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Many plant secondary substances are feeding deterrents for insects and play a key role in the selection of host plants. The taste sensilla of phytophagous insects contain gustatory sensory neurons sensitive to deterrents but the molecular basis of deterrent chemoreception remains unknown. We investigated the function of Gr180, the most highly expressed bitter gustatory receptor in the maxillary galea of Helicoverpa armigera larvae. Functional analyses using the Xenopus oocyte expression system and two-electrode voltage clamp revealed that the oocytes expressing Gr180 responded to coumarin. Tip recording results showed that the medial sensilla styloconica of the maxilla of fifth instar larvae exhibited electrophysiological responses to coumarin. Two-choice feeding bioassays confirmed that coumarin inhibited larval feeding. A homozygous mutant strain of H. armigera with truncated Gr180 proteins (Gr180-/-) was established using the CRISPR-Cas9 system. The responses of the medial sensilla styloconica in Gr180-/- to coumarin were almost abolished, and the responses to sinigrin and strychnine were also significantly decreased. Knockout of Gr180 alleviated the feeding deterrent effects of coumarin, sinigrin, and strychnine. Thus, we conclude that Gr180 is a receptor responding to coumarin,and also participates in sensing sinigrin and strychnine. These results enhance our understanding of the gustatory sensing mechanisms of phytophagous insects to deterrents.
Figure 2. Fig 2. Functional analysis of Helicoverpa armigera Gr180 in Xenopus oocytes. (A) Representative inward current responses of Xenopus oocytes expressing Gr180 in response to compounds. (B) Response profiles of Xenopus oocytes expressing Gr180 in response to compounds (n = 8–14). (C) Representative inward current responses of Xenopus oocytes expressing Gr180 in response to coumarin at a range of concentrations. (D) Dose responses of Xenopus oocytes expressing Gr180 to coumarin (n = 8). Data are mean ± SEM. Different letters are significantly different at p < 0.05 (one-way ANOVA followed by post-hoc analysis with Tukey’s HSD test). https://doi.org/10.1371/journal.pgen.1010455.g002
S2 Fig. Responses of Xenopus oocytes expressing HarmGr67, HarmGr68, or injected with distilled water to stimulated compounds. No inward current responses of Xenopus oocytes injected with (A) HarmGr67, (B) HarmGr68, or (C) distilled water to tested compounds. https://doi.org/10.1371/journal.pgen.1010455.s002
Fig 1. Expression patterns of gustatory receptors (GRs) in Helicoverpa armigera.(A) The TPM value of the top 20 highly expressed GRs in the larval maxillary galea of H. armigera via transcriptome sequencing. (B) Relative transcript levels of Gr180 in the organs of the fifth instar larvae by qRT-PCR. (C) Relative transcript levels of Gr180 in the organs of virgin female and male adults. Data are mean ± SEM, n = 3. Columns with different letters are significantly different at p < 0.05 (one-way ANOVA followed by post-hoc analysis with Tukey’s HSD test).
Fig 2. Functional analysis of Helicoverpa armigera Gr180 in Xenopus oocytes.(A) Representative inward current responses of Xenopus oocytes expressing Gr180 in response to compounds. (B) Response profiles of Xenopus oocytes expressing Gr180 in response to compounds (n = 8–14). (C) Representative inward current responses of Xenopus oocytes expressing Gr180 in response to coumarin at a range of concentrations. (D) Dose responses of Xenopus oocytes expressing Gr180 to coumarin (n = 8). Data are mean ± SEM. Different letters are significantly different at p < 0.05 (one-way ANOVA followed by post-hoc analysis with Tukey’s HSD test).
Fig 3. Electrophysiological response of sensilla styloconica on the maxillary galea of Helicoverpa armigera larvae to coumarin.(A) Representative responses and (B) spike frequencies of lateral and medial sensilla styloconica to coumarin and double distilled water (ddH2O) at 10−3 M (n = 8). *** and ns indicate significant difference (p < 0.001) and no significant difference (p > 0.05), respectively (two-tailed independent samples t-test). (C) Representative responses of medial sensilla styloconica to coumarin at a series of concentrations. (D) Dose responses of medial sensilla styloconica to coumarin (n = 8–10). Different letters indicate significant difference (one-way ANOVA followed by post-hoc analysis with Tukey’s HSD test). Data are mean ± SEM.
