Functional characterization of four antenna-enriched odorant binding proteins in Rhaphuma horsfieldi reveals the importance of RhorOBP1 in odorant recognition and insecticide resistance.

The cerambycid beetles are key players for the sustenance of biodiversity in the forest ecosystem, but in most cases are well known due to their harmfulness to agricultural and forest plants. Here, we characterized the odorant binding protein (OBP) gene family in Rhaphuma horsfieldi, emphasizing the roles of RhorOBP1 in odorant reception and insecticide sequestering. A homology-based search led to the identification of 35 RhorOBP genes with a major distribution in the Minus-C OBPs clade (21/35 genes). Expression profiles showed that RhorOBP1-RhorOBP4 had the abundant expression in antennae. Binding assays revealed that the four RhorOBPs exhibited diverse odorant response profiles tuned differentially to various classes of plant odorants, comprising walnut-derived host volatiles and ordinary floral scents. Two broadly tuned RhorOBP1 and RhorOBP2 exhibited different chain length-dependent binding properties to 10C12C alcohols, aldehydes or acetates. Compared with other three proteins, RhorOBP1 reduced the binding to ligands with high affinities at pH 5.0 (1.27-6.72-fold differences relative to pH 7.4). Molecular docking and point-mutation experiments confirmed that Ser107, Tyr118, Tyr119 and Phe120 situated in the binding pocket of RhorOBP1 were critical determinants for the recognition of 14, 15, 10 and 10 compounds, respectively. On the other hand, RhorOBP1 could strongly bind six insecticides, particularly chlorpyrifos (dissociation constant, K = 3.69 ± 0.74 μM). This study has provided insights into different binding properties of four antenna-enriched RhorOBPs in R. horsfieldi and identifies a dual role of RhorOBP1 in the binding of odorants and insecticides.