Fig 1.
Frankliniella intonsa prefers volatiles emitted by TZSV-infected pepper plants.
(A) Choice of F. intonsa on pepper plants that were mock-inoculated (MP) or virus-infected (IP). Significant differences were determined by chi-squared (χ2) test (P < 0.05). (B) Electroantennogram (EAG) responses of F. intonsa antennae to volatile compounds. Different lowercase letters indicate significant differences between doses (P < 0.05). Ten individuals were assayed per dose and per compound. Data are means ± SE. (C) Preference tests of F. intonsa to volatile compounds. Data are means ± SE, expressed as a percentage of the number of thrips (n = 60) in a Y-tube olfactometer exposed to one of six volatile compounds induced by TZSV infection (right) or liquid paraffin (control, left). Significant differences were determined by χ2 tests (P < 0.05).
Fig 2.
Odorant responses of olfactory genes of Frankliniella intonsa after exposure to TZSV-infected pepper plant odors.
(A) Relative transcript levels of FintCSP1, FintCSP2, FintOBP, and FintOR in F. intonsa after exposure to TZSV-infected (IP) or mock-inoculated (MP) pepper plants. *, P < 0.05, as determined by t-test between MP and IP treatments; ns, not significant. Each histogram bar represents the mean (± standard error) of three replicates. (B) Relative transcript levels of FintCSP1, FintCSP2, FintOBP, and FintOR in F. intonsa injected with dsFintCSP1, dsFintCSP2, dsFintOBP, dsFintOR, and dsEGFP (control). Each gene contained three replicates. *, P < 0.05, as determined by t-test between control and treatment. Each histogram bar represents the mean (± standard error) of three replicates. (C) Behavioural responses of F. intonsa to plants after injection with dsFintCSP1, dsFintCSP2, dsFintOBP, dsFintOR, and dsEGFP (control). Significant differences were determined by chi-squared (χ2) test (P < 0.05). (D) Behavioural responses of thrips to volatile compounds induced by TZSV infection (right) or liquid paraffin (control, left) after injection with the indicated dsRNA. Significant differences between the treatment (dsFintCSP1) and the control (dsEGFP) were determined by χ2 test (P < 0.05). (E) EAG responses of dsRNA-injected F. intonsa to individual volatile compounds. *, P < 0.05, as determined by t-test between the treatment (dsFintCSP1) and the control (dsEGFP); ns, not significant.
Fig 3.
FintCSP1 and FintCSP1 abundance in antennae of Frankliniella intonsa.
(A) FinCSP1 relative transcript levels in antennae at various developmental stages of F. intonsa. Data are means ± SE. Different lowercase letters indicate significant differences, as determined by one-way ANOVA (P < 0.05). 1st: 1 instar nymphs; 2nd: 2 instar nymphs; Pu: pupae; Fe-2, Fe-5, Fe-10: 2-, 5-, and 10-day-old female adults; Ma-2, Ma-5, Ma-10: 2-, 5-, and 10-day-old male adults. (B) Distribution of FintCSP1 in female antennae of F. intonsa. Thrips were immunostained with FintCSP1-FITC (green) and observed under a confocal laser scanning microscope. FI-V: Flagellomeres I-V; P: pedicel; S: scape. Scale bars, 50 μm. (C) Immunolocalization of FintCSP1 in female adult antennae of F. intonsa. Sensilla basiconica in flagellum I was immunolabeled with FintCSP1-specific IgG as the primary antibody, followed by a specific secondary antibody that had been conjugated with 12-nm gold particles (white arrows). Scale bar, 200 nm.
Fig 4.
SDS-PAGE analysis of recombinant target proteins.
M, Molecular weight marker; 1, non-induced pET30a/FintCSP1; 2, induced crude extract from pET30a/FintCSP1; 3, supernatant of pET30a/FintCSP1; 4, purified recombinant wild-type FintCSP1 protein; 5−10, purified recombinant variants harboring the individual mutations Lys26Ala, Phe27Ala, Thr28Ala, Glu67Ala, Ser84Ala, and Val132Ala, respectively.
Fig 5.
Binding of 1-NPN to FintCSP1 and competitive binding of FintCSP1 with ligands.
