Antenna-Specific TabsOBP45 and TabsOBP46 Mediate Plant Volatile Recognition in Tuta absoluta (Lepidoptera: Gelechiidae)

Abstract

The tomato leaf miner, Tuta absoluta (Lepidoptera: Gelechiidae), is a destructive pest of Solanaceae crops worldwide. Its olfactory system plays an important role in locating mating partners and recognizing host plants. Understanding its olfactory recognition mechanism, particularly the function of odorant-binding proteins (OBPs), may reveal potential targets for pest management. In this study, we characterized two antenna-specific OBPs, TabsOBP45 and TabsOBP46, which were identified from the T. absoluta genome. Sequence analysis revealed that both TabsOBPs belong to the classic OBP subfamily, which is characterized by the presence of six conserved cysteine residues and an N-terminal signal peptide. Both TabsOBPs showed predominant antennal expression in quantitative real-time PCR (qRT-PCR) assays, suggesting their key roles in olfactory perception. Fluorescence competitive binding assays with a total of 63 tested volatiles revealed that 13 compounds exhibited strong binding affinities (Ki < 22 µM) to TabsOBP45, with the highest binding affinity to β-ionone, β-caryophyllene, terpinolene, and cinnamaldehyde. Nine compounds showed strong binding affinities to TabsOBP46, with the strongest binding to 4-anisaldehyde, 4-methoxybenzaldehyde, cinnamaldehyde, and β-ionone. Molecular docking analysis revealed the key residues involved in β-ionone binding: TabsOBP45 interacted with ILE8, ALA9, PHE12, TRP37, ILE92, PHE94, THR115, and PHE118, while TabsOBP46 interacted with ILE8, PHE12, PHE36, TRP37, ILE92, LEU94, PHE118, and VAL134. These results provide new insights into the olfactory mechanism of T. absoluta and potential molecular targets for the development of olfactory-based pest control strategies.

1. Introduction

An accurate olfactory system plays a pivotal role in mediating various behaviors such as foraging, mating, and oviposition, in insects [1]. The sophisticated chemosensory process, which involves multiple functional proteins in a highly coordinated molecular network, enables insects to discriminate between thousands of volatile compounds [2]. Olfactory sensory neurons (OSNs) express a variety of olfactory-related functional proteins, including odorant binding proteins (OBPs), odorant receptors (ORs), chemosensory proteins (CSPs), gustatory receptors (GRs), ionotropic receptors (IRs), sensory neuron membrane proteins (SNMPs), and odorant degrading enzymes (ODEs) [3,4]. It is hypothesized that, once captured by the sensilla in the antennae, hydrophobic volatiles are initially bound by water-soluble OBPs or CSPs, which facilitate their transport through the lymph to the chemosensory receptors. The odorants are then recognized by ORs, GRs, or IRs, and converted into electrical signals. Finally, ODEs in the lymphatic cavity or sensory cells deactivate the odorants and terminate the signal [1,5].

During odor recognition, OBPs are responsible for capturing and transporting volatile odorants to chemosensory receptors [6]. Insect OBPs possess a barrel-shaped binding pocket with a central hydrophobic cavity that can specifically bind to hydrophobic volatile molecules [7]. These binding pockets, typically formed by conserved cysteine residues, vary in shape and polarity among insect species, conferring selective binding capacities for specific odorant molecules [8,9]. Based on the pattern of conserved cysteine residues, OBPs are generally classified into four subfamilies: classic, plus-C, minus-C, and atypical [6,10]. Among them, classic OBPs represent the most abundant and widespread subfamily in insects [10]. They are characterized by six conserved cysteine residues that form three disulfide bridges, which are essential for maintaining protein stability and creating a functional binding cavity [3,11]. In Lepidoptera, classic OBPs display antenna-specific expression patterns and show selective binding affinities for different host plant volatiles [12]. The structural diversity of their binding pockets allows them to recognize specific plant-derived volatiles, positioning them as key mediators in host plant selection behavior [13,14,15]. These structural and functional characteristics make classic OBPs promising targets for pest management strategies [12,16].

