNA (dsRNA) mediated gene knockdown, which likely involves proteins that regulate these pathways, such as Argonaute 1 (Ago1), Argonaute 2 (Ago2), Dicer 1 (Dcr1), Dicer 2 (Dcr2), and Drosha in insects. We previously performed functional characterization of Ago2 and Dcr2 of western corn rootworm (WCR), Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae) and observed that knockdown of Ago2 and Dcr2 ameliorated the lethal effect induced by the dsRNA-mediated knockdown of an essential gene in WCR, thereby confirming the involvement of Ago2 and Dcr2 in the dsRNA pathway. In the current study, we identified and characterized additional members of the Argonaute and Dicer gene families, namely Ago1, Ago3, Aubergine, and Dcr1, in a previously developed WCR transcriptome. We also identified a Drosha homolog in the same transcriptome. We evaluated the impacts on WCR adult fitness associated with the dsRNA-mediated knockdown of Ago1, Ago2, Dcr1, Dcr2, and Drosha genes. Among these putative RNAi pathway genes, only the knockdown of Ago1 incurred significant fitness costs such as reduced survival and oviposition rate, as well as decreased egg viability. The present study, to our knowledge, represents the first report showing that Ago1 is critical to the survival of insect adults. Our findings suggest that Ago1 plays an essential role in broader life stages of an insect than previously thought. Importantly, since fitness costs were not observed, downregulation or loss of function of RNAi pathway genes such as Ago2 or Dcr2 may confer resistance to pest control measures that rely on the normal functions of these genes. However, the precise roles of these genes under field conditions (i.e., in the presence of possible viral pathogens) requires further investigation.

Highlights

RNA interference (RNAi) can be broadly defined as regulation of gene expression directed by small non-coding RNA molecules [1]
Argonaute 1 (Ago1), Dicer 1 (Dcr1), and Drosha are likely involved in the miRNA pathway; Ago3 and Aubergine are likely involved in the Piwi-interacting RNA (piRNA) pathway
Because we were interested in evaluating the possibility that western corn rootworm (WCR) may develop resistance to doublestranded RNA (dsRNA)- or miRNA-based pest control measures through downregulating the expression of RNAi pathway components (i.e., Ago1, Argonaute 2 (Ago2), Dcr1, Dicer 2 (Dcr2), and Drosha), we performed loss-of-function analyses on these putative RNAi pathway genes using an RNAi-based approach

Summary

Introduction

RNA interference (RNAi) can be broadly defined as regulation of gene expression directed by small non-coding RNA molecules [1]. The dsRNA pathway is induced by endogenous or exogenous dsRNA that are recognized by Dicer, a RNase III protein, which cuts these molecules into double stranded small interfering RNAs (siRNAs) of 21–23 nucleotides [15]. Using siRNAs as guides, RISCs identify target mRNAs that are cleaved by RISC-bound Argonaute, achieving post-transcriptional gene silencing [12, 16,17,18]. The miRNA pathway involves endogenous miRNAs that are processed by the RNase III-like enzyme Drosha in the nucleus and a Dicer protein in the cytosol. The mature miRNAs are loaded onto an Argonaute family protein and together, they regulate the expression of target mRNAs by blocking ribosomes and reducing mRNA translation elongation. The miRNA-Ago complex may promote mRNA decay by affecting the polyadenylation status of the mRNAs [19, 20]

Methods

Results

Conclusion