RNA interference and validation of reference genes for gene expression analyses using qPCR in southern pine beetle, Dendroctonus frontalis

Bethany R. Kyre; Thais B. Rodrigues; Lynne K. Rieske

doi:10.1038/s41598-019-42072-6

RNA interference and validation of reference genes for gene expression analyses using qPCR in southern pine beetle, Dendroctonus frontalis

Bethany R. Kyre, Thais B. Rodrigues, Lynne K. Rieske

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32 Scopus citations

Abstract

RNA interference (RNAi) is a highly specific gene-silencing mechanism that can cause rapid insect mortality when essential genes are targeted. RNAi is being developed as a tool for integrated pest management of some crop pests. Here we focus on an aggressive forest pest that kills extensive tracts of pine forests, the southern pine beetle (SPB), Dendroctonus frontalis. We sought to identify reference genes for quantitative real-time PCR (qPCR) and validate RNAi responses in SPB by mortality and gene silencing analysis. Using an adult beetle feeding bioassay for oral ingestion of dsRNA, we measured the expression and demonstrated knockdown of target genes as well as insect mortality after ingestion of target genes. Our study validates reference genes for expression analyses and demonstrates highly effective RNAi responses in SPB, with RNAi response to some target dsRNAs causing 100% beetle mortality after ingestion.

Original language	English
Article number	5640
Journal	Scientific Reports
Volume	9
Issue number	1
DOIs	https://doi.org/10.1038/s41598-019-42072-6
State	Published - Dec 1 2019

Bibliographical note

Publisher Copyright:
© 2019, The Author(s).

Funding

The authors thank David Coyle, Wood Johnson, and William Shepherd (USDA Forest Service) for assistance obtaining beetles, and Bruce Webb (University of Kentucky) for comments on an early version of this manuscript. This is publication number 18-08-070 from the Kentucky Agricultural Experimental Station and is published with the approval of the director. This work was supported by the University of Kentucky, the USDA Forest Service Forest Health Research and Education Center, and the Kentucky Agricultural Experiment Station under McIntire-Stennis 2351197000.

Funders	Funder number
USDA Forest Service Forest Health Research and Education Center
University of Kentucky
Kentucky Agricultural Experiment Station	2351197000

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abstract = "RNA interference (RNAi) is a highly specific gene-silencing mechanism that can cause rapid insect mortality when essential genes are targeted. RNAi is being developed as a tool for integrated pest management of some crop pests. Here we focus on an aggressive forest pest that kills extensive tracts of pine forests, the southern pine beetle (SPB), Dendroctonus frontalis. We sought to identify reference genes for quantitative real-time PCR (qPCR) and validate RNAi responses in SPB by mortality and gene silencing analysis. Using an adult beetle feeding bioassay for oral ingestion of dsRNA, we measured the expression and demonstrated knockdown of target genes as well as insect mortality after ingestion of target genes. Our study validates reference genes for expression analyses and demonstrates highly effective RNAi responses in SPB, with RNAi response to some target dsRNAs causing 100% beetle mortality after ingestion.",

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RNA interference and validation of reference genes for gene expression analyses using qPCR in southern pine beetle, Dendroctonus frontalis

Abstract

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