The tomato leaf miner, Tuta absoluta, is a highly destructive pest that threatens tomato production worldwide [17,18]. Its larvae infest the leaves, stems, and fruits of Solanaceae cultivars, resulting in significant losses in yield and quality [18]. It is estimated that the infestations of this pest cause annual losses of six billion USD. Over the past decade, T. absoluta has rapidly invaded more than 100 countries, covering approximately 2.8 million hectares [17,19]. Field observations and laboratory studies have shown that T. absoluta relies heavily on olfactory cues for host location, particularly volatile compounds emitted by tomato plants [20,21]. Several volatile compounds including β-phellandrene, 2-carene, α-pinene, limonene, and (E)-β-caryophyllene have been identified as primary cues that guide T. absoluta to its host plants [22,23,24]. Another document suggests that 1-nonanol and ethyl octanoate are potential attractants, while E,E-2,4-nonadienal and E-2-nonenal are repellents for T. absoluta [25]. In contrast, 1-fluorododecane, predominantly found in non-preferred hosts such as eggplant and datura, acts as a natural deterrent [23]. Essential oils from Ocimum species that contain thymol, p-cymene, and γ-terpinene exhibit promising repellent activity [26]. In addition, plant defense elicitors such as methyl jasmonate and methyl salicylate can induce changes in plant volatile profiles that significantly affect the host preference and oviposition behavior of T. absoluta [27,28], providing valuable insights for the development of sustainable management strategies against this pest.

Recent advances in genomic and molecular techniques have significantly improved our understanding of the olfactory system of T. absoluta. From the genome, 97 putative TabsOBPs have been identified, including a notable expansion of the plus-C subfamily [29]. Through transcriptional response analysis, TabsOBP45 and TabsOBP46 are significantly up-regulated in adults after exposure to the repellent plant Plectranthus tomentosa volatiles, showing higher expression levels compared to both pure air control and tomato volatiles treatment groups [29]. These two TabsOBPs are also highly expressed in the adult stage, suggesting their critical role in olfactory recognition [29]. Although these TabsOBPs have been identified and their developmental and volatile-induced expression patterns have been characterized, their critical binding affinities to plant volatiles remain uncharacterized. In this study, we aimed to determine the tissue-specific expression patterns and binding properties of two TabsOBPs, as well as predict the key amino residues that bind to plant volatiles. These results could improve our understanding of the odorant-binding properties of insect OBPs and provide a basis for the development of odorant-based management against T. absoluta.

2. Materials and Methods

2.1. Insect Rearing and RNA Extraction

Approximately 1000 larvae of T. absoluta were obtained from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences (Beijing, China). All developmental stages were maintained under the following conditions: 28 °C, 60–70% relative humidity (RH), and a 14:10 light:dark photoperiod. The larvae were reared on tomato seedlings until pupation. The pupae were separated by sex and individually housed in 1.5 mL centrifuge tubes until emergence [30]. Three-day-old female and male moths were dissected to collect heads, thoraxes, abdomens, legs, wings, and antennae. The number of individuals used for each tissue collection ranged from 15 to 250, depending on the size of tissue.

Total RNA was extracted from each sample using the Trizol Up reagent (TransGen Biotech, Beijing, China). The quality of collected RNA was evaluated using 1% agarose gel electrophoresis, and the concentration and purity were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Carlsbad, CA, USA). First-strand cDNA synthesis was performed using an All-in-One First Strand cDNA Synthesis SuperMix kit (TransGen Biotech, Beijing, China), following the manufacturer’s instructions. All tissue collections were performed in three biological replicates.

2.2. Sequence Characterization and Multiple Sequence Alignment of TabsOBP45 and TabsOBP46

The sequences of TabsOBP45 and TabsOBP46 were retrieved from the NCBI GenBank database (accession numbers KAJ2948746.1 and KAJ2948745.1, respectively). Signal peptides were predicted using SignalP-6.0 (https://services.healthtech.dtu.dk/services/SignalP-6.0/, accessed on 16 May 2024). The molecular weights and isoelectric points of the proteins were calculated using the ExPASy Compute pI/Mw tool (https://web.expasy.org/compute_pi/, accessed on 20 May 2024). Multiple sequence alignments of orthologous lepidopteran OBPs were conducted using MEGA7 software.

2.3. Tissue Expression Pattern of TabsOBP45 and TabsOBP46

The tissue-specific expression levels of TabsOBP45 and TabsOBP46 were determined using a QuantStudio 6 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and a TransStart Tip Green qPCR SuperMix Kit (TransGen Biotech, Beijing, China). Gene-specific primers (Table S1) were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Each 20 μL qRT-PCR reaction system contained 1 μL of diluted cDNA, 1.2 μL of forward and reverse primer mixture (10 μM), 10 μL of FastFire qPCR PreMix (TransGen Biotech), and 7.8 μL of double-distilled water. The qRT-PCR thermal cycling conditions were as follows: initial at 94 °C for 30 s, followed by 40 cycles (94 °C for 5 s, 55 °C for 15 s, and 72 °C for 10 s). TabsRPS13 and TabsTBP were used as reference genes for normalization [31]. The relative expression levels of TabsOBPs were calculated using the comparative 2^−ΔΔCT method [32]. Each sample was analyzed in three independent biological replicates and three technical replicates to ensure reproducibility. Reactions without a cDNA template were performed as negative controls in all assays.

