Fluorescence Competitive Binding Research Articles

Chemosensory Proteins Protect Nilaparvata lugens from Imidacloprid by Sequestering the Insecticide and Facilitating Metabolic Detoxification.

The involvement of chemosensory proteins (CSPs) in binding to insecticides has been implicated. However, our understanding of CSP-mediated insecticide resistance remains limited. Herein, 15 CSP genes were identified and characterized from Nilaparvata lugens. Expression analysis identified six CSPs with overexpression in the imidacloprid-resistant strain, whose involvement in imidacloprid resistance was validated by RNA interference. Among them, four CSPs were successfully expressed using a prokaryotic expression system, and their binding affinities to imidacloprid were confirmed through fluorescence competitive binding assays. Knockdown of them impaired the capacity of N. lugens to metabolize imidacloprid and inhibited the activity of metabolic detoxification pathways, while their overexpression in Escherichia coli enhanced bacterial metabolic efficiency toward imidacloprid. Furthermore, the transcriptional regulation of CSP2 and CSP15 was found to be mediated by AhR/ARNT and CncC/MafK. These findings suggest that the overexpression of CSPs in N. lugens promotes imidacloprid resistance by sequestering the insecticide and enhancing metabolic detoxification.

Key amino acids in odorant-binding protein OBP7 enable Bradysia odoriphaga to recognize host plant volatiles

Bradysia odoriphaga (Diptera: Sciaridae) is a devastating underground pest that can cause serious economic losses. Odorant binding proteins (OBPs) are crucial components of the insect olfactory system, playing key roles in locating host plants, oviposition sites, and mates. Therefore, they are considered potential targets for pest control. Here, we obtained one OBP gene (BodoOBP7) from the antennal transcriptome of B. odoriphaga, and observed that the expression level of BodoOBP7 was primarily in the antennae of both sexes, with significantly higher expression level in females than in males. Fluorescence competitive binding assays indicated that BodoOBP7 exhibited strong binding affinities for the six host plant volatiles, including propyl disulfide, dipropyl trisulfide, dimethyl trisulfide, 2-tridecanone, 2-undecanone and alpha-ionone. Subsequently, homology modeling, molecular docking and site-directed mutagenesis revealed that four key amino acid residues (Phe79, Phe99, Ile96, Leu100) participate in the binding of BodoOBP7 with six host plant volatiles. Our results demonstrate that BodoOBP7 is involved in olfactory recognition in B. odoriphaga. These findings may enhance our understanding of the interaction mechanisms between host plants and B. odoriphaga, potentially offering new perspectives for the development of effective green control strategies.

Odorant binding protein TcOBPC02 contributes to phytochemical defense in the red flour beetle, Tribolium castaneum

AbstractThe red flour beetle, Tribolium castaneum, is an agricultural and storage pest with a global distribution. Studies have shown that eucalyptol has strong contact toxicity against larvae of this beetle, whereas odorant binding proteins (OBPs) are known to contribute to larval defenses against this phytochemical toxin. However, the mechanisms underlying the protective effect of insect OBPs against eucalyptol remain unclear. Here, TcOBPC02 from T. castaneum was cloned and characterized. Gene expression profile analysis showed that TcOBPC02 is highly expressed at early larval and early pupal stages. Additionally, tissue expression profiling revealed that, in the adult, TcOBPC02 was most highly expressed in the head, followed by the epidermis, whereas in larvae, TcOBPC02 was mainly expressed in hemolymph and the epidermis. These developmental stages and tissues that exhibit high TcOBPC02 expression are closely related to the detoxification of heterologous substances. Furthermore, the mRNA level of TcOBPC02 was significantly increased after exposure to eucalyptol, whereas TcOBPC02‐targeted RNA interference increased the susceptibility of T. castaneum to eucalyptol, indicating that TcOBPC02 participates in the tolerance of this beetle to eucalyptol. Additionally, recombinant TcOBPC02 was expressed in Escherichia coli and isolated, enabling a straightforward fluorescence competition binding assay. In combination, these results have demonstrated that TcOBPC02 is required for defenses against phytochemicals in T. castaneum. This study provides a theoretical basis for understanding the mechanisms underlying the degradation of exogenous toxicants in insects and adds to the repertoire of potential target genes for pest control.

