Functional characterization of heterologously expressed Codling Moth Olfactory Receptors. Alberto Maria Cattaneo. Chemical Ecology research group ...
Functional characterization of heterologously expressed Codling Moth Olfactory Receptors
Alberto Maria Cattaneo Chemical Ecology research group
Piana rotaliana – Trentino, Italy
The Codling Moth Tortricid pest
http://ipmnet.org/codlingmoth/
Targeting Olfaction: Mating disruption
Insect olfaction at the molecular level
(A) A classical model of insect olfactory transduction that involves a G-protein-mediated PLC-IP3 pathway. (B) Alternative model in which the odorant receptor (OR) forms a heteromeric odorant-gated non-selective cation channel with an Orco family protein. (C) Alternative model postulating two pathways, both of which depend on the cation channel function of Orco. An ionotropic pathway involves the direct activation of Orco by an OR with a bound ligand inducing a rapid but transient cation influx. A metabotropic pathway is coupled to the G protein, and induces slow but prolonged cation currents. Takeshi et al. 2014
Isolation of OR-Coding Sequences 5’(3’)-rapid amplification of cDNA ends (RACE) mRNAs
ATG
TAA 454-sequencings
Assembling results
Partial contig, from 454-sequencing of transcripts 5’-RACE
3’-RACE
ATG ATG
TAA CDS of PRs and other SPs
TAA
Codling Moth Pheromone Receptors
6 candidate PRs within 43 candidate ORs
Bengtsson J. et al 2012
CpomOR1 ? CpomOR3 --- pear ester (Bengtsson J. et al 2014) CpomOR4 ? CpomOR5 ? CpomOR6 ?
Heterologous expression in HEK293T cells XGal- staining
CMV-BFP pEBFP plasmid (Clontech), cotransfected for Ca++-imaging
IHC of PRs in HEK293T cells Bright field
DAPI
Anti-V5tag
Merged
Control (-)
V5ORCO (+)
ORCOV5OR1
V5ORCO
V5OR1
ORCOV5OR3
V5OR3
ORCO+V5OR
ORCOV5OR4
V5OR4
V5OR
Ca++-Imaging equipment Inverted fluorescence microscope (Olympus IX-71), equipped with a cooled CCD camera (ORCA R2, Hamamatsu) Perfusion (CH1)
Gravity-fed perfusion contour (250 µL/min) Stimulus (CH3)
Wash CH3 Tube switch
Multi channel rapid solution changer (RSC-160, Bio-Logic)
Whole-cell Patch clamp apparatus
Petri (HEK cells)
Multi channel rapid solution changer (RSC-160, Bio-Logic)
Vacuum
Perfusion system Whole-cell Patch clamp apparatus
Whole-cell Patch clamp apparatus
Perfusion system Vacuum
Stimulus (CH3)
Stimulus (CH3) Perfusion (CH1) Perfusion (CH1)
The Clampex 9 software (Molecular Devices) regulate persusion according with establishment of proper stimulation protocols: i.e. Wait 10 seconds (10 s channel 1) stimulates 10 seconds (10 s channel 3) finish trial (channel 1) up to 120 s
Bright field image of HEK cells: HEK cells are incubated with a Calciumsensitive fluorescent dye (i.e. Fluo 4 - AM (Invitrogen) )
HEK cells are cotransfected with a Blue Fluorescent protein regulated by the same promoter of our gene of interest (CMV): Blue positives are «Positive» cells
HEK cells expressing Blue Fluorescent protein are selected
Ca++Imaging of CpomOrco, Stimulation with 250 µM VUAA1
Insect ORs are ionostropic receptors, activation of the receptor opens a Ca++-channel and the cytoplasmic Ca++ increasement activates the fluorescent dye
Cell fluorescence
Background fluorescence
Background fluorescence
Cell fluorescence
ΔFx = [Max(3-4) – min(1-2)] / min(1-2) Stimulus
Normalization • Find saturating concentration ΔFsat (Ligand concentration giving the same or slightly lower responses, compared to lower concentrations)
Divide ΔFx to ΔFsat (ΔFNx = ΔFx/ΔFsat) i.e. If ΔFsat = ΔF500 ΔFN10 = ΔF10/ΔF500 ΔFN50 = ΔF50/ΔF500 ΔFN100 = ΔF100/ΔF500 ΔFN250 = ΔF250/ΔF500 ΔFN500 = ΔF500/ΔF500
0.6
0.4
0.2
• Plot (SigmaPlot 11.0) 0.0 10
100
1000
Whole-cell and outside-out patch clamp recordings
VUAA
Functional expression of CpomOrco Bar: 20 µm
VUAA1: acetamide, N-(4ethylphenyl)-2-[[4-ethyl-5-(3pyridinyl)-4H-1,2,4-triazol-3yl]thio]-
VUAA3: acetamide, 2[[4-ethyl-5-(4-pyridinyl)4H-1,2,4-triazol-3yl]thio]-N-[4-(1methylethyl)phenyl]-
DmelOrco vs CpomOrco
• • • • •
C409, C429, C449: if mutated, increased sensitivity to VUAA1; (Turner et al. 2014) C228, C446: if mutated, decreased sensitivity to VUAA1; C87, C216, C221: if mutated, no change; D357, D466 in TM5 and TM7: critical for VUAA-sensitivity (Kumar et al 2013); Y478: critical for K+ selectivity in Bombyx mori ORCO (Nakagawa et al 2012).
