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.

众多基因可通过可变外显子使用提升编码能力。编码昆虫烟碱型乙酰胆碱受体（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。本研究证实，番茄潜叶蛾（Tuta absoluta）对多杀菌素的抗性与Taα6的剪接调控异常相关，由此产生一种由外显子跳跃介导的新型杀虫剂抗性机制。对经多杀菌素筛选及未筛选的番茄潜叶蛾品系的α6亚基互补DNA（complementary DNA, cDNA）进行测序发现，筛选品系的所有Taα6转录本均缺失外显子3；对比基因组DNA与mRNA序列可知，该现象源于外显子跳跃。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；利用改造后的人源nAChR α7（昆虫α6的模型体系）对该变异进行功能表征，证实外显子3对受体功能至关重要，进而影响多杀菌素敏感性。DNA与RNA测序分析显示，外显子跳跃并非源于内含子或外显子顺式调控元件的遗传变异，而是与外显子3a下游的单一处表观遗传修饰，以及已知参与可变剪接调控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，可在抗性进化过程中被轻易利用，并明确外显子跳跃是赋予杀虫剂抗性的分子变异。