Mechanistic exploration of odorant binding protein-mediated chlorpyrifos resistance in Nilaparvata lugens: Insights from insecticide sequestration and transcriptional regulation

The effectiveness and sustainable application of insecticides are severely threatened by the rapid evolution of resistance in agricultural pests. Recent research indicates that odorant binding proteins (OBPs) may be involved in facilitating insecticide resistance, while the specific mechanisms remain poorly understood. Herein, 11 OBPs were identified from Nilaparvata lugens. Among them, OBP5 exhibited high and specific expression in the head, and showed constitutive overexpression in the chlorpyrifos-resistant strain. Knockdown of OBP5 notably restored susceptibility to chlorpyrifos in N. lugens, while overexpression of OBP5 in Escherichia coli significantly enhanced bacterial tolerance to chlorpyrifos. Fluorescence competitive binding assay confirmed the strong binding affinities of OBP5 to chlorpyrifos and its active metabolite chlorpyrifos-oxon. Molecular docking studies proposed a critical interacting amino acid (Lys147) in the binding site, which was further validated by comparative binding studies between wildtype OBP5 and the mutated protein OBP5K147A. Furthermore, Lim1β that also presented overexpression pattern in the resistant strain, was found to regulate expression of OBP5 through a dual-luciferase reporter assay. Our findings demonstrate that the overexpression of OBP5 contributes to chlorpyrifos resistance by binding and sequestering the insecticides, shedding light on the sequestration resistance mechanism conferred by OBPs and offering potential targets for resistance management.

Functional Role of Odorant-Binding Proteins in Response to Sex Pheromone Component Z8-14:Ac in Grapholita molesta (Busck).

The plum fruit moth (PFM), Grapholita funebrana, and the oriental fruit moth (OFM), G. molesta, are closely related fruit moth species that severely damage fruit trees in Rosaceae. Both species share common primary sex pheromone components Z8-12:Ac and E8-12:Ac. The secondary sex pheromone components of PFMs consist of Z8-12:OH, Z8-14:Ac, and Z10-14:Ac, while those of OFMs include Z8-12:OH and 12:OH. Previous researchers have proved that the inclusion of Z8-14:Ac and Z10-14:Ac did not augment PFM catches but inhibited OFM catches in orchards in Europe, thereby maintaining the species-specificity of the PFM sex attractant. However, which of these components, Z8-14:Ac or Z10-14:Ac, plays the major role in inhibiting OFM attraction remains unclear. In the current study, electroantennogram (EAG) assays indicated that both OFM and PFM males exhibited a moderate EAG response to Z8-14:Ac and Z10-14:Ac. Rubber septa loaded with varying ratios of Z8-14:Ac (1% to 30%) or Z10-14:Ac (5% to 110%) combined with a constant dose of Z8-12:Ac and E8-12:Ac produced diverse trapping effects. Sex attractants containing Z8-14:Ac did not significantly affect the trapping of PFM males but drastically reduced the capture of OFM males, with the reduction reaching up to 96.54%. Attractants containing more than 10% of Z10-14:Ac simultaneously reduced the number of OFM and PFM males captured. Z8-14:Ac was indispensable for maintaining the specificity of sex pheromones. Fluorescence competitive binding assays of recombinant GmolPBP2 showed the lowest Ki value (0.66 ± 0.02 μM) among the PBPs/GOBPs from OFMs, suggesting that it is the most likely target for Z8-14:Ac. Molecular dynamic simulation and site-directed mutagenesis assays confirmed that the Phe12 residue, which forms a π-alkyl interaction with Z8-14:Ac, was crucial for GmolPBP2 binding to Z8-14:Ac. In conclusion, Z8-14:Ac is vital to the specificity of PFM sex pheromones inhibiting OFM attractants when added to Z8-12:Ac and E8-12:Ac. This could be potentially used to develop species-specific sex attractants for the PFM.

