Tomato Spotted Wilt Virus Promotes Offspring and Egg Production of Its Vector, Frankliniella occidentalis, by Suppressing Plant Defences Induced by a Thrips Salivary Elicitor

Abstract

Western flower thrips (Frankliniella occidentalis) are among the most significant invasive pests worldwide. In addition to causing direct plant damage, they transmit tomato spotted wilt virus (TSWV) (species Orthotospovirus tomatomaculae; genus Orthotospovirus), a member of the genus Orthotospovirus. Although numerous studies have examined virus-insect-host plant interactions, research on the TSWV-thrips-plant tripartite interaction remains limited. In this study, we found that F. occidentalis can induce plant defence responses. FoCSP1, a chemosensory protein from F. occidentalis, was identified as a salivary elicitor capable of inducing serial plant defence responses in Nicotiana benthamiana. Our results revealed that the FoCSP1-induced plant defence responses did not affect thrips feeding preference but significantly inhibited both offspring and egg production. Moreover, TSWV impairs these defence responses through its encoded proteins, N and NSs, thereby alleviating the FoCSP1-mediated suppression of thrips offspring and egg production. Collectively, these findings indicate that TSWV promotes the offspring and egg production of its thrips vector by inhibiting plant defences induced by FoCSP1, providing new insights into the TSWV-thrips-plant tripartite interaction.

Keywords: Frankliniella occidentalis; CSP1; plant defence responses; salivary elicitor; tomato spotted wilt virus.

FIGURE 1

Identification of FoCSP1 as a putative salivary elicitor of Frankliniella occidentalis . (a) Phylogenetic analysis of the amino acid sequence of the identified chemosensory protein (CSP) with known thrips CSPs. The CSP identified in this study is highlighted with a red star. Focc represents F. occidentalis , and Fint represents F. intonsa . (b) Sequence alignment of FoCSP1 with CSPs from Myzus persicae and Sitobion miscanthi , highlighting the signal peptide and four conserved cysteine residues (CX₆CX₁₈CX₂C). (c) FoCSP1 expression in the head, anterior thorax (containing the salivary gland), and abdomen of adult thrips was analysed by reverse transcription‐quantitative PCR and normalised to FoActin. Different letters (a, b, c) indicate statistically significant differences (p < 0.05) based on one‐way ANOVA with Tukey's test; error bars represent SEM (n = 4). The experiment was replicated three times with similar results. (d) FoCSP1‐FLAG was transiently expressed through agroinfiltration, with an empty vector (EV) as a control. Chlorosis became visible at 2 days post‐infiltration (dpi) and was photographed at 5 dpi. Numbers indicate leaves displaying the observed phenotype versus the total leaves expressing FoCSP1‐FLAG or EV. (e) Western blot confirmed FoCSP1‐FLAG expression using FLAG‐specific antibodies. Leaves infiltrated with EV served as the negative control.

FIGURE 2

FoCSP1‐triggered plant defence responses in Nicotiana benthamiana. (a) FoCSP1 was transiently expressed in N. benthamiana leaves via agroinfiltration, with empty vector (EV) as the negative control and INF1 as the positive control. 3,3'‐Diaminobenzidine (DAB) staining was performed at 1 day post‐infiltration (dpi). Numbers indicate leaves displaying the observed phenotype versus total leaves assessed for reactive oxygen species (ROS) accumulation. (b) PVX‐FoCSP1 was inoculated via agroinfiltration, with PVX‐GFP as the control. The dwarfing phenotype was photographed at 7 dpi. Numbers indicate PVX‐FoCSP1‐inoculated plants displaying the dwarfing phenotype versus total PVX‐FoCSP1‐inoculated plants. (c) FoCSP1 or EV was transiently expressed in N. benthamiana, and leaves were collected at 24 and 48 hpi. Reverse transcription‐quantitative PCR was used to analyse defence‐related gene expression, normalised to NbActin. Asterisks indicate significant differences based on Student's t tests (*p < 0.05, **p < 0.01); error bars represent SEM (n = 4). The experiment was replicated three times with similar results. (d) FoCSP1‐YFP and YFP were transiently expressed in N. benthamiana leaves, and YFP fluorescence was observed at 24 h post‐infiltration (scale bars, 25 μm). (e) Western blot using YFP‐specific antibodies detected FoCSP1‐YFP and YFP expression. Leaves infiltrated with EV served as the negative control.

