Fig 1.
TSWV infection enhances plant attractiveness to the thrips vector and suppresses plant terpene synthesis.
(A) Thrips preference (as percentage recaptured WFT out of 50 released) on different pepper plants. Four-week-old pepper plants were infected with TSWV (TSWV) or inoculated with buffer (mock). Plants of a similar size were used for thrips bioassay at 14 days post viral infection (dpi). Data are mean + SE, n = 6. **P < 0.01, Wilcoxon matched pairs tests. (B) Relative expression levels of TPS genes in pepper with or without thrips infestation for 6 h. Values are means + SE, n = 3. P < 0.05, one-way ANOVA plus Duncan’s multiple range tests. (C) Thrips preference (as percentage recaptured WFT out of 50 released) on N. benthamiana plants. Three-week-old N. benthamiana plants were infected with TSWV (TSWV) or inoculated with buffer (mock). Leaves of a similar size were used for the thrips bioassay at 14 dpi. Data are mean + SE, n = 6. **P < 0.01, Wilcoxon matched pairs tests. (D) Relative expression levels of various TPS genes in mock or TSWV-infected N. benthamiana after MeJA treatment. N. benthamiana plants were sprayed with 100 μM MeJA (Sigma-Aldrich) containing 0.01% (v/v) Tween 20. Values are means + SE, n = 3. *P < 0.05 **P < 0.01, Student’s t-test.
Fig 2.
TSWV infection increases attractiveness to the thrips vector in a terpene-dependent manner.
(A) Representative GC/MS ion chromatograms of the headspace volatile compounds of control (mock-thrips) and TSWV-infected peppers (TSWV-thrips) after thrips infestation for 6 h. The peaks of specific products are marked with arrows in the left panel. Relative abundance of terpenes emitted after thrips infestation are showed in the right panel. Values are means + SE, n = 4. **P < 0.01, Student’s t-test. (B) Terpenes emitted by N. benthamiana after TSWV infection (under MeJA treatment). Values are mean relative amounts (percentage of internal standard peak area) ± SE, n = 4. **P < 0.01, Student’s t-test. (C) Thrips preference (as percentage recaptured WFT out of 50 released) on the pure monoterpenes (linalool, α-pinene, β-pinene) and solvent control (n-hexane) in a two-choice assay. Data are mean percentages + SE, n = 6. *P < 0.05 **P < 0.01, Wilcoxon matched pairs tests.
Fig 3.
NSs from TSWV is a vector behavior manipulator.
(A) Phenotype of pepper leaves inoculated with recombinant Potato virus X (PVX) vectors. PVX-NSs, PVX-NSm or PVX-Ncp was transformed into peppers via agroinfiltration. PVX-GFP was used as the control. Bar = 2 cm. (B) Attractiveness of different infiltrated peppers. Agrobacteria carrying individual recombinant PVX vectors were infiltrated into peppers. Plants of a similar size were used for thrips two-choice assays at 10 dpi. Data are mean choice percentages + SE, n = 6. *P < 0.05, Wilcoxon matched pairs tests.
Fig 4.
(A) Yeast two-hybrid assay between NSs and AtMYC2. Yeast cotransformed with the indicated plasmids was spotted onto synthetic medium (SD-Leu-Trp-His) containing 0.04 mg/mL X-α-gal and 10 mM 3-amino-1,2,4-triazole (3-AT). The empty vectors pGBKT7 (BD) and pGADT7 (AD) were used as negative controls. (B) Bimolecular fluorescence complementation (BiFC) assay. NSs-cEYFP and nEYFP-AtMYC2 were transiently expressed in H2B-RFP transgenic N. benthamiana leaf epidermal cells via agroinfiltration. Bars = 15 μm. (C) GST pull-down assays between NSs and AtMYC2. (D) Interaction between NSs and AtMYC2 in Co-immunoprecipitation (Co-IP) assay. Total protein was extracted from N. benthamiana leaves transiently expressing 35S:MYC2-Myc together with 35S:YFP-NSs or 35S:YFP alone. GFP-trap beads were used to precipitate the interaction complex, Anti-GFP and Anti-Myc antibodies were using to detect the immunoprecipitates.
Fig 5.
MYC2 and its homologs in Arabidopsis are essential regulators of host immunity responses against WFT.
(A) GUS staining of AtMYC2p-GUS and AtTPS10p-GUS seedlings after 24 h of thrips feeding. An untreated line was used as a control. Arrows indicate thrips feeding sites. Bars = 2 mm. (B) Thrips preference (as percentage recaptured WFT out of 50 released) between the mutants and WT control in a two-choice assay. Three-week-old Arabidopsis plants cultured in MS medium were used for the thrips two-choice assay. Data are mean percentages + SE, n = 6. **P < 0.01, Wilcoxon matched pairs tests. (C) β-ocimene is less attractive to thrips than mock treatment in a two-choice assay. Data are mean percentages + SE, n = 6. **P < 0.01, Wilcoxon matched pairs tests. (D) Thrips preference (as percentage recaptured WFT out of 50 released) between the 35S:YFP-NSs transgenic Arabidopsis lines (NSs-1; NSs-2) and mock control in a two-choice assay. Three-week-old transgenic Arabidopsis plants cultured in MS medium were used. Data are mean percentages + SE, n = 6. **P < 0.01, Wilcoxon matched pairs tests.
Fig 6.
NSs promotes WFT performance by targeting MYC-mediated host defense.
(A-C) Effects of different genes on the number of WFT offspring. Seven adult females fed on each three-week-old Arabidopsis line. After 2 weeks, new larvae and adults were counted. Values are means ± SE, n = 8. *P < 0.05, **P < 0.01, Student’s t-test.
Fig 7.
A conserved protein interaction between Orthotospovirus NSs and plant MYC2 proteins.
(A) BiFC assays of the interaction between TSWV NSs and CaMYC2. H2B-RFP transgenic N. benthamiana plants, which express a nucleus marker were used in this assay. Bars = 15 μm. (B) Co-immunoprecipitation (Co-IP) assay of the interaction between TSWV NSs and CaMYC2. GFP-trap beads were used to precipitate the interaction complex. (C) BiFC assays of the interaction between TZSV NSs–AtMYC2 and TZSV NSs–CaMYC2. H2B-RFP transgenic N. benthamiana plants were used in this assay. Bars = 15 μm. (D) Co-immunoprecipitation (Co-IP) assay of the interaction between TZSV NSs–AtMYC2 (left panel) and TZSV NSs–CaMYC2 (right panel). GFP-trap beads were used to precipitate the interaction complex.