Binding properties of olfactory proteins to host volatiles, free fatty acids and cuticular hydrocarbons in the termite Reticulitermes aculabialis

As eusocial insects prevalent in tropical and subtropical regions, termites are characterized by highly organized behaviors and exceptional adaptability, rooted in caste differentiation and chemical communication. These traits make them excellent models for studying insect social structures and ecological interactions. Investigating how termites use chemical signals to perceive and respond to their environment provides insights into their coordination and adaptation within complex ecosystems. This study delved into the chemosensory mechanisms of Reticulitermes aculabialis, examining the interactions of four olfactory proteins with 70 ligands, including host volatiles, cuticular hydrocarbons (CHCs), and free fatty acids (FFAs). Molecular docking simulations revealed varied affinities of the olfactory proteins for long-chain hydrocarbons (n-C23 to n-C28), suggesting a nuanced chemical communication system through specific hydrocarbon detection. RacuCSP1 and RacuCSP2 exhibited specific binding to linoleic acid and undecanoic acid, respectively, highlighting the significance of FFAs in the physiological and behavioral processes of termites. The four olfactory proteins showed a strong affinity for longifolene in fluorescence competitive binding experiments. Notably, RacuOBPs exhibited unique affinities for terpenoid volatiles such as β-lonone and neocembrene, while RacuCSPs specifically bound with terpenoids like 3-carene, myrtenol, α-pinene oxide and β-pinene indicating their critical roles in host detection. Behavioral observations following gene silencing revealed that RacuOBP5 was essential for recognizing longifolene and α-lonone recognition, while RacuCSP1 was key for detecting α-pinene in termites. These findings enhance our understanding of the termite chemosensory system and offer insights for developing precise pest management strategies.

Characterization of two glutathione S-transferase genes involved in clothianidin resistance in Bradysia odoriphaga.

Glutathione S-transferase (GST) is a key phase II detoxification enzyme involved in xenobiotics metabolism, and plays a pivotal role in the evolution of resistance to various types of insecticides. However, the specific functions of GST genes in clothianidin resistance remain obscure in Bradysia odoriphaga. Here, a specific GST inhibitor, diethyl maleate (DEM), significantly increased the mortality of Bradysia odoriphaga larvae following exposure to clothianidin, and the activity of GST enzyme in clothianidin-resistant (CL-R) strain of Bradysia odoriphaga was markedly greater than that in the SS strain. Two sigma BoGSTs (BoGSTs1 and BoGSTs2) were markedly overexpressed in the CL-R strain and exhibited a higher abundance in the Malpighian tubules or midgut. Exposure to clothianidin resulted in a significant increased expression of BoGSTs1 and BoGSTs2. The knockdown of BoGSTs1 and BoGSTs2 increased sensitivity of larvae to clothianidin in the resistant strain. Furthermore, overexpression of BoGSTs1 and BoGSTs2 led to a significant increase in Escherichia coli cells tolerance to clothianidin. In vitro metabolic assays indicate that these two GSTs cannot directly metabolize clothianidin and its secondary metabolite desmethyl-clothianidin. Disk diffusion assays and fluorescence competitive binding assays indicated that BoGSTs1 and BoGSTs2 play a critical role in clothianidin resistance by antioxidant activity and non-catalytic binding activity. The docking results showed that BoGSTs1 and BoGSTs2 have strong binding affinity toward clothianidin. Collectively, these findings pinpoint the potential role of BoGSTs1 and BoGSTs2 in conferring insecticide resistance in Bradysia odoriphaga and contribute to our understanding of the underlying mechanisms of insecticide resistance. © 2024 Society of Chemical Industry.

A larval expressed chemosensory protein involved in recognition of anthocyanins in Spodoptera litura (Lepidoptera: Noctuidae).

Anthocyanins are secondary metabolites which act as diverse functions during plant growth. Insects can discriminate host plants by their sensitive gustatory systems. It is hypothetical that chemosensory proteins (CSPs) play a crucial role in regulating this behavioral process. However, the underlying molecular mechanisms remain obscure. In the present study, we characterized a CSP SlitCSP8 from the Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae). Quantitative real-time-polymerase chain reaction analysis demonstrated that SlitCSP8 was mainly expressed in the head of the 7th S. litura larvae, especially labrum. Further, recombinant SlitCSP8 was obtained using bacterial expression system. Fluorescence competitive binding assays demonstrated that the purified SlitCSP8 exhibited a strong binding affinity to anthocyanins, a natural compound derived from the host plant. Silencing SlitCSP8 through RNAi significantly reduced the sensitivity of S. litura larvae to anthocyanins-treated leaf disks, the development from larva to pupae was not affected. These data provide insight into the molecular basis that CSP8 can detect anthocyanins in host plants by chemosensory system of insects. It can be further used in designing novel optimal food attractant targeting to the CSPs for pest control.

