Functional characterization of antennae-enriched chemosensory protein 4 in emerald ash borer, <i>Agrilus planipennis</i>

<i>Agrilus planipennis</i> is an invasive species that inflicts substantial harm on ash trees (<i>Fraxinus</i> spp.) globally. Elucidating its olfactory mechanisms is essential for devising effective pest management approaches. In this research, we identified chemosensory protein 4 (AplaCSP4) in <i>A. planipennis</i>, which is highly expressed in the antennae of both male and female individuals. Notably, the mRNA expression level of <i>AplaCSP4</i> in females is 1.9 times higher than that in males. Fluorescence competition binding assays revealed that recombinant AplaCSP4 has a broad binding spectrum, capable of interacting with 11 compounds from various chemical classes such as esters, alkanes, terpenes, terpenoids, and terpenols. The dissociation constants (<i>K</i> _D) for these binding affinities range from 0.25 to 11.47 µM. AplaCSP4 shows binding affinity for volatiles from <i>Fraxinus</i> species, including dodecane, myrcene, ocimene, farnesene, (+)-limonene, and nerolidol, with the highest affinity observed for farnesene (<i>K</i> _D = 0.25 µM). Molecular docking and dynamics simulation were employed to elucidate the binding mode of farnesene, which exhibited the strongest binding affinity with AplaCSP4. The results indicated that farnesene binds within the hydrophobic pocket of AplaCSP4, with a binding energy of -31.830 ± 2.015 kcal/mol and -32.585 ± 2.011 kcal/mol in dual-replicate molecular dynamics simulations, and primarily driven by van der Waals interactions. Importantly, during the two molecular dynamics simulations, the centroid distances between farnesene and the key residues in the binding pocket of AplaCSP4 were maintained relatively stable. The combined results from <i>in vitro</i> experiments and computational modeling suggest that AplaCSP4 is critically involved in plant volatile detection. This study offers insights into the molecular basis of olfactory perception in <i>A. planipennis</i> and may provide a foundation for developing novel olfactory-based pest control strategies targeting chemosensory proteins.