2.4. Expression and Purification of TabsOBP45 and TabsOBP46

The coding sequences of TabsOBP45 and TabsOBP46 were cloned into the pCzn1 expression vector between the NdeI and XbaI restriction sites. The recombinant plasmids were subsequently transformed into Escherichia coli Rosetta (DE3) cells for expression. Positive clones were cultured in LB medium (containing 50 μg/mL ampicillin) at 37 °C and 220 rpm. When the culture reached an OD₆₀₀ of approximately 0.6, 1mM of isopropyl β-D-thiogalactoside (IPTG) solution was added to induce protein expression. After an overnight incubation at 15 °C, bacterial cells were harvested by centrifugation at 8000× g and 4 °C, and then resuspended in lysis buffer (pH 7.0). An empty vector control was included and processed under identical conditions.

After ultrasonic cell lysis, the lysate was centrifuged at 14,000 rpm for 20 min to separate the supernatant and inclusion bodies. For vitro protein refolding, inclusion bodies were washed with a buffer solution (20 mM Tris-HCl, 1 mM EDTA, 2 M urea, 1 M NaCl, and 1% Triton X-100, pH = 8). The washed pellets were then dissolved in a denaturing buffer (20 mM Tris-HCl, 5 mM DTT, 0.15 M NaCl, 8 M urea, pH = 8.0). The soluble proteins were then purified using a Ni-IDA 6FF Sefinose™ Resin Kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s protocol. The purity of the recombinant TabsOBP45 and TabsOBP46 was assessed by 12% SDS-PAGE, and protein concentrations were determined using the Bradford assay.

2.5. Fluorescence Competitive Binding Assays

The binding affinities of recombinant TabsOBP45 and TabsOBP46 to ligands were evaluated using a multifunctional microplate reader [33]. A total of 63 chemical compounds were tested, including sex pheromone components and volatiles identified from host plants, flowers, and microorganisms (Table S2) [14,19,20,22]. A 4,4′-Bis (1-anilinonaphthalene-8-sulfonate) (bis-ANS) was used as a fluorescent probe to assess binding activity. Stock solutions of bis-ANS and the test ligands (1 mM) were prepared in HPLC-grade methanol. To determine the binding properties of bis-ANS to each recombinant protein, a titration assay was conducted as previously described [33]. Briefly, a solution of TabsOBP45 or TabsOBP46 (2 μM, dissolved in PBS, pH = 7.4) was incrementally titrated with a bis-ANS solution (final concentration range: 2–20 μM). Fluorescence emission spectra were recorded from 490 to 600 nm upon excitation at 295 nm.

For the competitive binding assays, the mixtures containing 2 μM recombinant TabsOBP and 2 μM bis-ANS were titrated with the individual ligands (500 μM) until the fluorescence quenching reached a plateau. Each assay was performed in triplicate to ensure reproducibility. The dissociation constant (Ki), calculated by the formula: Ki = [IC₅₀]/(1 + [bis-ANS]/K_bis-ANS), was employed to evaluate the binding affinity between ligands and TabsOBPs [34]. In the formula, IC₅₀ is the ligand concentration that halves the initial fluorescence value, [bis-ANS] represents the free concentration of bis-ANS, and Kbis-ANS is the dissociation constant between TabsOBP and bis-ANS. Generally, ligands with Ki values lower than 22 μM are considered to have strong binding affinity to the protein [35].

2.6. Molecular Docking

The 3D structures of odorant ligands were obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/, accessed on 16 February 2025). The tertiary structures of TabsOBP45 and TabsOBP46 were predicted via homology modeling using the SWISS-MODEL server, with silkworm pheromone-binding protein (BmorPBP, PDB ID: 1DQE) and pheromone-binding protein 1 of Sesamia inferens (SinsPBP1, UniProt ID: A0A7D5YVB7) serving as templates, respectively. The quality and reliability of the predicted protein structures were evaluated using the SAVES v6.1 server (https://saves.mbi.ucla.edu/, accessed on 20 February 2025). We employed ERRAT for analyzing non-bonded atomic interactions and generated Ramachandran plots to assess the stereochemical quality of the protein structures by examining backbone dihedral angles of amino acid residues [14]. Structural visualization and docking site analysis were conducted using Pymol (https://autodock.scripps.edu/, accessed on 22 February 2025). Molecular docking between the modeled TabsOBPs and candidate ligands was performed using AutoDock Vina [13]. The binding energies and interaction residues were used to assess affinity between TabsOBPs and ligands [13].

