Yang J et al. (2021), Identification of a gustatory receptor tuned to...

XB-ART-58570

PLoS Genet 2021 Jul 06;177:e1009527. doi: 10.1371/journal.pgen.1009527.

Show Gene links Show Anatomy links

Identification of a gustatory receptor tuned to sinigrin in the cabbage butterfly Pieris rapae.

Yang J , Guo H , Jiang NJ , Tang R , Li GC , Huang LQ , van Loon JJA .

???displayArticle.abstract???
Glucosinolates are token stimuli in host selection of many crucifer specialist insects, but the underlying molecular basis for host selection in these insects remains enigmatic. Using a combination of behavioral, electrophysiological, and molecular methods, we investigate glucosinolate receptors in the cabbage butterfly Pieris rapae. Sinigrin, as a potent feeding stimulant, elicited activity in larval maxillary lateral sensilla styloconica, as well as in adult medial tarsal sensilla. Two P. rapae gustatory receptor genes PrapGr28 and PrapGr15 were identified with high expression in female tarsi, and the subsequent functional analyses showed that Xenopus oocytes only expressing PrapGr28 had specific responses to sinigrin; when ectopically expressed in Drosophila sugar sensing neurons, PrapGr28 conferred sinigrin sensitivity to these neurons. RNA interference experiments further showed that knockdown of PrapGr28 reduced the sensitivity of adult medial tarsal sensilla to sinigrin. Taken together, we conclude that PrapGr28 is a gustatory receptor tuned to sinigrin in P. rapae, which paves the way for revealing the molecular basis of the relationships between crucifer plants and their specialist insects.

???displayArticle.pubmedLink??? 34264948
???displayArticle.pmcLink??? PMC8282186
???displayArticle.link??? PLoS Genet

???attribute.lit??? ???displayArticles.show???