Glutamine rather than Histidine after the VUAA1-critical C429 * * * * *
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Functional Expression studies of CpomOrco Dose/response experiments with VUAA1 and VUAA3
Functional Expression studies of CpomOrco Inhibitory experiments with the amiloride derivative 5-(N-methyl-N-isobutyl)amiloride (MIA)
Functional Expression studies of CpomOrco Whole-cell and outside-out patch clamp recordings
Monovalent cation permeability studies
CpomORs are potentially ionotropic
Rb+>K+>Cs+~Na+>Li+
Heterologous Expression of candidate CpomPRs Functional expression of CpomOR3
0.6
V5OR3
0.4
0.2
0.0 10
100
1000
Funtional expression of CpomOR3 Comparison of fluorescence variations
Heterologous Expression of candidate CpomPRs Deorphanization of CpomOR1 to Codlemone acetate Δ8,Δ10-dodecadienyl-1-yl-acetate 0.7 0.6 0.5 0.4 0.3 0.2
0.1 0.0 -0.1 1
10
100
1000
Heterologous Expression of candidate CpomPRs Deorphanization of CpomOR1 to Codlemone acetate
Bäckman et al 2000: Three receptor neurone types in Codling moth males: • I : responsive to codlemone [(E,E)-8,10-12OH]; codlemone geometric isomers [(Z,E);(E,Z);(Z,Z)] (10 fold less); codlemone acetate [(E,E)-8,10-12Ac]; codlemone acetate geometric isomers [(Z,E);(E,Z);(Z,Z)](but less then E,E isomer) • II : responsive to all codlemone acetate geometric isomers [Δ8,Δ10-12Ac] but mostly to (E,E); not responsive to any codlemone isomer
Ecological Importance of Codlemone acetate •
(E,Z+E,E)-8,10-12Ac isomers blend for Cydia splendana pheromone race from south Sweden (Oak pest) • (E,E+Z,E)-8,10-12Ac isomers blend for Cydia splendana pheromone race from South France, Switzerland, Hungary (Oak+Cestnut pest) Bengtsson M. et al 2014 •
(E,Z+E,E)-8,10-12Ac isomers blend for Cydia latiferrana (Acorns, Walnuts, Hazelnuts pest) Chambers et al 2011, Davis et al 1984 • Codlemone acetate is the main pheromone of Cydia pyrivora Makranczy et al 1998
•
Most tortricids of Eucosmini and Grapholitini tribes of the subfamily Olethreutinae use Codlemone acetate Witzgall et al 1996 •
Despite it is an antagonist to codlemone for Cydia pomonella (Hataway et al 1974) 1:1 blends with Codlemone stimulate fly upwinds (Witzgall et al 1999)
Current state CpomPRs
Bengtsson J. et al 2012
CpomOR1 E,E codlemone acetate :D CpomOR3 pear ester / methyl ester CpomOR4 ? CpomOR5 ? CpomOR6 ?