2.7. Statistics

Relative expression levels of TabsOBP45 and TabsOBP46 across different tissues were statistically analyzed via one-way analysis of variance (ANOVA) using SPSS Statistics 27.0 software (IBM, Chicago, IL, USA). Tukey’s tests were followed to determine the significance among groups (p < 0.05). Additionally, 95% confidence intervals (CIs) were calculated to assess the precision of the mean differences among groups.

3. Results

3.1. Sequence Analysis of TabsOBP45 and TabsOBP46

The coding sequence (CDS) of TabsOBP45 spanned 492 base pairs (bp), encoding a protein of 163 amino acids (aa) with a predicted molecular weight of 18.06 kilodaltons (kDa) and an isoelectric point (pI) of 4.73. TabsOBP46 had a CDS of 489 bp, encoding a 162 aa protein with a molecular weight of 17.88 kDa and pI = 5.00. Both proteins contained a 20 aa signal peptide at the N-terminus, indicating that they were secretory proteins (Figure 1). Multiple sequence alignment revealed that TabsOBP45 and TabsOBP46 had six conserved cysteine residues, which arranged according to the characteristic arrangement of lepidopteran OBPs: C1-X_22-32-C2-X₃-C3-X_36-46-C4-X₈-14-C5-X₈-C6 [36]. This conserved framework suggested that both TabsOBPs belong to the classic OBP family with a typical binding domain architecture.

3.2. Relative Expression Analysis of TabsOBP45 and TabsOBP46

A qRT-PCR analysis revealed distinct tissue-specific expression patterns of TabsOBP45 and TabsOBP46 in both male and female T. absoluta. TabsOBP45 exhibited significantly higher expression in the antennae of both sexes, with the highest expression in male antennae (F_11,24 = 5967.3, p < 0.001). In males, the second highest expression was detected in the legs, followed by the head, while lower expression levels were observed in the thorax, abdomen, and wings. In females, TabsOBP45 was also expressed in other tissues, particularly the head and legs. Similarly, TabsOBP46 showed predominant expression in the antennae of both sexes (F_11,24 = 127.6, p < 0.001) (Figure 2). In contrast to TabsOBP45, TabsOBP46 showed a more balanced expression profile across non-olfactory tissues, particularly in the legs and wings. These results suggest that both the antenna-enriched TabsOBP45 and TabsOBP46 are putatively involved in olfactory recognition.

3.3. Binding Properties of TabsOBP45 and TabsOBP46

SDS-PAGE analysis revealed that the His-tagged proteins TabsOBP45 (~18 kDa) and TabsOBP46 (~18 kDa) were predominantly present in the precipitate after IPTG induction (Figure S1A,B), indicating that both TabsOBPs were expressed as inclusion bodies. The molecular weights of the expressed proteins were in agreement with the theoretical values predicted by ExPASy: 17.88 kDa for TabsOBP45 and 18.06 kDa for TabsOBP46. Given the putative role of TabsOBP45 and TabsOBP46 in olfactory recognition, we assessed their binding specificity to 63 candidate ligands. First, we determined the dissociation constants (Kd) for the interaction between TabsOBPs and bis-ANS. The calculated Kd values were 4.46 μM for TabsOBP45 and 3.33 μM for TabsOBP46 (Figure 3A,B), indicating a suitable binding affinity for competitive assays. Competitive binding assays revealed that the two TabsOBPs exhibited different ligand-binding profiles (Table S2). TabsOBP45 showed high binding affinities (Ki < 22 μM) to 13 volatile compounds. The strongest binding was observed with β-ionone (Ki = 13.77 ± 1.14 μM), followed by β-caryophyllene (14.61 ± 1.51 μM), terpinolene (15.66 ± 1.14 μM), and cinnamaldehyde (16.13 ± 2.04 μM) (Figure 3C, Table S2). Similarly, TabsOBP46 bound effectively to nine compounds, showing the strongest affinity for 4-anisaldehyde (Ki = 12.64 ± 1.11 μM), followed by 4-methoxyacetophenone (12.87 ± 0.65 μM), cinnamaldehyde (13.71 ± 1.22 μM), and β-ionone (15.98 ± 1.59 μM) (Figure 3D, Table S2).