	Fig 1. Feeding preference of the fifth instar larvae of P. rapae to glucosinolates.Cowpea leaf discs were used as the substrate for the two choice assays of fifth instar P. rapae larvae. The upper surface of each disc was treated with 20 μL of glucosinolate solutions. Each control disc was supplied with the same volume of water. The concentration gradients of (A) sinigrin, (B) gluconapin, (C) glucoiberin, (D) glucobrassicin, and (E) gluconasturtiin all ranged from 10−6 to 10−2 M. When the total feeding area was larger than 25% or after 24 h feeding, the area of each disc consumed by larvae was measured, and the feeding preference index was calculated. Differences in feeding amounts on treated and control discs were tested by paired Student’s t-test. n represents the replicates of larvae and are labeled in the figures. Data are presented as mean ± SEM. * P < 0.05, P < 0.01, * P < 0.001.
	Fig 2. Response properties of sensilla styloconica on larval maxilla of P. rapae to glucosinolates.(A) Representative responses and (B) spike frequencies of lateral sensilla styloconica (n = 10); (C) representative responses and (D) spike frequencies of medial sensilla styloconica (n = 11). All tested glucosinolates were at 10 mM, and 2 mM KCl was used as control. (E-I) Dose-response curves of lateral sensilla styloconica to sinigrin (n = 10–11), gluconapin (n = 10–12), glucoiberin (n = 10), glucobrassicin (n = 5–7), and gluconasturtiin (n = 10), respectively; (J) dose-response curves of medial sensilla styloconica to glucobrassicin (n = 6–8). Data are presented as mean ± SEM. One-way ANOVA with Tukey HSD test was used. *** P < 0.001, compared with KCl control.
	Fig 3. Response properties of taste sensilla on the fifth foreleg-tarsi of female P. rapae to glucosinolates.(A) Representative responses and (B) spike frequencies of female lateral tarsal sensilla (n = 7–15); (C) representative responses and (D) spike frequencies of female medial tarsal sensilla (n = 10–17). All tested glucosinolates were at 10 mM, and 2 mM KCl was used as control. (E, F) Dose-response curves from female lateral tarsal sensilla to gradient concentration of glucobrassicin (n = 6) and gluconasturtiin (n = 5); (G-K) dose-response curves of female medial tarsal sensilla to sinigrin (n = 10), gluconapin (n = 8), glucoiberin (n = 7–8), glucobrassicin (n = 8), and gluconasturtiin (n = 8), respectively. Data are presented as mean ± SEM. One-way ANOVA with Tukey HSD test was used. Different letters labeled indicate significant differences. * P < 0.05, P < 0.01, * P < 0.001, compared with KCl control.
	Fig 4. Response properties of taste sensilla on the fifth foreleg-tarsi of male P. rapae to glucosinolates.(A) Representative responses and (B) spike frequencies of lateral tarsal sensilla (n = 9–16); (C) representative responses and (D) spike frequencies of medial tarsal sensilla (n = 12–18). All tested glucosinolates were at 10 mM, and 2 mM KCl was used as control. (E, F) Dose-response curves of lateral tarsal sensilla to glucobrassicin (n = 6–7) and gluconasturtiin (n = 3). (G-K) Dose-response curves of medial tarsal sensilla to sinigrin (n = 6–7), gluconapin (n = 6–8), glucoiberin (n = 5–7), glucobrassicin (n = 7–8), and gluconasturtiin (n = 7), respectively. Data are presented as mean ± SEM. One-way ANOVA with Tukey HSD test was used. Different letters labeled indicate significant differences. * P < 0.05, P < 0.01, * P < 0.001, compared with KCl control.
	Fig 5. Phylogenetic relationships and tissue expression patterns of GR genes in P. rapae.(A) Phylogenetic tree of candidate GRs from P. rapae and other Lepidoptera species. Phylogenetic tree was constructed using Maximum likelihood phylogenies with JTT + F + G4 model. The purple, brown, yellow and black arcs represent sugar receptors, fructose receptors, CO2 receptors and bitter receptors, respectively. Prap, Pieris rapae (red); Bm, Bombyx mori (green); Hm, Heliconius melpomene (blue). (B) Expression profiles of candidate GR genes. Transcript levels were detected by qRT-PCR and calculated based on the 2−ΔΔCt method. n = 3. Data are presented as mean ± SEM. One-way ANOVA with Tukey HSD test was used. * P < 0.05, P < 0.01, * P < 0.001, compared with female tarsi.
	Fig 6. Functional analysis of PrapGrs in Xenopus oocytes.(A, B) Response profiles of Xenopus oocytes expressing PrapGr28 (A) and PrapGr15 (B) in response to compounds at 1 mM. *** P < 0.001. n represents the number of oocytes and are labeled in the figures. (C-F) Inward current responses (C, E) and dose-response curve (D, F) of Xenopus oocytes expressing PrapGr28 (n = 5–6), and PrapGr15 (n = 5) stimulated with a range of sinigrin concentrations, respectively. Data are presented as mean ± SEM. Different letters labeled indicate significant differences. One-way ANOVA with Tukey HSD test was used.
	Fig 7. The presence of PrapGr28 confer sinigrin sensitivity to D. melanogaster sweet neuron.(A) Schematic diagram of PrapGr28 expressed in sweet neuron of D. melanogaster L-type sensilla. (B) Expression of PrapGr28 in the labellum of fly lines. Tubulin was used as reference gene. (C) Representative responses and (D) spike frequencies of L-type sensilla on the labellum of fly lines to glucosinolates. KCl, n = 10–11; sinigrin, n = 10–11; gluconapin, n = 8–11; glucoiberin, n = 9–11; glucobrassicin, n = 8–11; gluconasturtiin, n = 9–11. All tested glucosinolates were at 10 mM, and 1 mM KCl was used as control. (E) Dose-response curves of L-type sensilla to sinigrin. n = 7–12. Data are presented as mean ± SEM. One-way ANOVA with Tukey HSD test was used. * P < 0.05, *** P < 0.001, compared with control flies.
	Fig 8. RNA interference of PrapGr28 suppresses the response to glucosinolates in female butterflies.(A) Schematic of PrapGr28 and the regions used for dsRNA synthesis. Region a and b, named PrapGr28 a and PrapGr28 b, respectively, are portions of the coding region of PrapGr28 used for preparation of dsRNA. (B) Relative expression levels of PrapGr28 in dsRNA-injected adult butterflies. n = 4–5. WT, wild type without injection; dsGFP, GFP dsRNA; dsGr28 a, PrapGr28 a dsRNA; dsGr28 b, PrapGr28 b dsRNA; dsGr28 a+b, PrapGr28 a+b dsRNA. (C) Representative responses and (D) smaller amplitude spike frequencies elicited by glucosinolates in the medial sensilla of the fifth prothoracic tarsi. Sinigrin, n = 10–13; gluconapin, n = 9–14; glucoiberin, n = 7–13; glucobrassicin, n = 6–12; gluconasturtiin, n = 6–14. All tested glucosinolates were at 10 mM. (E, F) Dose-response curves from medial sensilla on the prothoracic tarsi of female butterflies to gradient concentration of sinigrin (n = 5–9) and gluconapin (n = 5–9), respectively. Data are presented as mean ± SEM. One-way ANOVA with Tukey HSD test was used. * P < 0.05, P < 0.01, * P < 0.001, compared with the control.

