Pheromone binding proteins enhance the sensitivity of olfactory receptors to sex pheromones in Chilo suppressalis

Abstract

Sexual communication in moths offers a simplified scenario to model and investigate insect sensory perception. Both PBPs (pheromone-binding proteins) and PRs (pheromone receptors) are involved in the detection of sex pheromones, but the interplay between them still remains largely unknown. In this study, we have measured the binding affinities of the four recombinant PBPs of Chilo suppressalis (CsupPBPs) to pheromone components and analogs and characterized the six PRs using the Xenopus oocytes expression system. Interestingly, when the responses of PRs were recorded in the presence of PBPs, we measured in several combinations a dramatic increase in signals as well as in sensitivity of such combined systems. Furthermore, the discrimination ability of appropriate combinations of PRs and PBPs was improved compared with the performance of PBPs or PRs alone. Besides further supporting a role of PBPs in the pheromone detection and discrimination, our data shows for the first time that appropriate combinations of PRs and PBPs improved the discrimination ability of PBPs or PRs alone. The variety of responses measured with different pairing of PBPs and PRs indicates the complexity of the olfaction system, which, even for the relatively simple task of detecting sex pheromones, utilises a highly sophisticated combinatorial approach.

Figure 1. Relative expression of CsupPRs and CsupPBPs in antennae of adults.

The expression levels are calculated relative to that of the housekeeping gene, CsupG3PDH and normalised on the values of male CsupPR2 and CsupPBP1 set to 100. Experiments were performed in triplicates. Error bars indicate SEM.

Figure 2. Ligand binding assay of CsupPBPs.

(a) Binding curves of 1-NPN to CsupPBPs (A) and competitive binding curves of CsupPBP1 to sex pheromone components (C) and analogs (D). Displacement curves for CsupPBP2, CsupPBP3 and CsupPBP4 are reported in Figure S1. Experiments with 1-NPN and with competitors were performed in triplicates and mean values and standard errors are reported. The Prism software was used to analyse the data and plot the binding curves Panel (B) reports in graphical form the affinities of the four PBPs to the seven ligands. For a more immediate visualisation, the reciprocal of dissociation constants have been plotted. Structures of the ligands utilised are also reported in the same figure.

Figure 3. Functional analysis of CsupPR genes in Xenopus oocytes.

In each panel: (Left) Inward current responses of CsupPR/CsupOrco-coexpressed Xenopus oocytes to 10⁻⁴ mol/L sex pheromone components and analogs. (Right) Response profiles of CsupPRs. Error bars indicate SEM (n = 6).

Figure 4. CsupPBPs can enhance the responses of CsupPRs to sex pheromone components.

(a) Inward current response of CsupPR2/CsupOrco-coexpressed Xenopus oocytes to 10⁻⁴ mol/L sex pheromone. (b) Response values of CsupPRs/CsupOrco-coexpressed Xenopus oocytes to 10⁻⁴ mol/L ligands with either DMSO or CsupPBP. 1–7 are Z9–16:Ald, Z11–16:Ald, Z13–18:Ald, Z9–14:OH, Z11–16:OH, Z9,E12–14:Ac and 16:Ald. Experiments were performed in quadruplicates and average values are reported.

Figure 5. CsupPBPs can enhance the sensitivity of CsupPRs to sex pheromone components.

(a) Dose-response of PR1 to Z11–16:Ald with or without PBP2. (b) Comparison of EC50 of CsupPRs to two sex pheromone components with and without CsupPBPs. The data were assessed by one-way analysis of variance (ANOVA). Error bars indicate SEM (n = 3 ~ 6)