Conclusions • We functionally expressed CpomOrco and candidate PRs • We identified a novel ligand of CpomOR3 • We deorphanized CpomOR1 for E,E Codlemone acetate
Next targets • • • •
Site directed mutagenesis of Q to H in CpomORCO TMVI-VII loop to test VUAAs Deorphanization of CpomOR4 and other candidates PRs Test of further ?-(E,Z)-2,4-decadienoates for CpomOR3 Test of activation and synergism of further E8,E10-12Ac isomers (E,Z ; Z,E ; Z,Z) on CpomOR1 • Sensitivity of OR1 and OR3 to ligands at thermical conditions • Insect behavior with novel ligands
Acknowledgments Prof. Peter Witzgall Dr. William B. Walker III
Dr. Emmanuelle Jacquin-Joly
Dr. Nicolas Montagné
Dr. Jonas M. Bengtsson
Ache lab: Prof. Barry W. Ache , Dr Yuriy V. Bobkov, Dr Elizabeth Corey, Dr Kirill Ukhanov Whitney lab Director: Dr Mark Q. Martindale
Dr Gianfranco Anfora, Umberto Salvagnin & CE-research group
Prof. Angela Bassoli & Dr. Gigliola Borgonovo
Thanks for your attention
References • • •
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Bäckman, A.C., Anderson, P., Bengtsson, M., Löfqvist, J., Unelius, C.R., Witzgall, P. (2000) Antennal response of codling moth males, Cydia pomonella L. (Lepidoptera: Tortricidae), to the geometric isomers of codlemone and codlemone acetate. J Comp Physiol A. Jun;186(6):513-9. Bengtsson, J.M., Trona, F., Montagné, N., Anfora, G., Ignell, R., Witzgall, P., Jacquin-Joly, E.(2012) Putative chemosensory receptors of the Codling Moth, Cydia pomonella, identified by antennal transcriptome analysis Plos ONE February 2012, Volume 7, Issue 2, e31620 Bengtsson, J.M., Gonzalez, F., Cattaneo, A.M., Montagné, N., Walker, W.B., Bengtsson, M., Anfora, G., Ignell, R., Jacquin-Joly, E. and Witzgall, P. (2014) A predicted sex pheromone receptor of codling moth Cydia pomonella detects the plant volatile pear ester. Frontiers in Ecology and Evolution; 2(33) doi: 10.3389/fevo.2014.00033 Bengtsson, M., Boutitie, A., Jósvai, J., Toth, m., Andreadis, S., Rauscher, S., Unelius, C.R., and Witzgall, P. (2014) Pheromone races of Cydia splendana (Lepidoptera, Tortricidae) overlap in host plant association and geographic distribution . Frontiers in Ecologoy and Evolution. August 2014, Volume 2, Article 46 Chambers, U.,Walton,V.M.,andMehlenbacher,S.A. (2011). Susceptibility of hazelnut cultivars to filbertworm, Cydia latiferreana. Hort science 46, 1377–1380. Davis, H.G., McDonough, L.M., Burditt, A.K., and Bieri-Leonhardt, B.A. (1984). Filbertworm sex pheromone. Identification and field tests of (E,E)- and (E,Z)- 8,10 dodecadien-1-ol acetates. J. Chem Ecol. 10,53-61 Hathaway, D.O., McGovern, T.P., Beroza, M., Moffitt, H.R., McDonough, L.M. and Butt, B.A. (1974). An inhibitor of sexual attraction of male Codling Moths to a synthetic sex pheromone and virgin females in traps. Environ. Entomol. 3: 522–524 Makranczy ,G., To’th, M., Chambon, J-P., Unelius, C.R., Bengtsson, M., Witzgall, P. (1998) Sex pheromone of pear moth, Cydia pyrivora Danil. (Lepidoptera, Tortricidae). BioControl 43:339–44 Kumar, B.N., Taylor, R.W., Pask, G.M., Zwiebel, L.J., Newcomb, R.D., Christie, D.L. (2013) A conserved aspartic acid is important for agonist (VUAA1) and odorant/tuning receptor-dependent activation of the insect odorant co-receptor (Orco). PLOS ONE July 2013, Volume 8, Issue 7, e70218 Nakagawa, T., Pellegrino, M., Sato, K., Vosshall, L.B., Touhara, K. (2012) Amino acid residues contributing to function of the heteromeric insect olfactory receptor complex. PLOS ONE March 2012, Volume 7, Issue 3, e32372 Takeshi S, Shigehiro N, and Ryohei K (2014) Molecular and neural mechanisms of sex pheromone reception and processing in the silkmoth Bombyx mori. Front. Physiol., 31 March 2014 | doi: 10.3389/fphys.2014.00125 Turner, R.M., Derryberry, S.L., Kumar, B.N., Brittain, T., Zwiebel, L.J., Newcomb, R.D., and Christie, D.L. (2014) Mutational analysis of cysteine residues of the insect odorant co-receptor (Orco) from Drosophila melanogaster reveals differential effects on agonist- and odorant-tuning receptordependent activation. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 46, pp. 31837–31845, November 14, 2014 Witzgall ,P., Bäckman, A-C., Svensson, M., Koch, U.T., Rama, F., El-Sayed, A., Strandh, M., Brauchli, J., Arn, H., Bengtsson, M., Löfqvist, J. (1999) Behavioral observations of codling moth, Cydia pomonella, in orchards permeated with synthetic pheromone. Bio Control 44: 211-237 Witzgall ,P., Chambon , J-P., Bengtsson, M., Unelius ,C.R., Appelgren, M., et al. (1996) Sex pheromones and attractants in the Eucosmini and Grapholitini (Lepidoptera, Tortricidae). Chemoecology 7:13–23