References [+] :

Agnihotri, Gustatory receptors in Lepidoptera: chemosensation and beyond. 2016, Pubmed

	Fig 1. Feeding preference of the fifth instar larvae of P. rapae to glucosinolates.Cowpea leaf discs were used as the substrate for the two choice assays of fifth instar P. rapae larvae. The upper surface of each disc was treated with 20 μL of glucosinolate solutions. Each control disc was supplied with the same volume of water. The concentration gradients of (A) sinigrin, (B) gluconapin, (C) glucoiberin, (D) glucobrassicin, and (E) gluconasturtiin all ranged from 10−6 to 10−2 M. When the total feeding area was larger than 25% or after 24 h feeding, the area of each disc consumed by larvae was measured, and the feeding preference index was calculated. Differences in feeding amounts on treated and control discs were tested by paired Student’s t-test. n represents the replicates of larvae and are labeled in the figures. Data are presented as mean ± SEM. * P < 0.05, P < 0.01, * P < 0.001.
	Fig 2. Response properties of sensilla styloconica on larval maxilla of P. rapae to glucosinolates.(A) Representative responses and (B) spike frequencies of lateral sensilla styloconica (n = 10); (C) representative responses and (D) spike frequencies of medial sensilla styloconica (n = 11). All tested glucosinolates were at 10 mM, and 2 mM KCl was used as control. (E-I) Dose-response curves of lateral sensilla styloconica to sinigrin (n = 10–11), gluconapin (n = 10–12), glucoiberin (n = 10), glucobrassicin (n = 5–7), and gluconasturtiin (n = 10), respectively; (J) dose-response curves of medial sensilla styloconica to glucobrassicin (n = 6–8). Data are presented as mean ± SEM. One-way ANOVA with Tukey HSD test was used. *** P < 0.001, compared with KCl control.
	Fig 3. Response properties of taste sensilla on the fifth foreleg-tarsi of female P. rapae to glucosinolates.(A) Representative responses and (B) spike frequencies of female lateral tarsal sensilla (n = 7–15); (C) representative responses and (D) spike frequencies of female medial tarsal sensilla (n = 10–17). All tested glucosinolates were at 10 mM, and 2 mM KCl was used as control. (E, F) Dose-response curves from female lateral tarsal sensilla to gradient concentration of glucobrassicin (n = 6) and gluconasturtiin (n = 5); (G-K) dose-response curves of female medial tarsal sensilla to sinigrin (n = 10), gluconapin (n = 8), glucoiberin (n = 7–8), glucobrassicin (n = 8), and gluconasturtiin (n = 8), respectively. Data are presented as mean ± SEM. One-way ANOVA with Tukey HSD test was used. Different letters labeled indicate significant differences. * P < 0.05, P < 0.01, * P < 0.001, compared with KCl control.
	Fig 4. Response properties of taste sensilla on the fifth foreleg-tarsi of male P. rapae to glucosinolates.(A) Representative responses and (B) spike frequencies of lateral tarsal sensilla (n = 9–16); (C) representative responses and (D) spike frequencies of medial tarsal sensilla (n = 12–18). All tested glucosinolates were at 10 mM, and 2 mM KCl was used as control. (E, F) Dose-response curves of lateral tarsal sensilla to glucobrassicin (n = 6–7) and gluconasturtiin (n = 3). (G-K) Dose-response curves of medial tarsal sensilla to sinigrin (n = 6–7), gluconapin (n = 6–8), glucoiberin (n = 5–7), glucobrassicin (n = 7–8), and gluconasturtiin (n = 7), respectively. Data are presented as mean ± SEM. One-way ANOVA with Tukey HSD test was used. Different letters labeled indicate significant differences. * P < 0.05, P < 0.01, * P < 0.001, compared with KCl control.
