期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2016
卷号：113
期号：51
页码：14692-14697
DOI：10.1073/pnas.1618258113
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceAn old enigma in insect toxicology, the mode of action (MoA) of selective chitin biosynthesis inhibitors in arthropods, is resolved. Benzoylureas, buprofezin, and etoxazole share a MoA by directly interacting with chitin synthase 1. The finding that a single mutation confers striking levels of insecticide resistance against three putative different MoAs has important ramifications on resistance management strategies and rational use of insecticides against major agricultural pests and vectors of human diseases. Our results also show that CRISPR/Cas9-mediated gain-of-function mutations in single-copy genes of highly conserved target sites in arthropods provide opportunities for comprehensive insecticide resistance investigations across species boundaries and against several insecticide classes. Despite the major role of chitin biosynthesis inhibitors such as benzoylureas (BPUs) in the control of pests in agricultural and public health for almost four decades, their molecular mode of action (MoA) has in most cases remained elusive. BPUs interfere with chitin biosynthesis and were thought to interact with sulfonylurea receptors that mediate chitin vesicle transport. Here, we uncover a mutation (I1042M) in the chitin synthase 1 (CHS1) gene of BPU-resistant Plutella xylostella at the same position as the I1017F mutation reported in spider mites that confers etoxazole resistance. Using a genome-editing CRISPR/Cas9 approach coupled with homology-directed repair (HDR) in Drosophila melanogaster, we introduced both substitutions (I1056M/F) in the corresponding fly CHS1 gene (kkv). Homozygous lines bearing either of these mutations were highly resistant to etoxazole and all tested BPUs, as well as buprofezin--an important hemipteran chitin biosynthesis inhibitor. This provides compelling evidence that BPUs, etoxazole, and buprofezin share in fact the same molecular MoA and directly interact with CHS. This finding has immediate effects on resistance management strategies of major agricultural pests but also on mosquito vectors of serious human diseases such as Dengue and Zika, as diflubenzuron, the standard BPU, is one of the few effective larvicides in use. The study elaborates on how genome editing can directly, rapidly, and convincingly elucidate the MoA of bioactive molecules, especially when target sites are complex and hard to reconstitute in vitro.
