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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 = 8â14). (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 Tukeyâs 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
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