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Mol Biol Evol
2024 Oct 04;4110:. doi: 10.1093/molbev/msae196.
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Functional Characterization Supports Multiple Evolutionary Origins of Pheromone Receptors in Bark Beetles.
Biswas T
,
Sims C
,
Yuvaraj JK
,
Roberts RE
,
Löfstedt C
,
Andersson MN
.
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Chemical communication using pheromones is thought to have contributed to the diversification and speciation of insects. The species-specific pheromones are detected by specialized pheromone receptors. Whereas the evolution and function of pheromone receptors have been extensively studied in Lepidoptera, only a few pheromone receptors have been identified in beetles, which limits our understanding of their evolutionary histories and physiological functions. To shed light on these questions, we aimed to functionally characterize potential pheromone receptors in the spruce bark beetle Ips typographus ('Ityp') and explore their evolutionary origins and molecular interactions with ligands. Males of this species release an aggregation pheromone comprising 2-methyl-3-buten-2-ol and (4S)-cis-verbenol, which attracts both sexes to attacked trees. Using two systems for functional characterization, we show that the highly expressed odorant receptor (OR) ItypOR41 responds specifically to (4S)-cis-verbenol, with structurally similar compounds eliciting minor responses. We next targeted the closely related ItypOR40 and ItypOR45. Whereas ItypOR40 was unresponsive, ItypOR45 showed an overlapping response profile with ItypOR41, but a broader tuning. Our phylogenetic analysis shows that these ORs are present in a different OR clade as compared to all other known beetle pheromone receptors, suggesting multiple evolutionary origins of pheromone receptors in bark beetles. Next, using computational analyses and experimental validation, we reveal two amino acid residues (Gln179 and Trp310) that are important for ligand binding and pheromone specificity of ItypOR41 for (4S)-cis-verbenol, possibly via hydrogen bonding to Gln179. Collectively, our results shed new light on the origins, specificity, and ligand binding mechanisms of pheromone receptors in beetles.
2022-03597 Swedish research councils VR, Foundation in Memory of Oscar and Lili Lamm, CTS17:25 Carl, Royal Physiographic Society in Lund, Max Planck center
Fig. 1: Approximately maximum likelihood tree showing phylogenetic relationships among coleopteran Group 7 odorant receptors (ORs) and the Orco lineage, which was used to root the
tree. The ORs included are from Ips typographus (‘Ityp’) (Yuvaraj et al. 2021), Dendroctonus
ponderosae (Dpon) (Andersson et al. 2019), Rhynchophorus ferrugineus (‘Rfer’) (B. Antony,
unpublished data, included with permission), Lissorhoptrus oryzophilus (‘Lory’) (Zhang et al.
2019), Phyllotreta striolata (‘Pstr’) (Wu et al. 2016), and Anoplophora glabripennis (‘Agla’)
(McKenna et al. 2016). The tree is based on a MAFT alignment of OR amino acid sequences and
constructed using FastTree. The small clade containing the ORs marked in yellow. Local support values were calculated using the Shimodaira-Hasegawa (SH) test
implemented in FastTree. The blue capital letters indicate the ORs that have been functionally
characterized and orange asterisks indicate the pheromone receptors. Main ligands for functionally characterized ORs: A lanierone: ItypOR36 (Yuvaraj et al. 2023), B (−)-verbenone, (+)-transverbenol, (−)-trans-verbenol: ItypOR45 (present study), C (4S)-cis-verbenol: ItypOR41 (present
study), D phenylacetaldehyde: LoryOR20 (Zhang et al. 2023), E ferrugineol and ferrugineone:
RferOR1 (Antony et al. 2021), F (−)-verbenone and (S)-cis-verbenol: PstrOR17 (Xu et al. 2024),
G (+)-isopinocamphone: ItypOR29 (Hou et al. 2021), H (R)-(−)-ipsdienol: ItypOR49 (Yuvaraj et
al. 2021), I (+)-3-carene: ItypOR25, J p-cymene: ItypOR27, K E-myrcenol: ItypOR28, L (+)-
trans-4-thujanol: ItypOR23 (I-L from Hou et al. 2021), and M (S)-(−)-ipsenol: ItypOR46 (Yuvaraj
et al. 2021).
Fig. 2: Functional characterization of ItypOR41 in HEK293 cells and Xenopus laevis oocytes. (A)
Response of TREx/HEK293 cells co-expressing ItypOrco and ItypOR41 to the test odor panel and
vehicle control in the screening experiment (30 µM concentration; n = 3 biological replicates, ntotal
= 9). (+)-Induction: response of cells induced to express ItypOR41 and ItypOrco; (-)-induction:
response of non-induced control cells. As a positive control for functional Orco expression, 50 µM
VUAA1 was also tested. Asterisks (***) indicate significantly higher responses in induced versus
non-induced cells at p < 0.001; GLMM). (B) Responses of X. laevis oocytes co-expressing
ItypOrco and ItypOR41 to the test odor panel at the 100 µM screening concentration (n = 3-5; see
Supplementary table S2 for details). Different lowercase letters indicate significant differences
between the active compounds at p < 0.05 (one-way ANOVA followed by Tukey’s post hoc test).
(C) Dose-dependent responses of TREx/HEK293 cells co-expressing ItypOrco and ItypOR41 to
the four active ligands (n= 3-5 biological replicates, ntotal= 9-15). Error bars (in A and B) indicate
the full (minimum-maximum) response range, with the dots (in A, B, and C) representing
individual data points. (D) Representative raw current trace from a single oocyte co-expressing
ItypOrco and ItypOR41, showing absolute responses (in nA) to successive stimulations with
different compounds (100 µM screening concentration), DMSO (negative control), and the Orco
agonist VUAA1 (positive control) used for response normalization. The arrow heads represent
compound stimulation for 20 s. All raw data is reported in Supplementary table S2.