Comparation of pheromone-binding proteins 1 and 2 of Spodoptera frugiperda in perceiving the three sex pheromone components Z9-14:Ac, Z7-12: Ac and Z11-16: Ac

Pheromone-binding proteins (PBPs) are mainly responsible for binding and transporting hydrophobic pheromone molecules across the aqueous sensilla lymph to the receptor proteins. The preference of each PBP is believed to be different for each pheromone component within a single species. Significantly higher expression level of PBP1 and PBP2 in the male antennae of Spodoptera frugiperda suggesting that SfruPBP1 and SfruPBP2 might play important roles in pheromone perception. However, the preference of these two PBP to the three main pheromone components Z9-14: Ac, Z7-12: Ac and Z11-16: Ac have not been determined. In this study, a fluorescence competitive binding assay revealed that the binding intensities of SfruPBP1 and SfruPBP2 to Z9-14: Ac or Z7-12: Ac was comparable. We then used the CRISPR/Cas9 system to individually or simultaneously knock out PBP1 and PBP2 in S.frugiperda. The result of courtship behavior indicated that SfruPBP1 and SfruPBP2 were indispensable and played equal roles in perceiving the pheromones Z9-14: Ac and Z7-12: Ac for orientation, wing vibration, and hair-pencil display. Compared with Z9-14:Ac and Z7-12: Ac, Z11-16: Ac showed higher or medium binding intensities with SfruPBP1 and SfruPBP2 but played a minor role in inducing the wing vibration behavior. The results of this study are valuable for elucidating the mechanisms involved in sex pheromone perception and may facilitate the development of PBP-targeted pest control techniques.

An expanded odorant-binding protein mediates host cue detection in the parasitic wasp Baryscapus dioryctriae basis of the chromosome-level genome assembly analysis

BackgroundBaryscapus dioryctriae (Chalcidodea: Eulophidae) is a parasitic wasp that parasitizes the pupae of many Pyralidae members and has been used as a biological control agent against Dioryctria pests of pinecones.ResultsThis B. dioryctriae assembly has a genome size of 485.5 Mb with a contig N50 of 2.17 Mb, and scaffolds were assembled onto six chromosomes using Hi-C analysis, significantly increasing the scaffold N50 to 91.17 Mb, with more than 96.13% of the assembled bases located on chromosomes, and an analysis revealed that 94.73% of the BUSCO gene set. A total of 54.82% (279.27 Mb) of the assembly was composed of repetitive sequences and 24,778 protein-coding genes were identified. Comparative genomic analysis demonstrated that the chemosensory perception, genetic material synthesis, and immune response pathways were primarily enriched in the expanded genes. Moreover, the functional characteristics of an odorant-binding protein (BdioOBP45) with ovipositor-biased expression identified from the expanded olfactory gene families were investigated by the fluorescence competitive binding and RNAi assays, revealing that BdioOBP45 primarily binds to the D. abietella-induced volatile compounds, suggesting that this expanded OBP is likely involved in locating female wasp hosts and highlighting a direction for future research.ConclusionsTaken together, this work not only provides new genomic sequences for the Hymenoptera systematics, but also the high-quality chromosome-level genome of B. dioryctriae offers a valuable foundation for studying the molecular, evolutionary, and parasitic processes of parasitic wasps.

Revealing the pH-dependent conformational changes in sol g 2.1 protein and potential ligands binding

Sol g 2, a major protein found in the venom of the tropical fire ant (Solenopsis geminata), is well-known for its ability to bind various hydrophobic molecules. In this study, we investigate the binding activity of recombinant Sol g 2.1 protein (rSol g 2.1) with potential molecules, including (E)-β-Farnesene, α-Caryophyllene, and 1-Octen-3-ol at different pH levels (pH 7.4 and 5.5) using fluorescence competitive binding assays (FCBA). Our results revealed that Sol g 2.1 protein has higher affinity binding with these ligands at neutral pH. Relevance to molecular docking and molecular dynamics simulations were utilized to provide insights into the stability and conformational dynamics of Sol g 2.1 and its ligand complexes. After simulation, we found that Sol g 2.1 protein has higher affinity binding with these ligands as well as high structural stability at pH 7.4 than at an acidic pH level, indicating by RMSD, RMSF, Rg, SASA, and principal component analysis (PCA). Additionally, the Sol g 2.1 protein complexes at pH 7.4 showed significantly lower binding free energy (∆Gbind) and higher total residue contributions, particularly from key non-polar amino acids such as Trp36, Met40, Cys62, and Ile104, compared to the lower pH environment. These explain why they exhibited higher binding affinity than the lower pH. Therefore, we suggested that Sol g 2.1 protein is a pH-responsive carrier protein. These findings also expand our understanding of protein–ligand interactions and offer potential avenues for the development of innovative drug delivery strategies targeting Sol g 2.1 protein.

