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Olfactory proteins mediating chemical communication in the navel orangeworm moth, Amyelois transitella.

Leal WS, Ishida Y, Pelletier J, Xu W, Rayo J, Xu X, Ames JB - PLoS ONE (2009)

Bottom Line: We have cloned nine cDNAs encoding olfactory proteins from the navel orangeworm, including two pheromone-binding proteins, two general odorant-binding proteins, one chemosensory protein, one glutathione S-transferase, one antennal binding protein X, one sensory neuron membrane protein, and one odorant receptor.Of these, AtraPBP1 is highly enriched in male antennae.Fluorescence, CD and NMR studies suggest a dramatic pH-dependent conformational change, with high affinity to pheromone constituents at neutral pH and no binding at low pH.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, University of California Davis, Davis, California, United States of America. wsleal@ucdavis.edu

ABSTRACT

Background: The navel orangeworm, Amyelois transitella Walker (Lepidoptera: Pyralidae), is the most serious insect pest of almonds and pistachios in California for which environmentally friendly alternative methods of control--like pheromone-based approaches--are highly desirable. Some constituents of the sex pheromone are unstable and could be replaced with parapheromones, which may be designed on the basis of molecular interaction of pheromones and pheromone-detecting olfactory proteins.

Methodology: By analyzing extracts from olfactory and non-olfactory tissues, we identified putative olfactory proteins, obtained their N-terminal amino acid sequences by Edman degradation, and used degenerate primers to clone the corresponding cDNAs by SMART RACE. Additionally, we used degenerate primers based on conserved sequences of known proteins to fish out other candidate olfactory genes. We expressed the gene encoding a newly identified pheromone-binding protein, which was analyzed by circular dichroism, fluorescence, and nuclear magnetic resonance, and used in a binding assay to assess affinity to pheromone components.

Conclusion: We have cloned nine cDNAs encoding olfactory proteins from the navel orangeworm, including two pheromone-binding proteins, two general odorant-binding proteins, one chemosensory protein, one glutathione S-transferase, one antennal binding protein X, one sensory neuron membrane protein, and one odorant receptor. Of these, AtraPBP1 is highly enriched in male antennae. Fluorescence, CD and NMR studies suggest a dramatic pH-dependent conformational change, with high affinity to pheromone constituents at neutral pH and no binding at low pH.

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Competitive binding assays with AtraPBP1.Two major constituents of the sex pheromone of the navel orangeworm, Z11Z13-16Ald and Z11Z13-16OH, and a behavioral antagonist, Z11Z13-16OAc, were incubated with AtraPBP1 at the same concentration. The two pheromone constituents bound to AtraPBP1 with nearly, equally high affinity, whereas the behavioral antagonist showed even higher apparent affinity.
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pone-0007235-g012: Competitive binding assays with AtraPBP1.Two major constituents of the sex pheromone of the navel orangeworm, Z11Z13-16Ald and Z11Z13-16OH, and a behavioral antagonist, Z11Z13-16OAc, were incubated with AtraPBP1 at the same concentration. The two pheromone constituents bound to AtraPBP1 with nearly, equally high affinity, whereas the behavioral antagonist showed even higher apparent affinity.

Mentions: Next, we tested binding affinity of other constituents of the navel orangeworm sex pheromone. Female-produced sex pheromones in moths are normally complex mixtures of straight chain acetates, alcohols and aldehydes, with 10–18 carbon atoms and up to three unsaturations, the so-called Type I pheromones. Type II sex pheromone is comprised of polyunsaturated hydrocarbons and epoxy derivatives with long straight chains. The navel orangeworm is unusual in that its sex pheromone system in composed of a complex mixture that includes constituents of both types: Z11Z13-16Ald, Z11Z13-16OH, Z11Z13-16OAc (behavioral antagonist), (Z,Z,Z,Z,Z)-3,6,9,12,15-tricosapentaene and (Z,Z,Z,Z,Z)-3,6,9,12,15-pentacosapentaene, and other minor constituents [17]. As opposed to Type I pheromones that gave very low background indicating negligible non-specific binding (see buffer in Fig. 12), it was difficult to assess binding of the pentaene compounds because their hydrophobicity led to high background levels. On the other hand, the secondary constituent, Z11Z13-16OH bound to AtraPBP1 with affinity comparable to that of the major constituent, but showed no affinity at low pH (data not shown). Interestingly, the behavioral antagonist, Z11Z13-16OAc showed the highest affinity to AtraPBP1 of all tested ligands (data not shown). Next, we performed competitive binding studies with AtraPBP1 incubated with the three ligands at the same concentration. These competitive binding assays mirrored what was observed with non-competitive binding assays, AtraPBP1 was bound with the highest affinity to Z11Z13-16OAc, whereas the aldehyde and alcohol showed similar affinity (Fig. 12). These results suggest that a single PBP may be involved in the reception of multiple constituents of sex pheromones.


Olfactory proteins mediating chemical communication in the navel orangeworm moth, Amyelois transitella.

