Abstract：
Antennae and legs (primarily the tarsal segments) of insects are the foremost sensory organs that contact a diverse range of toxic chemicals including insecticides. Binding proteins expressed in the two tissues are potential molecular candidates serving as the binding and sequestering of insecticides, like chemosensory proteins (CSPs). Insect CSPs endowed with multiple roles have been suggested to participate in insecticide resistance, focusing mainly on moths, aphids and mosquitos. Yet, the molecular underpinnings underlying the interactions of cerambycid CSPs and insecticides remain unexplored. Here, we present binding properties of three antenna- and tarsus-enriched RhorCSPs (RhorCSP1, CSP2 and CSP3) in Rhaphuma horsfieldi to eight insecticide classes totaling 15 chemicals. From the transcriptome of this beetle, totally 16 CSP-coding genes were found, with seven full-length sequences. In phylogeny, these RhorCSPs were distributed dispersedly in different clades. Expression profiles revealed the abundant expression of RhorCSP1, CSP2 and CSP3 in antennae and tarsi, thus as representatives for studying the protein-insecticide interactions. Binding assays showed that the three RhorCSPs were tuned differentially to insecticides but exhibited the highest affinities with hexaflumuron, chlorpyrifos and rotenone (dissociation constants <13 μM). In particular, RhorCSP3 could interact strongly with 10 of tested insecticides, of which four residues (Tyr25, Phe42, Val65 and Phe68) contributed significantly to the binding of six, four, three and four ligands, respectively. Of these, the binding of four mutated RhorCSP3s to a botanical insecticide rotenone was significantly weakened compared to the wildtype protein. Furthermore, we also evidenced that RhorCSP3 was a broadly-tuned carrier protein in response to a wide variety of plant odorants outside insecticides. Altogether, our findings shed light on different binding mechanisms and odorant-tuning profiles of three RhorCSPs in R. horsfieldi and identify key residues of the RhorCSP3-insecticide interactions.
摘要：
昆虫的触角和腿（主要是骨部分）是接触各种有毒化学物质（包括杀虫剂）的最重要的感觉器官。在两个组织中表达的结合蛋白是潜在的分子候选物，用作杀虫剂的结合和螯合。比如化学感应蛋白(CSP)。具有多种作用的昆虫CSP已被建议参与杀虫剂抗性，主要关注飞蛾，蚜虫和蚊子.然而，cerambycidCSP和杀虫剂相互作用的分子基础仍未被探索。这里,我们介绍了Rhaphumahorsfieldi中三种富含触角和tarsus的RhorCSP（RhorCS1，CSP2和CSP3）对八种杀虫剂类别的结合特性，总计15种化学物质。从这只甲虫的转录组中，共发现16个CSP编码基因，有七个全长序列。在系统发育中，这些RhorCSP分散分布在不同的进化枝中。表达谱揭示了RhorCSP1、CSP2和CSP3在触角和tarsi中的大量表达,因此作为研究蛋白质-杀虫剂相互作用的代表。结合测定表明，三种RhorCSP对杀虫剂进行了不同的调整，但与氟铃脲的亲和力最高，毒死蜱和鱼藤酮(解离常数<13μM)。特别是,RhorCSP3可以与10种测试的杀虫剂强烈相互作用，其中4个残基(Tyr25、Phe42、Val65和Phe68)对6个残基的结合有显著贡献,四,三个和四个配体，分别。其中,与野生型蛋白相比，四个突变的RhorCSP3与植物杀虫剂鱼藤酮的结合显着减弱。此外，我们还证明了RhorCSP3是一种广泛调节的载体蛋白，可响应杀虫剂以外的多种植物气味剂。总之,我们的发现揭示了三种RhorCSP在R.horsfieldi中的不同结合机制和气味调节特征，并确定了RhorCSP3-杀虫剂相互作用的关键残基。