3.4. Modelling and Molecular Docking

Structural evaluation confirmed that both models were highly reliable (Figure S2). Consistent with the characteristic architecture of insect OBPs, both TabsOBP45 and TabsOBP46 exhibited six α-helical domains (Figure 4A,B). Based on the results of the fluorescence competitive binding assays, five ligands with high binding affinities (β-caryophyllene, β-ionone, cuminaldehyde, 4-anisaldehyde, and 4-methoxyacetophenone) were selected for molecular docking to elucidate their binding mechanisms. The docking results were consistent with the experimental binding data. TabsOBP45 showed particularly strong affinities to β-caryophyllene (−8.0 kcal/mol) and β-ionone (−7.26 kcal/mol) (Table S3), and TabsOBP46 exhibited particular affinities to β-ionone (−7.03 kcal/mol) and cuminaldehyde (−5.42 kcal/mol) (Table S3). Detailed structural analysis of the docking complexes revealed that β-caryophyllene bound to TabsOBP45 through hydrophobic contacts with PHE12, PHE76, PHE94, PHE118, LEU68, LEU90, and ALA77, with intermolecular distances ranging from 3.2 to 4.0 Å. Furthermore, β- ionone interacted with TabsOBP45 primarily via ILE8, PHE12, TRP37, and PHE118, with the closest contact observed at 2.8 Å between β-ionone and TRP37. In TabsOBP46, cuminaldehyde formed interactions with PHE12, PHE36, TRP37, PHE118, and VAL134. Meanwhile, β-ionone engaged a similar set of residues, including ILE8, ILE52, and ILE94 (Figure 4).

4. Discussion

T. absoluta, an invasive pest native to South America, is notorious for its destructive capacity [17,18]. Understanding the molecular mechanisms of its olfactory system is crucial for developing effective and sustainable management strategies [14]. This study characterized the specific binding affinities between plant volatiles and two antenna-expressed TabsOBPs, TabsOBP45 and TabsOBP46. TabsOBP45 showed strong binding to 13 volatiles, especially β-ionone, and TabsOBP46 exhibited a high affinity for nine volatiles, particularly 4-anisaldehyde. These results suggest that TabsOBP45 and TabsOBP46 primarily mediate host plant recognition in T. absoluta, and could be used as potential olfactory-based targets for the development of novel control strategies.

The expression level and tissue distribution of insect OBPs often reflect their diverse physiological functions beyond conventional olfaction [6]. While OBPs expressed in non-olfactory tissues can play important roles in contact chemosensation and gustatory perception, such as food recognition and evaluation. For example, PopeOBP16 in the legs of Phthorimaea operculella and AcerOBP15 in the feet of Apis cerana are involved in taste recognition during nectar and pollen collection [37,38]. Similarly, PregOBP56a, which is highly expressed in the mouthparts of Phormia regina plays a role in the solubilization of dietary fatty acids [39]. In T. absoluta, TabsOBP45 and TabsOBP46 were predominantly expressed in the antennae of both sexes, with significantly higher levels in males, confirming their primary role in olfactory recognition. Notably, these TabsOBPs were also expressed in non-olfactory tissues, particularly in the legs and wings, albeit at lower levels. Their presence in the legs suggests a potential involvement in contact chemosensation for substrate evaluation, while their expression in wings may be related to the detection of airborne chemicals during flight via wing sensilla [40,41].

OBPs play a crucial role in insect olfactory recognition and are essential for host seeking, mate location, and predator avoidance [42,43]. Extensive research on Lepidoptera, particularly the family Gelechiidae, has revealed the diverse functions of OBPs in detecting both host plant volatiles and sex pheromones [14,37,44]. For instance, ScerOBP15 and ScerOBP23 from Sitotroga cerealella show strong binding affinities to wheat volatiles [44], and PopeOBP16 from Phthorimaea operculella shows high affinity to nerol and 2-phenylethanol, which are emitted from host plants [37]. Similar patterns have been observed in other moth species whose OBPs are involved in recognizing β-ionone, such as DabiOBP5 and DabiOBP14 in Dioryctria abietella, MpOBP8 in M. pallidipes, and multiple CsupOBPs in Chilo suppressalis [45,46,47]. Previous studies have reported that, of the 97 putative TabsOBPs identified in the T. absoluta genome, TabsOBP45 and TabsOBP46 likely play crucial roles in host plant recognition and behavioral regulation [29]. In this study, TabsOBP45 showed strong binding affinities for terpenoids (β-caryophyllene, α-pinene, 3-carene, terpinolene) and aromatic compounds (cinnamaldehyde, methyl salicylate). Notably, TabsOBP45 showed a particularly high affinity for β-ionone, a carotenoid degradation product found in mature tomato fruits that serves as an important insect attractant [48]. TabsOBP46 bound to aromatic aldehydes including 4-anisaldehyde, cinnamaldehyde, and cuminaldehyde, as well as phenolic compounds such as eugenol. Many of these compounds are known constituents of Solanaceae plants [49,50], and several of them, including 4-anisaldehyde, cinnamaldehyde, and eugenol, have demonstrated insecticidal or repellent properties against various insect species [51,52,53].