Fig. 2: Functional characterization of ItypOR41 in HEK293 cells and Xenopus laevis oocytes. (A)
Response of TREx/HEK293 cells co-expressing ItypOrco and ItypOR41 to the test odor panel and
vehicle control in the screening experiment (30 µM concentration; n = 3 biological replicates, ntotal
= 9). (+)-Induction: response of cells induced to express ItypOR41 and ItypOrco; (-)-induction:
response of non-induced control cells. As a positive control for functional Orco expression, 50 µM
VUAA1 was also tested. Asterisks (***) indicate significantly higher responses in induced versus
non-induced cells at p < 0.001; GLMM). (B) Responses of X. laevis oocytes co-expressing
ItypOrco and ItypOR41 to the test odor panel at the 100 µM screening concentration (n = 3-5; see
Supplementary table S2 for details). Different lowercase letters indicate significant differences
between the active compounds at p < 0.05 (one-way ANOVA followed by Tukey’s post hoc test).
(C) Dose-dependent responses of TREx/HEK293 cells co-expressing ItypOrco and ItypOR41 to
the four active ligands (n= 3-5 biological replicates, ntotal= 9-15). Error bars (in A and B) indicate
the full (minimum-maximum) response range, with the dots (in A, B, and C) representing
individual data points. (D) Representative raw current trace from a single oocyte co-expressing
ItypOrco and ItypOR41, showing absolute responses (in nA) to successive stimulations with
different compounds (100 µM screening concentration), DMSO (negative control), and the Orco
agonist VUAA1 (positive control) used for response normalization. The arrow heads represent
compound stimulation for 20 s. All raw data is reported in Supplementary table S2.
Fig. 3: Responses of Xenopus laevis oocytes co-expressing ItypOrco and ItypOR45. (A)
Responses to the tested odor panel at the 100 µM screening concentration (n = 11 for the four most
active compounds; n = 3-8 for remaining compounds; see Supplementary table S2 for details). Different lowercase letters indicate significant differences between the four most active
compounds at p < 0.05 (one-way ANOVA followed by Tukey’s post hoc test; the less active
compounds were excluded from the analysis). Error bars indicate the full (minimum-maximum)
response range, with grey dots representing individual data points. (B) Dose-dependent response
of ItypOR45 to the four most active ligands (n = 5-6), with individual data points shown as
differently colored dots. In (A) and (B), responses were normalized as the proportion of the
response to VUAA1 (not shown). (C) Representative raw current trace from a single oocyte
showing absolute responses (in nA) to successive stimulations with different pheromone
compounds at the 100 µM screening concentration, DMSO (negative control), and the Orco
agonist VUAA1 (positive control) used for response normalization. The arrow heads represent
compound stimulation for 20 s. Raw data is reported in Supplementary table S2.
Fig. 4: Protein models of ItypOR41 and ItypOR45. (A) Overview cartoon model of ItypOR41
with binding cavity shown in grey mesh. Key residues Gln179, Trp310 and Phe313 are displayed
as blue sticks. (B) Close-up of the binding site of ItypOR41 identified in this study. (C)
Overview cartoon model of ItypOR45 with binding cavity shown in grey mesh. Residues
Gln177, Trp314 and Phe317 are displayed as red sticks. (D) Close-up of the binding site of
ItypOR45 identified in this study. TM = transmembrane domain, EL = extracellular loop.
Fig. 5 Molecular docking results for ItypOR41 and ItypOR45. (A) Predicted binding of (4S)-cis verbenol (yellow) to ItypOR41 (Site I), and (B) to ItypOR45 (Site I). (C) Predicted binding of
(4S)-cis-verbenol (yellow) to ItypOR41 (Site I) from a different angle, and (D) to ItypOR45 (Site
II). Binding Site I is found at the same location in both receptors. Predicted binding of (4S)-cis24 verbenol also in Site II was only found in ItypOR45. New positions of flexible residues are shown
in yellow and predicted hydrogen bonds between the compound and Gln179 in ItypOR41 and
Tyr318 in ItypOR45 are indicated as black dashed lines.
Fig. 6: Responses of HEK293 cells co-expressing ItypOrco and four different ItypOR41 mutants.
Upper panels show the responses in the four receptors to select compounds at the 30 µM screening
concentration (n = 3 biological replicates, ntotal = 9); lower panels show dose-response data for the
same receptors (n = 3-5 biological replicates, ntotal = 9-15). Screening data for all 37 tested compounds are shown in Supplementary table S2. (+)-Induction: response of cells induced to
express ItypOR41 and ItypOrco; (-)-induction: response of non-induced control cells. As a control
for functional Orco expression, VUAA1 (50 µM) was also tested in the screening experiments.
Asterisks (***) indicate significantly higher responses in induced versus non-induced cells at p <
0.001; GLMM). Data is shown for (A) ItypOR41Gln179Ala
, (B) ItypOR41Gln179Glu , (C)
ItypOR41Trp310Ala, and (D) ItypOR41Phe313Ala . Error bars in the upper panels (screening data) show
the full (minimum-maximum) response range, with grey dots representing individual data points.
Individual data points are represented by the differently colored dots in the lower panels (dose response data). Raw data is reported in Supplementary table S2.
