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

Insect culture

The collection of samples referred to the method of Wang et al. (2020), which is specifically described as follows. In April 2019, Fraxinus trees affected by the larvae of A. planipennis were felled in the suburban ash forests of Beijing and cut into approximately 50 cm segments. These segments were then brought indoors and placed within rearing cages. After the larvae pupated and the adults emerged, they were transferred to rearing boxes and fed with ash tree leaves for experimental purposes. The adult rearing conditions were maintained at a temperature of 25 ± 0.5 °C, a relative humidity of 60% ± 5%, and a photoperiod of 16L:8D.

RNA isolation and first-strand cDNA synthesis

Tissues from 100 antennae, six heads (without antennae), and four bodies (a mixture of thoraces, abdomens, legs, and wings) were dissected from 1- to 3-day-old female and male beetles, separately. RNA extraction and reverse transcription were performed according to the method of Wang et al. (2021), as detailed below. The tissues were immediately frozen in liquid nitrogen and stored at −80 °C until RNA isolation. Total RNA from different tissues was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the protocol. The integrity and purity of RNA were assessed using 1.2% agarose gel electrophoresis and a NanoPhotometer N60 (Implen, München, Germany), respectively. First-strand cDNA was synthesized from 1 µg of RNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Beijing, China), following the manufacturer’s instructions.

Sequence and multiple sequence alignment

Gene-specific primers (Table S1) for amplifying the open reading frame (ORF) of the A. planipennis chemosensory protein 4 (AlpaCSP4) were designed using the Primer 3 program (http://primer3.ut.ee/) according to the reference sequence (accession: XM_018478165.1). Cloning of the sequences was carried out following the method described by Wang et al. (2021). Specifically, polymerase chain reaction (PCR) amplification was conducted using one unit of KOD DNA polymerase (Taihe, Beijing, China) and 200 ng of cDNA template. The PCR cycling parameters were as follows: initial denaturation at 94 °C for 2 min, followed by 30 cycles consisting of denaturation at 94 °C for 20 s, annealing at 58 °C for 30 s, and extension at 68 °C for 1 min. A final extension step was performed at 68 °C for 5 min. The resulting PCR products were inserted into the pClone EZ-Blunt vector (Taihe, Beijing, China). Subsequently, the cloned products were sequenced using the M13 primer for further analysis.

The amino acid sequence of AlpaCSP4 was aligned with those of CSPs from Agrilus mali (AXG21596.1), Helicoverpa armigera (AIW65103.1), Glossina morsitans (CBA11330.1), Encarsia formosa (QJT73564.1), and Subpsaltria yangi (AXY87875.1) using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). The result of the multiple sequence alignment was visualized using ESPript 3.0 (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi).

Reverse transcription PCR

RT-PCR was completed according to the method of Wang et al. (2020), as follows. The spatial distributions of AplaCSP4 across the head (without antennae), body (mixtures of thoraxes, abdomens, legs, and wings), and antennae in males and females were characterized by semiquantitative RT-PCR, employing Taq DNA polymerase (Biomed, Beijing, China). Each PCR was performed in 25 µL reaction volume, containing 200 ng of cDNA from different tissues. The thermal cycling conditions for the PCR were initiated with an initial denaturation at 94 °C for 4 min, followed by 30 cycles comprising a denaturation step at 94 °C for 30 s, an annealing step at 55 °C for 30 s, and an extension step at 72 °C for 45 s. A final extension was conducted at 72 °C for 5 min to complete the amplification process. The integrity of the cDNA samples was assessed using β-actin (accession: XM_018479924.1) as a reference gene. For each amplification, negative controls with a water template were included to ensure specificity. The resulting amplification products were electrophoresed on 1.2% agarose gels to verify their size and integrity. To authenticate the identity of each gene, one amplification product per gene was subjected to sequencing. The gene-specific primers utilized in these reactions are detailed in Table S1.

Quantitative real-time PCR

Quantitative real-time reverse transcription PCR (qRT-PCR) was performed following the protocol described by Wang et al. (2021), with specific details as follows. The relative mRNA expression level of AplaCSP4 in the antennae of males and females was measured by qRT-PCR. The qRT-PCR was conducted using the ABI Prism 7500 System (Applied Biosystems, Carlsbad, CA, USA) and SYBR Green SuperReal PreMix Plus (TianGen, Beijing, China) in a 20 µL reaction mixture. The mixture comprised 10 µL of 2 × SuperReal PreMix Plus, one µL (200 ng) of sample cDNA, 0.4 µL of 50 × ROX Reference Dye, 0.4 µL of each forward and reverse primers, and 7.8 µL of sterilized ultrapure water. Each qRT-PCR experiment was performed with three biological replicates, and each replicate was assessed in triplicate. Both β-actin and elongation factor 1-α (EF-1α, accession: XM_018476784.2) EF-1α served as endogenous controls to normalize the target gene expression and account for sample-to-sample variability. Primers for performing the qRT-PCR were listed in Table S1. The specificity of each primer set was confirmed through melting curve analysis, while the amplification efficiency was determined by evaluating the standard curves generated from a 5-fold dilution series of cDNA. The expression level of AplaCSP4 in the antennae of female was compared relative to the antennae of male using the 2^−ΔΔCT method. The AplaCSP4 gene expression level in antennae of male and female individuals was compared using T-test of GraphPad prime 10 software (GraphPad Software, San Diego, CA, USA).