Molecular recognition between OBPs and their ligands is primarily mediated by specific structural features within the binding cavity [44,54,55]. These binding cavities are typically lined with nonpolar residues, particularly aromatic amino acids, which facilitate ligand recognition through π–π interactions and hydrophobic forces [56]. Our molecular docking analysis of TabsOBP45 and TabsOBP46 revealed binding patterns consistent with these established mechanisms. These binding characteristics are similar to those observed in other lepidopteran OBPs; for example, PopeOBP16 utilizes IEU4 and ILE44 to recognize host volatiles [37], AlepGOBP1 depends on ILE34, VAL96, ILE76, and PHE100 [13], and BmorPBP1 relies on phenylalanine residues to bind pheromones [57]. The remarkable conservation of aromatic residues, particularly phenylalanine and tryptophan, in the binding pockets of TabsOBP45 and TabsOBP46, was consistent with observations in ScerOBP15/23, in which similar residues play a crucial role in ligand recognition [44]. However, since molecular docking only provides static structural insights, further experimental validation through site-directed mutagenesis and binding assays will be essential to confirm the functional significance of these predicted key residues in TabsOBP45 and TabsOBP46 [58].

Understanding the molecular basis of olfactory recognition in T. absoluta can benefit the development of new pest management strategies. Plant volatiles that bind strongly to TabsOBP45 and TabsOBP46 could be utilized to develop attractants or repellents for behavioral manipulation [25,59]. The practical implementation of these findings could involve several field management approaches. The identified high-affinity ligands, such as β-ionone and 4-anisaldehyde, could be formulated into slow-release dispensers for mass trapping systems around tomato fields or incorporated into attract-and-kill devices to reduce population density with minimal pesticide use [60]. Repellent compounds like cinnamaldehyde, eugenol, and β-caryophyllene could be applied as foliar sprays to create protective barriers around crops or incorporated into push–pull strategies, where repellents push the pests away from crops while attractants in trap crops at field borders pull them into traps [61]. Many of these compounds are already known to have repellent or insecticidal properties against other insect species, which supports their potential efficacy against T. absoluta. Furthermore, the characterized OBPs themselves represent potential targets for RNAi-based control strategies. Silencing these genes could disrupt the pest’s ability to locate host plants, thereby reducing infestation rates. These olfactory-based approaches offer environmentally friendly alternatives to conventional pesticides, in line with the growing demand for sustainable pest management solutions. However, it is important to acknowledge the inherent limitations of the fluorescence competitive binding assays and molecular docking techniques employed in this study. While these in vitro and in silico methods provide valuable insights, they cannot fully replicate the physiological environment within the insect or capture dynamic protein–ligand interactions [58]. Therefore, functional validation through RNAi experiments, behavioral assays, and electrophysiological studies is essential to confirm the specific roles of these OBPs in T. absoluta olfactory recognition and to translate these findings into effective field applications [7,58]. Future research will focus on evaluating the behavioral effects of identified ligands in field trials to develop practical applications.

5. Conclusions

This study characterized the binding affinities of TabsOBP45 and TabsOBP46 to the volatiles emitted by host plants of T. absoluta. Our results highlight the critical roles of two TabsOBPs in recognizing host plant volatiles, as both proteins exhibit strong binding affinities for solanaceous volatiles. The antenna-predominant expression patterns and selective binding profiles suggest that these TabsOBPs are integral components of the chemosensory system that controls host plant location. Their evolutionary conservation and specialized binding properties further underscore their central role in T. absoluta’s adaptation to its host plants. These findings not only deepen our understanding of insect–plant interactions but also provide promising leads for the development of lure- or repellent-based strategies for sustainable T. absoluta management.