Fig 4. Feeding deterrence of the fifth instar Helicoverpa armigera larvae to coumarin by contact chemoreception.(A) Two-choice feeding assays with cowpea leaf discs: the feeding area of coumarin-treated discs (grey bars) and control discs (white bars) were measured (n = 19–20). (B) Modified two-choice feeding assays with ‘sandwich’ leaf discs: in the contact two-choice feeding assay, coumarin or control discs were painted on the upper leaf discs (n = 25); in the non-contact feeding assay, coumarin or control discs were painted on the lower leaf discs that prevented larvae from feeding (n = 24); the feeding area of the consumed upper leaf discs was measured. Feeding deterrence index (D.I.) = (consumed areas of the control discs—consumed areas of the treated discs) / (consumed areas of the control discs + consumed areas of the treated discs). Data are mean ± SEM. *** and ns indicate significant difference (p < 0.001) and no significant difference (p > 0.05), respectively (two-tailed paired samples t-test).
Fig 5. Establishment of Gr180 homozygous mutants of Helicoverpa armigera via CRISPR-Cas9.(A) Genomic structure of Gr180 and designation of sgRNA. Exons are shown as boxes and the lines between two exons indicate the introns. The sgRNA are located on the antisense strand of exon-1 (green box). The sgRNA targeting sequence is shown in blue and the PAM sequence is shown in red. (B) Various mutant genotypes of Gr180 identified by sequencing of the G1 adult PCR products. Purple inverted triangle indicates the cleavage site of Cas9 nuclease. Dashes indicate the deleted bases; lowercase letters are the inserted bases. The numbers of inserted or deleted bases are displayed at the right of each allele (+ insertion;–deletion). Red asterisks indicate the selected genotype to establish the homozygous mutant strain. (C) Representative chromatograms of direct sequencing of the PCR products obtained from wild types (Gr180+/+, upper graph), heterozygous mutants (Gr180+/−, middle graph), and homozygous mutants (Gr180−/−, lower graph). The start site of overlapping peaks is marked by a purple arrow. (D) Secondary structure prediction of wild type and truncated Gr180 protein. TOPCONS (topcons.net) models were used to predict secondary structure, and TOPO2 software (http://www.sacs.ucsf.edu/TOPO2/) was used to construct the images. In WT, the Gr180 protein consists of seven transmembrane domains, the truncated protein consists of three transmembrane domains in the mutants.
Fig 6. Electrophysiological responses of medial sensilla styloconica in larvae of Helicoverpa armigera wild type (WT) and Gr180−/− mutants.Representative responses (A, C, E, G) and spike frequencies (B, D, F, H) of medial sensilla styloconica to 10−2 M coumarin (A, B), 10−2 M sinigrin (C, D), 10−2 M strychnine (E, F), 10−3 M inositol (G, H). Data are mean ± SEM, n = 7–11. ** and *** indicate significant differences at the level of p<0.01 and p<0.001, respectively; ns indicates no significant difference (p>0.05) (two-tailed independent samples).
Fig 7. Feeding responses of Helicoverpa armigera wild type (WT) and Gr180−/− mutant larvae to four deterrent compounds.(A) Control vs 10−2 M coumarin (WT: n = 19; Gr180−/−: n = 19). (B) Control vs 10−2 M sinigrin (WT: n = 20; Gr180−/−: n = 21). (C) Control vs 10−2 M strychnine (WT: n = 17; Gr180−/−: n = 21). (D) Control vs 10−3 M azadirachtin (WT: n = 9; Gr180−/−: n = 8). Data are mean ± SEM. ** and *** indicate significant difference at the level of p < 0.01 and p < 0.001, respectively; ns indicates no significant difference (p > 0.05).
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