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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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Gene expression analysis by RT-PCR.Expression of AtraPBP1, AtraPBP2, AtraGOBP1, AtraGOBP2, and AtraCSP genes in control tissue (ML, male hindlegs) and olfactory tissues (MA, male antennae and FA, female antennae). Actin gene was used as endogenous control.
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pone-0007235-g002: Gene expression analysis by RT-PCR.Expression of AtraPBP1, AtraPBP2, AtraGOBP1, AtraGOBP2, and AtraCSP genes in control tissue (ML, male hindlegs) and olfactory tissues (MA, male antennae and FA, female antennae). Actin gene was used as endogenous control.

Mentions: To compare transcript patterns with protein profiles (Fig. 1), RT-PCR experiments were performed using gene-specific primers. First, we compared expression of AtraPBP1, AtraPBP2, AtraGOBP1, AtraGOBP2, and AtraCSP in non-olfactory tissues (male legs) with olfactory tissues (male and female antennae) (Fig. 2). In general, gene expression mirrored protein profiles, except for AtraCSP, which was detected not only in male and female antennae, but also in non-olfactory tissues (legs). AtraPBP2, AtraGOBP1 and AtraGOBP2 genes were detected in both male and female antennae, but not in legs, whereas AtraPBP1 was apparently expressed exclusively in male antennae. Next, we assessed gene expression during antennal development. Contrary to our previous experience with the wild silkworm moth, A. polyphemus [32], sampling antennal pockets from pupae and day 0 adults of the navel orangeworm and extracting RNA were very challenging due to high RNAse activity at this developmental stage as reflected in the irregular amplifications of actin control gene (Fig. 3). Indeed, we were unable to extract RNA sample just the day before adult eclosion (day -1). Despite the unavoidable fluctuation in template titers, these experiments suggest that gene expression of most olfactory proteins starts at least two days before adult emergence (Fig. 3). Expression of the male antennae-specific AtraPBP1 starts at day 0 of adult stage or the day prior to adult emergence.


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)

Gene expression analysis by RT-PCR.Expression of AtraPBP1, AtraPBP2, AtraGOBP1, AtraGOBP2, and AtraCSP genes in control tissue (ML, male hindlegs) and olfactory tissues (MA, male antennae and FA, female antennae). Actin gene was used as endogenous control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0007235-g002: Gene expression analysis by RT-PCR.Expression of AtraPBP1, AtraPBP2, AtraGOBP1, AtraGOBP2, and AtraCSP genes in control tissue (ML, male hindlegs) and olfactory tissues (MA, male antennae and FA, female antennae). Actin gene was used as endogenous control.
Mentions: To compare transcript patterns with protein profiles (Fig. 1), RT-PCR experiments were performed using gene-specific primers. First, we compared expression of AtraPBP1, AtraPBP2, AtraGOBP1, AtraGOBP2, and AtraCSP in non-olfactory tissues (male legs) with olfactory tissues (male and female antennae) (Fig. 2). In general, gene expression mirrored protein profiles, except for AtraCSP, which was detected not only in male and female antennae, but also in non-olfactory tissues (legs). AtraPBP2, AtraGOBP1 and AtraGOBP2 genes were detected in both male and female antennae, but not in legs, whereas AtraPBP1 was apparently expressed exclusively in male antennae. Next, we assessed gene expression during antennal development. Contrary to our previous experience with the wild silkworm moth, A. polyphemus [32], sampling antennal pockets from pupae and day 0 adults of the navel orangeworm and extracting RNA were very challenging due to high RNAse activity at this developmental stage as reflected in the irregular amplifications of actin control gene (Fig. 3). Indeed, we were unable to extract RNA sample just the day before adult eclosion (day -1). Despite the unavoidable fluctuation in template titers, these experiments suggest that gene expression of most olfactory proteins starts at least two days before adult emergence (Fig. 3). Expression of the male antennae-specific AtraPBP1 starts at day 0 of adult stage or the day prior to adult emergence.

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