Chemosensory protein 22 in Riptortus pedestris is involved in the recognition of three soybean volatiles

Riptortus pedestris (Hemiptera: Alydidae), a common agricultural pest, is the major causative agent of “soybean staygreen.” However, the interactions between chemosensory proteins (CSPs) in R. pedestris and host plant volatiles have yet to be comprehensively studied. In this study, we performed real-time fluorescence quantitative polymerase chain reaction (PCR) to analyze the antennal expression of RpedCSP22 and subsequently analyzed the interactions between 21 soybean volatiles, five aggregation pheromones, and RpedCSP22 protein in vitro using a protein expression system, molecular docking, site-directed mutagenesis, and fluorescence competitive binding experiments. The RpedCSP22 protein showed binding affinity to three soybean volatiles (benzaldehyde, 4-ethylbenzaldehyde, and 1-octene-3-ol), with optimal binding observed under neutral pH conditions, and lost binding ability after site-directed mutagenesis. In subsequent RNA interference (RNAi) studies, gene silencing was more than 90 %, and in silenced insects, electroantennographic responses were reduced by more than 75 % compared to non-silenced insects. Moreover, Y-tube olfactory behavioral assessments revealed that the attraction of R. pedestris to the three soybean volatiles was significantly attenuated. These findings suggest that RpedCSP22 plays an important role in the recognition of host plant volatiles by R. pedestris andprovides a theoretical basis for the development of novel inhibitors targeting pest behavior.

Binding characterization of odorant-binding protein BhorOBP29 in Batocera horsfieldi (Hope) with host-plant volatiles

Odorant binding proteins (OBPs) are involved in odorant discrimination and act as the first filter in the peripheral olfactory system. Previous studies have shown that BhorOBP29 is potentially involved in olfactory perception in an important wood-boring pest Batocera horsfieldi (Hope) (Coleoptera: Cerambycidae), however, its function remains unclear. Here, we investigated the ligand-binding profiles of recombinant BhorOBP29 with 22 compounds from its host plant using fluorescence competitive binding assays and fluorescence quenching assays. The results showed that BhorOBP29 could bind to five ligands relying mainly on hydrophobic interactions. Molecular docking analysis indicated that residues Ile48, Leu51, Met52, Trp57, Asn105, and Val119 were extensively involved in the interactions between BhorOBP29 and the five ligands. Furthermore, the site-directed mutagenesis analysis revealed that Leu51 and Met52 residues were indispensable for BhorOBP29-ligands binding. Finally, electroantennogram (EAG) assays confirmed that hexanal, (−)-limonene, and 2-methylbutyraldehyde elicited a concentration-dependent EAG response with a maximum at the concentration of 1/10 v/v. These findings suggest that BhorOBP29 may play a significant role in the perception of host plant volatiles by B. horsfieldi. This study may help to discover novel behavioral regulation and environmentally friendly strategies for controlling B. horsfieldi in the future.

OBP83b and OBP49a Involved in the Perception of Female-Derived Pheromones in Bactrocera dorsalis (Hendel).

In Bactrocera dorsalis, both males and females release chemical signals to attract mates. In our previous study, we identified ethyl laurate, ethyl myristate, and ethyl palmitate as potent female-derived pheromones that contribute to mate attraction. However, the mechanisms underlying the olfactory recognition remain unclear. In this study, we observed strong antennal and behavioral responses in male B. dorsalis to these female-derived pheromones, and further investigation revealed significant upregulation of OBP49a and OBP83b following exposure to these compounds. Through fluorescence competitive binding assays and RNA interference techniques, we demonstrated the crucial roles of OBP49a and OBP83b in detecting female-derived pheromones. Finally, molecular docking analysis identified key residues, including His134 in OBP83b and a lysine residue in OBP49a, which formed hydrogen bonds with female-derived pheromones, facilitating their binding. These findings not only advance our understanding of olfactory recognition of pheromones in B. dorsalis but also offer potential targets for developing olfaction-interfering techniques for pest control.

