Data from: 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 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.多数基因可通过选择性外显子使用方式拓展编码容量。编码昆虫烟碱型乙酰胆碱受体（nicotinic acetylcholine receptor, nAChR）α6亚基的基因便是其中一例——该基因作为生物杀虫剂多杀菌素（spinosad）的作用靶点，通过互斥外显子的可变剪接拓展蛋白质多样性。本研究发现，番茄潜叶蛾（Tuta absoluta）对多杀菌素的抗性与Taα6基因剪接的异常调控相关，由此产生了一种由外显子跳跃介导的新型杀虫剂抗性类型。对经多杀菌素筛选及未筛选的番茄潜叶蛾菌株的α6亚基互补DNA（complementary DNA, cDNA）进行测序，结果显示筛选菌株的全部Taα6转录本均缺失外显子3；通过比对基因组DNA与信使RNA（messenger RNA, mRNA）序列证实，该现象由外显子跳跃导致。存活生物测定结果表明，外显子跳跃与多杀菌素抗性共分离；以改造后的人源烟碱型乙酰胆碱受体α7作为昆虫α6的模型，对该变异进行功能表征后证实，外显子3对受体功能不可或缺，因而直接影响对多杀菌素的敏感性。DNA与RNA测序分析表明，外显子跳跃并非由内含子或外显子区域的顺式调控元件发生遗传变异所致，而是与外显子3a下游的一处表观遗传修饰，以及在可变剪接调控中发挥已知功能的反式作用蛋白的表达量变化相关。本研究结果证实，α6基因通过可变剪接产生转录本多样性的内在能力，可在抗性演化过程中被轻易利用；同时明确了外显子跳跃是一种赋予杀虫剂抗性的分子变异类型。