	Fig 5. Phylogenetic relationships and tissue expression patterns of GR genes in P. rapae.(A) Phylogenetic tree of candidate GRs from P. rapae and other Lepidoptera species. Phylogenetic tree was constructed using Maximum likelihood phylogenies with JTT + F + G4 model. The purple, brown, yellow and black arcs represent sugar receptors, fructose receptors, CO2 receptors and bitter receptors, respectively. Prap, Pieris rapae (red); Bm, Bombyx mori (green); Hm, Heliconius melpomene (blue). (B) Expression profiles of candidate GR genes. Transcript levels were detected by qRT-PCR and calculated based on the 2−ΔΔCt method. n = 3. Data are presented as mean ± SEM. One-way ANOVA with Tukey HSD test was used. * P < 0.05, P < 0.01, * P < 0.001, compared with female tarsi.
	Fig 6. Functional analysis of PrapGrs in Xenopus oocytes.(A, B) Response profiles of Xenopus oocytes expressing PrapGr28 (A) and PrapGr15 (B) in response to compounds at 1 mM. *** P < 0.001. n represents the number of oocytes and are labeled in the figures. (C-F) Inward current responses (C, E) and dose-response curve (D, F) of Xenopus oocytes expressing PrapGr28 (n = 5–6), and PrapGr15 (n = 5) stimulated with a range of sinigrin concentrations, respectively. Data are presented as mean ± SEM. Different letters labeled indicate significant differences. One-way ANOVA with Tukey HSD test was used.
	Fig 7. The presence of PrapGr28 confer sinigrin sensitivity to D. melanogaster sweet neuron.(A) Schematic diagram of PrapGr28 expressed in sweet neuron of D. melanogaster L-type sensilla. (B) Expression of PrapGr28 in the labellum of fly lines. Tubulin was used as reference gene. (C) Representative responses and (D) spike frequencies of L-type sensilla on the labellum of fly lines to glucosinolates. KCl, n = 10–11; sinigrin, n = 10–11; gluconapin, n = 8–11; glucoiberin, n = 9–11; glucobrassicin, n = 8–11; gluconasturtiin, n = 9–11. All tested glucosinolates were at 10 mM, and 1 mM KCl was used as control. (E) Dose-response curves of L-type sensilla to sinigrin. n = 7–12. Data are presented as mean ± SEM. One-way ANOVA with Tukey HSD test was used. * P < 0.05, *** P < 0.001, compared with control flies.
	Fig 8. RNA interference of PrapGr28 suppresses the response to glucosinolates in female butterflies.(A) Schematic of PrapGr28 and the regions used for dsRNA synthesis. Region a and b, named PrapGr28 a and PrapGr28 b, respectively, are portions of the coding region of PrapGr28 used for preparation of dsRNA. (B) Relative expression levels of PrapGr28 in dsRNA-injected adult butterflies. n = 4–5. WT, wild type without injection; dsGFP, GFP dsRNA; dsGr28 a, PrapGr28 a dsRNA; dsGr28 b, PrapGr28 b dsRNA; dsGr28 a+b, PrapGr28 a+b dsRNA. (C) Representative responses and (D) smaller amplitude spike frequencies elicited by glucosinolates in the medial sensilla of the fifth prothoracic tarsi. Sinigrin, n = 10–13; gluconapin, n = 9–14; glucoiberin, n = 7–13; glucobrassicin, n = 6–12; gluconasturtiin, n = 6–14. All tested glucosinolates were at 10 mM. (E, F) Dose-response curves from medial sensilla on the prothoracic tarsi of female butterflies to gradient concentration of sinigrin (n = 5–9) and gluconapin (n = 5–9), respectively. Data are presented as mean ± SEM. One-way ANOVA with Tukey HSD test was used. * P < 0.05, P < 0.01, * P < 0.001, compared with the control.