Key Laboratory of Forest Disaster Warning and Control of Yunnan Province, Southwest Forestry University, Kunming 650224, China.
Key Laboratory of Forest Disaster Warning and Control of Yunnan Province, Southwest Forestry University, Kunming 650224, China.
Pestic Biochem Physiol. 2024 Feb;199:105797. doi: 10.1016/j.pestbp.2024.105797. Epub 2024 Jan 19.
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.
昆虫的触角和腿（主要是跗节）是接触包括杀虫剂在内的各种有毒化学物质的最重要的感觉器官。两种组织中表达的结合蛋白是作为杀虫剂结合和隔离的潜在分子候选物，如化学感觉蛋白（CSPs）。具有多种作用的昆虫 CSP 被认为参与了杀虫剂抗性，主要集中在鳞翅目、蚜虫和蚊子上。然而，鳞翅目昆虫 CSP 与杀虫剂相互作用的分子基础仍未得到探索。在这里，我们展示了 3 种触角和跗节富含 RhorCSPs（RhorCSP1、CSP2 和 CSP3）在 Rhaphuma horsfieldi 中对 8 种杀虫剂类别的结合特性，共 15 种化学物质。从这种甲虫的转录组中，总共发现了 16 个 CSP 编码基因，其中有 7 个全长序列。在系统发育中，这些 RhorCSPs 分散分布在不同的分支中。表达谱显示，RhorCSP1、CSP2 和 CSP3 在触角和跗节中大量表达，因此可以作为研究蛋白质-杀虫剂相互作用的代表。结合实验表明，这 3 种 RhorCSP 对杀虫剂的结合具有不同的选择性，但对六氟隆、毒死蜱和鱼藤酮的亲和力最高（解离常数<13 μM）。特别是，RhorCSP3 可以与测试的 10 种杀虫剂中的 10 种强烈相互作用，其中 4 个残基（Tyr25、Phe42、Val65 和 Phe68）分别对 6、4、3 和 4 种配体的结合有显著贡献。其中，与野生型蛋白相比，4 种突变的 RhorCSP3 对植物杀虫剂鱼藤酮的结合显著减弱。此外，我们还证明 RhorCSP3 是一种广泛调节的载体蛋白，能够对各种植物气味剂（而非杀虫剂）产生反应。总之，我们的研究结果揭示了 R. horsfieldi 中 3 种 RhorCSPs 的不同结合机制和气味调节特性，并确定了 RhorCSP3 与杀虫剂相互作用的关键残基。
