Insecticide resistance mediated by an exon skipping event

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 s...

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Published in	Molecular ecology Vol. 25; no. 22; pp. 5692 - 5704
Main Authors	Berger, Madeleine, Puinean, Alin Mirel, Randall, Emma, Zimmer, Christoph T., Silva, Wellington M., Bielza, Pablo, Field, Linda M., Hughes, David, Mellor, Ian, Hassani-Pak, Keywan, Siqueira, Herbert A. A., Williamson, Martin S., Bass, Chris
Format	Journal Article
Language	English
Published	England Blackwell Publishing Ltd 01.11.2016 John Wiley and Sons Inc
Subjects	alpha7 Nicotinic Acetylcholine Receptor - genetics Alternative Splicing Animals Bioassays complementary DNA Deoxyribonucleic acid DNA Drug Combinations epigenetics evolution Exons Genetics Humans Insecta - genetics insecticide resistance Insecticide Resistance - genetics Insecticides Insects leafminers Lycopersicon esculentum Macrolides messenger RNA nicotinic acetylcholine receptor nicotinic receptors Original ORIGINAL ARTICLES Protein expression regulatory sequences spinosad Tomatoes Tuta absoluta spinosad alternative splicing Tuta absoluta nicotinic acetylcholine receptor
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ISSN	0962-1083 1365-294X 1365-294X
DOI	10.1111/mec.13882

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Summary:	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.
Bibliography:	Biotechnology and Biological Sciences Research Council of the UK - No. BB/G023352/1 ArticleID:MEC13882 REA - No. PIRSES-GA-2012 - 318246 BBSRC - No. 1096240 Fig. S1 (A) cDNA and predicted protein sequence of the Tuta absoluta nAChR α6 subunit (with exon 3a and 8a). (B) Alternative exons 3a and 3b and exons 8a, 8b and 8c of the Tuta absoluta nAChR α6 subunit.Fig. S2 Comparison of the amino acid sequence of the Tuta absoluta α6 subunit with that of Bombyx mori (GenBank ABL67934.1) and Plutella xylostella (GenBank GU207835.1).Fig. S3 Alignment of a 4000 bp genomic sequence encompassing the Taα6 exon 3 cluster from the Spin, SpinSel and TA4 strains.Fig. S4 Amino acid alignment of the exon 2-4 region of Taα6 from 21 sequenced clones generated from four different Tuta absoluta spinosad susceptible strains (TA1, TA3, TA4, GA).Fig. S5 Alignment of contigs encoding suppressor of white apricot from two de novo Tuta absoluta transcriptome assemblies.Table S1 Exon three usage in sequenced clones of Taα6 in the Spin and SpinSel strains.Table S2 Larval mortality of F2 hybrids of the Spin and SpinSel strains to a discriminating dose of spinosad (120 mg/L).Table S3 Table of CpG sites and corresponding methylation ratios in the exon 3 cluster for the SpinSel, Spin and TA4 Tuta absoluta strains as revealed by bisulphite sequencing.Table S4 Transcripts identified by RNAseq as significantly differentially expressed between the Spin and SpinSel Tuta absoluta strains using assembly one as a reference (see methods).Table S5 Transcripts identified by RNAseq as significantly differentially expressed between the Spin and SpinSel Tuta absoluta strains using assembly two as a reference (see methods).Table S6 Expression of isoforms encoding suppressor of white apricot in the Tuta absoluta transcriptome.Table S7 Sequences of primers used in this study.Table S8 Sequence of primers used for Bisulphite PCR.Table S9 Assembly statistics of Tuta absoluta transcriptomes. European Union Seventh Framework Programme FP7/2007-2013 istex:D04C5D9C21B152A129109B5CAA1FD8D4A0839C6C ark:/67375/WNG-GVCHRVW1-K ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23
ISSN:	0962-1083 1365-294X 1365-294X
DOI:	10.1111/mec.13882