Leal WS, Ishida Y, Pelletier J, Xu W, Rayo J, Xu X, Ames JB - PLoS ONE (2009)

Competitive binding assays with AtraPBP1.Two major constituents of the sex pheromone of the navel orangeworm, Z11Z13-16Ald and Z11Z13-16OH, and a behavioral antagonist, Z11Z13-16OAc, were incubated with AtraPBP1 at the same concentration. The two pheromone constituents bound to AtraPBP1 with nearly, equally high affinity, whereas the behavioral antagonist showed even higher apparent affinity.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2749207&req=5

pone-0007235-g012: Competitive binding assays with AtraPBP1.Two major constituents of the sex pheromone of the navel orangeworm, Z11Z13-16Ald and Z11Z13-16OH, and a behavioral antagonist, Z11Z13-16OAc, were incubated with AtraPBP1 at the same concentration. The two pheromone constituents bound to AtraPBP1 with nearly, equally high affinity, whereas the behavioral antagonist showed even higher apparent affinity.
Mentions: Next, we tested binding affinity of other constituents of the navel orangeworm sex pheromone. Female-produced sex pheromones in moths are normally complex mixtures of straight chain acetates, alcohols and aldehydes, with 10–18 carbon atoms and up to three unsaturations, the so-called Type I pheromones. Type II sex pheromone is comprised of polyunsaturated hydrocarbons and epoxy derivatives with long straight chains. The navel orangeworm is unusual in that its sex pheromone system in composed of a complex mixture that includes constituents of both types: Z11Z13-16Ald, Z11Z13-16OH, Z11Z13-16OAc (behavioral antagonist), (Z,Z,Z,Z,Z)-3,6,9,12,15-tricosapentaene and (Z,Z,Z,Z,Z)-3,6,9,12,15-pentacosapentaene, and other minor constituents [17]. As opposed to Type I pheromones that gave very low background indicating negligible non-specific binding (see buffer in Fig. 12), it was difficult to assess binding of the pentaene compounds because their hydrophobicity led to high background levels. On the other hand, the secondary constituent, Z11Z13-16OH bound to AtraPBP1 with affinity comparable to that of the major constituent, but showed no affinity at low pH (data not shown). Interestingly, the behavioral antagonist, Z11Z13-16OAc showed the highest affinity to AtraPBP1 of all tested ligands (data not shown). Next, we performed competitive binding studies with AtraPBP1 incubated with the three ligands at the same concentration. These competitive binding assays mirrored what was observed with non-competitive binding assays, AtraPBP1 was bound with the highest affinity to Z11Z13-16OAc, whereas the aldehyde and alcohol showed similar affinity (Fig. 12). These results suggest that a single PBP may be involved in the reception of multiple constituents of sex pheromones.

Bottom Line: We have cloned nine cDNAs encoding olfactory proteins from the navel orangeworm, including two pheromone-binding proteins, two general odorant-binding proteins, one chemosensory protein, one glutathione S-transferase, one antennal binding protein X, one sensory neuron membrane protein, and one odorant receptor.Of these, AtraPBP1 is highly enriched in male antennae.Fluorescence, CD and NMR studies suggest a dramatic pH-dependent conformational change, with high affinity to pheromone constituents at neutral pH and no binding at low pH.

View Article: PubMed Central - PubMed

Affiliation: Department of Entomology, University of California Davis, Davis, California, United States of America. wsleal@ucdavis.edu

ABSTRACT

Background: The navel orangeworm, Amyelois transitella Walker (Lepidoptera: Pyralidae), is the most serious insect pest of almonds and pistachios in California for which environmentally friendly alternative methods of control--like pheromone-based approaches--are highly desirable. Some constituents of the sex pheromone are unstable and could be replaced with parapheromones, which may be designed on the basis of molecular interaction of pheromones and pheromone-detecting olfactory proteins.

Methodology: By analyzing extracts from olfactory and non-olfactory tissues, we identified putative olfactory proteins, obtained their N-terminal amino acid sequences by Edman degradation, and used degenerate primers to clone the corresponding cDNAs by SMART RACE. Additionally, we used degenerate primers based on conserved sequences of known proteins to fish out other candidate olfactory genes. We expressed the gene encoding a newly identified pheromone-binding protein, which was analyzed by circular dichroism, fluorescence, and nuclear magnetic resonance, and used in a binding assay to assess affinity to pheromone components.

Conclusion: We have cloned nine cDNAs encoding olfactory proteins from the navel orangeworm, including two pheromone-binding proteins, two general odorant-binding proteins, one chemosensory protein, one glutathione S-transferase, one antennal binding protein X, one sensory neuron membrane protein, and one odorant receptor. Of these, AtraPBP1 is highly enriched in male antennae. Fluorescence, CD and NMR studies suggest a dramatic pH-dependent conformational change, with high affinity to pheromone constituents at neutral pH and no binding at low pH.

Show MeSH
Related in: MedlinePlus