Expression and purification of recombinant AplaCSP4

AplaCSP4 was amplified by PCR using specific primers (Table S1). The resulting PCR products were inserted into a T vector (Taihe, Beijing, China) and subsequently cloned into the bacterial expression vector pET30a (+) (Novagen, Madison, WI, USA), and the sequence was verified by sequencing. The plasmids of harboring the correct insert sequence were transferred into BL21 (DE3) competent cells for subsequent protein expression. Protein expression was induced in LB medium at 18 °C for 16 h by the addition of one mM isopropyl-β-D-thiogalactopyranoside (IPTG). The bacterial cultures were harvested through centrifugation and subsequently resuspended in a 50 mM Tris–HCl buffer (pH 7.4). Following sonication and centrifugation, the recombinant proteins were predominantly present in the supernatant, and were purified by a standard Ni column (GE Healthcare, Waukesha, WI, United States). The His-tag was selectively cleaved from the recombinant proteins using a recombinant enterokinase (Novagen, Madison, WI, USA) according to the manufacturer’s protocol. Purified AplaCSP4 was dialyzed in a 50 mM Tris, and the protein concentration was then accurately determined using the Bradford protein assay.

Fluorescence competitive binding assays

Fluorescence competitive binding assays were conducted according to the method described by Wang et al. (2021). Specifically, the binding affinities of AplaCSP4 for 43 volatile organic compounds were determined using F-380 fluorescence spectrophotometer (Tianjin, China) with a 10 nm slits and a 1 cm light path. These 43 substances are common green leaf volatiles, among which 16 volatiles originate from the ash tree. As the fluorescent probe, N-phenyl-1-naphthylamine (1-NPN) was excited at the wavelength of 337 nm, and emission spectra were recorded between 390 and 530 nm. Utilizing N-phenyl-1-naphthylamine (1-NPN) as the fluorescent probe, excitation was set at 337 nm, with emission spectra captured in the range from 390 nm to 530 nm. To quantify the binding affinity of 1-NPN to AplaCSP4, a 2 µM solution of the purified protein in 50 mM Tris–HCl buffer (pH 7.4) was titrated with aliquots of 1-NPN (one mM in methanol) to achieve final concentrations between 2 and 16 µM.

Competitive binding assays were conducted by titrating a solution containing both AplaCSP4 protein and 1-NPN, each at a concentration of two µM, with aliquots of a 1 mM methanol solution of the ligand, achieving final concentrations ranging from 2 to 20 mM. The dissociation constants of the competitors were determined using the equation K_D = IC₅₀/(1 + [1-NPN]/K_1−NPN), in which IC₅₀ represents the concentration of the ligand required to reduce the initial fluorescence intensity of 1-NPN by half, [1-NPN] denotes the free concentration of 1-NPN, and K_1−NPN indicates the dissociation constant characterizing the AplaCSP4/1-NPN complex. The experiments were executed in triplicate for ligands that demonstrated significant binding affinity, while those ligands exhibiting minimal binding were subjected to a single experiment.

Molecular docking and dynamic simulation

The signal peptide of AplaCSP4 was removed and subsequently used to construct a 3D protein model using AlphaFold3 (https://alphafoldserver.com/). The AplaCSP4 structure minimization was performed using the ff14sb force field on Wemol (https://wemol.wecomput.com/ui/#/). A Ramachandran plot was generated using the online tool PROCHECK to assess the quality of the constructed 3D model. Molecular docking of AplaCSP4 with farnesene was performed using CB-Dock 2 (https://cadd.labshare.cn/cb-dock2/php/index.php). The conformation with the highest score is used for molecular dynamics simulation. The GROMACS 2024 was used to perform the protein-ligand complex molecular dynamics simulation, and the AMBER03 force field was selected in conjunction with the TIP3P water model. Energy minimization was performed using the steepest descent method (Steep), with the energy convergence threshold set at 10 kJ/mol/nm. The simulation was conducted under the NPT ensemble, with a coupling reference pressure of one bar and a temperature maintained at 300 K. The simulation time step was 0.002 ps, and the total simulation duration was 200 ns. MMPBSA was used to calculate the binding energy. With different initial velocity random assignments, the molecular dynamics simulation was repeated using the same parameters. The conformation of the last frame was used for the analysis of the interactions between the protein and the ligand by applying PyMOL 2.6.0 (https://pymol.org/) and Maestro 14.1 (https://www.schrodinger.com/).