(A) Binding curve of 1-NPN to FintCSP1. Inset: Scatchard plot analysis. (B) Binding assays between recombinant FintCSP1 and cis-3-hexenal or trans-2-hexenal. A mixture of recombinant FintCSP1 protein and 1-NPN in 50 mM Tris-HCl buffer (pH 7.4) both at a concentration of 2 μM was titrated with each competing ligand over a final concentration range of 2–20 μM. Data represent the means of three independent replicates. Error bars indicate SE.
Fig 6.
Homology modeling and molecular docking analysis for FintCSP1.
(A) Structure-based sequence alignment between FintCSP1 and the template (CSPsg4) structure. Identical or similar residues are highlighted in blue, and dissimilar ones are highlighted in red; darker colors indicate more similar or dissimilar residues. Residues corresponding to α-helix regions are marked by horizontal red lines; random coil or turn regions are marked by horizontal blue lines. Conserved cysteines are marked by orange box. (B) Predicted 3D model of FintCSP1 (a) and superposed FintCSP1 model onto the template structure (b). FintCSP1 is shown in cyan, and the template structure is shown in violet. (C) Interaction diagram between FintCSP1 amino acid residues and cis-3-hexenal. Cis-3-hexenal is shown in cyan. Surrounding residues in the binding pocket are colored in yellow. Hydrogen bond is depicted as a green dashed line.
Fig 7.
Binding curves of wild-type FintCSP1 and selected mutants to cis-3-hexenal.
(A) Binding curve of 1-NPN to wild-type FintCSP1 or each of the six variants. (B) Competitive binding curves between wild-type FintCSP1 or variants and cis-3-hexenal. A mixture of recombinant wild-type FintCSP1 and mutant protein and 1-NPN in 50 mM Tris-HCl buffer (pH 7.4) both at 2 μM was titrated with each competing ligand over the concentration range of 2–20 μM. Data represent means of three independent replicates. Error bars indicate SE.
Table 1.
Binding affinities of cis-3-hexenal to wild-type FintCSP1 and mutants.
Fig 8.
Frankliniella occidentalis prefers volatile cues from TZSV-infected pepper plants.
(A) Choice of F. occidentalis on pepper plants that were mock-inoculated (MP) or virus–infected (IP). Significant differences were determined by chi-squared (χ2) test (P < 0.05). (B) Relative transcript levels of FoccCSP in F. occidentalis after exposure to TZSV-infected (IP) or mock-inoculated (MP) pepper plants. *, P < 0.05, as determined by t-test between MP and IP treatments. Each histogram bar represents the mean (± standard error) of three replicates. (C) Relative transcript levels of FoccCSP in F. occidentalis injected with dsFoccCSP and dsEGFP (control). Each histogram bar represents the mean (± standard error) of three replicates. *, P < 0.05, as determined by t-test between control and treatment. (D) Behavioural responses of F. occidentalis to mock-inoculated (MP) or virus-infected (IP) plants after injection with dsFoccCSP or dsEGFP (control). Significant differences were determined by χ2 test (P< 0.05). (E) Behavioural responses of thrips to cis-3-hexenal (right) or liquid paraffin (control, left) after injection with dsFoccCSP or dsEGFP. Significant differences between the treatment (dsFoccCSP) and the control (dsEGFP) were determined by χ2 test (P < 0.05). (F) EAG responses of dsFoccCSP-injected F. occidentalis to cis-3-hexenal. *, P <0.05, as determined by t-test between the treatment (dsFoccCSP) and the control (dsEGFP).
Table 2.
Binding affinities of cis-3-hexenal to wild-type FoccCSP and mutants.
Fig 9.
Binding curves of wild-type FoccCSP and selected mutants to cis-3-hexenal.
(A) Binding curve of 1-NPN to wild-type FoccCSP or each of the nine variants. (B) Competitive binding curves between wild-type FoccCSP or variants and cis-3-hexenal. A mixture of recombinant wild-type FoccCSP and mutant protein and 1-NPN in 50 mM Tris-HCl buffer (pH 7.4) both at 2 μM was titrated with each competing ligand over the concentration range of 2–20 μM. Data represent means of three independent replicates. Error bars indicate SE.