Insecticide resistance mediated by an exon skipping event

Madeleine Berger,Linda M Field,David Hughes,Herbert A A Siqueira,Pablo Bielza,Alin Mirel Puinean,Keywan Hassani‐Pak,Christoph T Zimmer,Martin S Williamson,Wellington M Silva,Ian Mellor,Emma Randall,Chris Bass

doi:10.1111/mec.13882

Abstract

Many genes increase coding capacity by alternate exon usage. The gene encoding the insect nicotinic acetylcholine receptor (nAChR) α6 subunit, target of the bio‐insecticide spinosad, is one example of this and expands protein diversity via alternative splicing of mutually exclusive exons. Here, we show that spinosad resistance in the tomato leaf miner, Tuta absoluta is associated with aberrant regulation of splicing of Taα6 resulting in a novel form of insecticide resistance mediated by exon skipping. Sequencing of the α6 subunit cDNA from spinosad selected and unselected strains of T. absoluta revealed all Taα6 transcripts of the selected strain were devoid of exon 3, with comparison of genomic DNA and mRNA revealing this is a result of exon skipping. Exon skipping cosegregated with spinosad resistance in survival bioassays, and functional characterization of this alteration using modified human nAChR α7, a model of insect α6, demonstrated that exon 3 is essential for receptor function and hence spinosad sensitivity. DNA and RNA sequencing analyses suggested that exon skipping did not result from genetic alterations in intronic or exonic cis‐regulatory elements, but rather was associated with a single epigenetic modification downstream of exon 3a, and quantitative changes in the expression of trans‐acting proteins that have known roles in the regulation of alternative splicing. Our results demonstrate that the intrinsic capacity of the α6 gene to generate transcript diversity via alternative splicing can be readily exploited during the evolution of resistance and identifies exon skipping as a molecular alteration conferring insecticide resistance.

Highlights

Alternative splicing enables a single gene to code for multiple proteins and is a key driver of proteome diversity
We report the discovery of a novel mechanism of spinosad resistance in T. absoluta that exploits the intrinsic capacity of the nicotinic acetylcholine receptor (nAChR) a6 gene to generate transcript diversity via alternative splicing and describe an example of insecticide resistance mediated by an exon skipping event
piperonyl butoxide (PBO) was selected as an inhibitor of cytochrome P450s and esterases as previous biochemical characterization of spinosad resistance in T. absoluta have implicated these enzyme systems in resistance (Reyes et al 2012)

Summary

Introduction

Alternative splicing enables a single gene to code for multiple proteins and is a key driver of proteome diversity. Several insect species have developed resistance to this compound and a range of genetic alterations have been described in the a6 subunit of spinosad resistant strains including point mutations (Puinean et al 2013; Bao et al 2014) and other, more profound, alterations that result in truncated nonfunctional proteins (Baxter et al 2010; Rinkevich et al 2010; Hsu et al 2012). Such loss-of-function mutations do not result in catastrophic loss of fitness as insects without a functional copy of the a6 gene are still viable (Perry et al 2007)

Methods

Results

Conclusion