Identification of attractants for adult Spodoptera litura based on the interaction between odorant-binding protein 34 and host volatiles

Odorant-binding proteins (OBPs) play key roles in host plant location by insects, and can accordingly serve as important targets for the development of attractants. In this study, we detected the high expression of SlitOBP34 in male antennae of Spodoptera litura. Subsequently, the fluorescence competitive binding experiments displayed that the SlitOBP34 protein has binding affinity for different ligands. Then, protein-ligand interaction analyses found the presence of six amino acid residues may serve as key recognition sites. Further electroantennographic and biobehavioral assessments revealed that the electrophysiological responses of male antennae were evoked in response to stimulation with the six identified host volatiles, and that these volatiles attracted male moths to varying extents. Notably, low concentrations of benzaldehyde, 1-hexanol, and cis-3-hexenyl acetate were found to have significant attractant effects on male moths, thereby identifying these three host volatiles as potential candidates for the development of male attractants. These findings advance our current understanding of the olfactory-encoded mechanisms of host plants selection in S. litura and have enabled us to develop novel adult attractants for controlling the pest in the future.

Exploring the binding affinity and characteristics of DcitOBP9 in citrus psyllids

Odorant-binding proteins (OBPs) are crucial in insect olfaction. The most abundant expressed OBP of citrus psyllids, DcitOBP9 encodes 148 amino acids. DcitOBP9 lacks a transmembrane structure and possesses a 17-amino acid signal peptide at the N-terminus. Characterized by the six conserved cysteine sites, DcitOBP9 is classified as the Classical-OBP family. RT-qPCR experiments revealed ubiquitous expression of DcitOBP9 across all developmental stages of the citrus psyllid, with predominant expression in adults antennae. Fluorescence competitive binding assays demonstrated DcitOBP9′s strong affinity for ocimene, linalool, dodecanoic acid, and citral, and moderate affinity for dimethyl trisulfide. Additionally, it binds to myrcia, (−)-trans-caryophyllene, (±)-Citronellal, nonanal, and (+)-α-pinene. Among them, ocimene, linalool, and dodecanoic acid were dynamically bound to DcitOBP9, while citral was statically bound to DcitOBP9. Molecular docking simulations with the top five ligands indicated that amino acid residues V92, S72, P128, L91, L75, and A76 are pivotal in the interaction between DcitOBP9 and these odorants. These findings suggest DcitOBP9′s involvement in the citrus psyllid's host plant recognition and selection behaviors, thereby laying a foundation for elucidating the potential physiological and biological functions of DcitOBP9 and developing attractants.

Identification of odorant-binding proteins and functional analysis of antenna-specific BhorOBP28 in Batocera horsfieldi (Hope).

The important wood-boring pest Batocera horsfieldi has evolved a sensitive olfactory system to locate host plants. Odorant-binding proteins (OBPs) are thought to play key roles in olfactory recognition. Therefore, exploring the physiological function of OBPs could facilitate a better understanding of insect chemical communications. In this research, 36 BhorOBPs genes were identified via transcriptome sequencing of adults' antennae from B. horsfieldi, and most BhorOBPs were predominantly expressed in chemosensory body parts. Through fluorescence competitive binding and fluorescence quenching assays, the antenna-specific BhorOBP28 was investigated and displayed strong binding affinities forming stable complexes with five volatiles, including (+)-α-Pinene, (+)-Limonene, β-Pinene, (-)-Limonene, and (+)-Longifolene, which could also elicit conformation changes when they were interacting with BhorOBP28. Batocera horsfieldi females exhibited a preference for (-)-Limonene, and a repellent response to (+)-Longifolene. Feeding dsOBP19 produced by a bacteria-expressed system with a newly constructed vector could lead to the knockdown of BhorOBP28, and could further impair B. horsfieldi attraction to (-)-Limonene and repellent activity of (+)-Longifolene. The analysis of site-directed mutagenesis revealed that Leu7, Leu72, and Phe121 play a vital role in selectively binding properties of BhorOBP28. By modeling the molecular mechanism of olfactory recognition, these results demonstrate that BhorOBP28 is involved in the chemoreception of B. horsfieldi. The bacterial-expressed dsRNA delivery system gains new insights into potential population management strategies. Through the olfactory process concluded that discovering novel behavioral regulation and environmentally friendly control options for B. horsfieldi in the future. © 2024 Society of Chemical Industry.