Sequence analysis of AplaCSP4

The nucleotide sequence of AplaCSP4 was verified by molecular cloning and sequencing. Analysis of the AplaCSP4 sequence revealed a full-length ORF consisting of 390 nucleotides that encode 130 amino acid residues (Fig. S1). The results of multiple sequences alignment displayed that the AplaCSP4 possessed the typical chemosensory protein conserved domain of C1-X6-C2-X18-C3-X2-C4 and contained seven α helices (Fig. 1).

The expression level of AplaCSP4 in various tissues

RT-PCR was employed to assess the expression of AplaCSP4 in the antennae, heads, and bodies of both female and male individuals. The findings revealed that AplaCSP4 was specifically and prominently expressed in the antennae of both sexes (Fig. 2). The qRT-PCR results demonstrated that the mRNA expression level of AplaCSP4 in female antennae was 1.91-fold higher than that in male antennae (P = 0.0002) (Fig. 2).

Binding characteristic of recombinant AplaCSP4

AplaCSP4 was expressed in a bacterial system and subsequently utilized to screen for potential ligands of AplaCSP4. The protein was purified using affinity chromatography on Ni columns and employed for ligand-binding experiments. The size and purity of the recombinant protein were assessed by SDS-PAGE (Fig. 3).

The binding affinities of AplaCSP4 for 43 volatile compounds were quantified using fluorescence competition binding assays, employing 1-NPN as a fluorescent probe. The binding affinity constant (K_D) between 1-NPN and AplaCSP4 was determined to be 9.36 µM, thereby validating 1-NPN as a suitable fluorescent reporter (Fig. 4). The binding experiments demonstrated that AplaCSP4 exhibited binding interactions with 11 volatile compounds, encompassing esters, alkanes, terpenes, terpenoids, and terpenols, with K_D values ranging from 0.25 to 11.47 µM. Among the 11 plant volatiles, six are derived from the ash tree, including dodecane, myrcene, ocimene, farnesene, (+)-limonene, and nerolidol. Notably, farnesene displayed the strongest binding affinity, with a K_D value of 0.25 µM (Fig. 4 and Table 1). Additionally, AplaCSP4 exhibited moderate binding affinities with cis-3-hexenyl benzoate, n-octane, decane, dodecane, myrcene, nerolidol, and β-ionone, with K_D values ranging from 1.38 to 9.90 µM (Fig. 4 and Table 1). Conversely, ocimene, (+)-limonene and hexyl butyrate exhibited relatively low binding affinities, with K_D values of 10.43, 11.34 and 11.47 µM, respectively (Fig. 4 and Table 1).

Molecular docking and dynamic simulation

The Ramachandran plot was used to assess the rationality of the minimized structure of AplaCSP4 and revealed that 100% of residues were in the allowed region (Fig. S2), indicating the predicted model of AplaCSP4 was reasonable and reliable. The AplaCSP4 contained 7 helices (α 1–7) (Fig. S2).

The volatile compound farnesene, which exhibited the highest binding affinity in the fluorescence competition binding assay, was selected for molecular docking with AplaCSP4. The conformation with the highest docking score was subsequently employed for molecular dynamics (MD) simulation with dual-replicate. The results of the two molecular dynamics simulations indicate that the root mean square deviation (RMSD) reaches a stable state after 125 ns (Fig. 5). Additionally, the root mean square fluctuations (RMSF) were displayed in Fig. 5. The results of the MMPBSA analysis showed that the binding energy between AplaCSP4 and farnesene was −31.830 ± 2.015 kcal/mol and −32.585 ± 2.011 kcal/mol in dual-replicate molecule dynamics simulations (Table 2). Both molecular dynamics simulations demonstrated that farnesene binds within the same hydrophobic binding pocket of AplaCSP4 (Fig. 6), with binding site coordinates at (30.558, 30.106, 31.277) and (31.075, 29.304, 31.781), respectively. The energy contributions of the amino acid residues in the active site are depicted in Fig. 7, where van der Waals forces represent the predominant interaction forces between the amino acids and farnesene. The centroid distance between farnesene and binding pocket of AplaCSP4 was analyzed by two molecular dynamics simulations, and the results indicated that the average centroid distance of farnesene from AplaCSP4 active center was 0.13 ± 0.004 nm and 0.16 ± 0.004 nm in both simulations, and the average centroid distance of carbon atom between farnesene and the side chain of hydrophobic amino acids was 1.41 ± 0.003 nm and 1.39 ± 0.002 nm (Fig. 8), it suggested that the farnesene has stable interaction with AplaCSP4.