Binding properties of chemosensory protein 4 in Riptortus pedestris to aggregation pheromones

In insects, chemosensory proteins (CSPs) play an important role in the perception of the external environment and have been widely used for protein-binding characterization. Riptortus pedestris has received increased attention as a potential cause of soybean staygreen syndrome in recent years. In this study, we found that RpedCSP4 expression in the antennae of adult R. pedestris increased with age, with no significant difference in expression level observed between males and females, as determined through quantitative real-time polymerase chain reaction (qRT-PCR). Subsequently, we investigated the ability of RpedCSP4 to bind various ligands (five aggregated pheromone components and 13 soybean volatiles) using a prokaryotic expression system and fluorescence competitive binding assays. We found that RpedCSP4 binds to three aggregated pheromone components of R. pedestris, namely, ((E)-2-hexenyl (Z)-3-hexenoate (E2Z3), (E)-2-hexenyl (E)-2-hexenoate (E2E2), and (E)-2-hexenyl hexenoate (E2HH)), and that its binding capacities are most stable under acidic condition. Finally, the structure and protein-ligand interactions of RpedCSP4 were further analyzed via homology modeling, molecular docking, and targeted mutagenesis experiments. The L29A mutant exhibited a loss of binding ability to these three aggregated pheromone components. Our results show that the olfactory function of RpedCSP4 provides new insights into the binding mechanism of RpedCSPs to aggregation pheromones and contributes to discover new target candidates that will provide a theoretical basis for future population control of R. pedestris.

Binding Properties of Odorant Binding Protein 37 in Plagiodera versicolora to Host Volatile, o-Cymene.

The chemosensory system plays an important role in the host plants location. Plagiodera versicolora (Coleoptera: Chrysomelidae) is a worldwide leaf-eating forest pest that feeds exclusively on salicaceous trees. There is no function study of odorant binding proteins (OBPs) in P. versicolora. In the current study, we found that PverOBP37 has a high expression in male and female antennae, heads, and legs by quantitative real-time PCR. The binding properties of PverOBP37 to 18 host plant volatiles were determined by fluorescence competition binding assays. The results showed that PverOBP37 could bind to the host plant volatile, o-cymene. Furthermore, four candidate key amino acid residues (F8, Y50, F103, and R107) of PverOBP37 to o-cymene were identified by molecular docking. The functional assay to confirm Y50, F103, and R107 mutations were key amino acid residues of PverOBP37 involved in the binding to o-cymene. Knockdown of PverOBP37 and Y-tube behavioral bioassays of mated females led to a significantly reduced attraction to o-cymene. This study not only revealed the molecular mechanism of PverOBP37 but also suggested that PverOBP37 is essential to detect host plant volatiles as cues to search for egg-laying sites in P. versicolora.

EsigPBP3 Was the Important Pheromone-Binding Protein to Recognize Male Pheromones and Key Eucalyptus Volatiles.

Pheromone-binding proteins (PBPs) are specific odorant-binding proteins that can specifically recognize insect pheromones. Through transcriptional analysis of the antennae of adult Endoclita signifer, EsigPBP3 was discovered and identified, and EsigPBP3 was found to be highly expressed in the antennae of male moths. Based on the binding characteristics and ability of EsigPBP3, we can find the key ligands and binding site to consider as a target to control the key wood bore E. signifier. In this study, the fluorescence competitive binding assays (FCBA) showed that EsigPBP3 had a high binding affinity for seven key eucalyptus volatiles. Molecular docking analysis revealed that EsigPBP3 had the strongest binding affinity for the sexual pheromone component, (3E,7E)-4,7,11-trimethyl-1,3,7,10-dodecatetraene. Furthermore, same as the result of FCBA, the EsigPBP3 exhibited high binding affinities to key eucalyptus volatiles, eucalyptol, α-terpinene, (E)-beta-ocimene, (-)-β-pinene, and (-)-α-pinene, and PHE35, MET7, VAL10, PHE38, ILE52, and PHE118 are key sites. In summary, EsigPBP3 exhibits high binding affinity to male pheromones and key volatile compounds and the crucial binding sites PHE35, MET7, VAL10, PHE38, ILE52, and PHE118 can act as targets in the recognition of E. signifier pheromones.