FIGURE 3

FoCSP1 reduced Frankliniella occidentalis offspring and egg production. (a) Phenotypes of FoCSP1 transgenic Arabidopsis. (b) Reverse transcription‐quantitative PCR analysis of defence‐related genes expression in wild‐type (WT) and FoCSP1‐transgenic Arabidopsis, normalised to AtActin. Asterisks indicate significant differences based on Student's t tests (*p < 0.05 **p < 0.01); error bars represent SEM (n = 4). The experiment was replicated three times with similar results. (c) and (d) Two‐choice assay assessing F. occidentalis feeding preference between WT and FoCSP1‐transgenic Arabidopsis. The proportion of thrips per leaf type relative to the total number of thrips was recorded at 24 and 48 h post‐infestation. Statistically analysis was performed using χ ² tests, ns indicates no significant difference; error bars represent SEM (n = 50). (e) and (f) Ten female F. occidentalis of the same age were reared on WT or FoCSP1‐transgenic Arabidopsis. Adult and offspring numbers were recorded daily for up to 9 days post‐feeding (dpf). (g) Fifteen female F. occidentalis of the same age were reared on WT or FoCSP1‐transgenic Arabidopsis. Leaves were collected at 3 dpf, and the total number of eggs was counted. The mean number of eggs laid per female adult was calculated. Asterisks indicate significant differences based on Student's t tests (*p < 0.05 **p < 0.01), ns indicates no significant difference; error bars represent SEM. All experiments were repeated three times with three biological replicates, yielding consistent results.

FIGURE 4

Tomato spotted wilt virus (TSWV) inhibits FoCSP1‐induced plant defence responses. (a) Nicotiana benthamiana plants were inoculated with TSWV or phosphate buffer (PB, mock). Photographs were taken at 5 days post‐inoculation (dpi). (b) FoCSP1 or an empty vector (EV) was transiently expressed in TSWV‐ or mock‐inoculated leaves at 5 dpi. At 24 h post‐agroinfiltration, TSWV infection was confirmed by western blot using TSWV N‐specific antibodies. R1 to R4 represent four biological replicates. (c) Reverse transcription‐quantitative PCR analysis of defence‐related gene expression, normalised to NbActin. Different letters (a, b, c) indicate statistically significant differences (p < 0.05) based on one‐way ANOVA with Tukey's test; error bars represent SEM (n = 4). The experiment was repeated three times with similar results.

FIGURE 5

Tomato spotted wilt virus (TSWV) N and NSs counteract FoCSP1‐induced plant defence responses. (a) and (c) Reverse transcription‐quantitative PCR analysis of defence‐related gene expression in plants expressing an empty vector (EV), FoCSP1‐FLAG+EV, or FoCSP1‐FLAG+N/NSs. Gene expression was normalised to NbActin. Different letters (a, b, c) indicate statistically significant differences (p < 0.05) based on one‐way ANOVA with Tukey's test; error bars represent SEM (n = 4). The experiment was repeated three times with similar results. (b) and (d) Western blot analysis of FoCSP1‐FLAG accumulation with or without N or NSs using FLAG‐specific antibodies. The presence of N and NSs was confirmed via western blot using their respective antibodies.

FIGURE 6

Tomato spotted wilt virus (TSWV) mitigates the suppressive effect of FoCSP1 on Frankliniella occidentalis offspring and egg production. (a) Wild type (WT) and FoCSP1‐transgenic Arabidopsis were inoculated with TSWV or phosphate buffer (PB, mock). Photographs were taken at 12 days post‐inoculation (dpi). (b) TSWV infection was confirmed by western blot using TSWV N‐specific antibodies. R1 and R2 represent two of eight biological replicates. (c) and (d) Ten female F. occidentalis of the same age were reared on WT or FoCSP1‐transgenic Arabidopsis leaves pre‐inoculated with TSWV or PB buffer. Adult and offspring numbers were recorded daily for up to 9 days post‐feeding (dpf). Asterisks indicate statistically significant differences based on Student's t tests (*p < 0.05 **p < 0.01), ns indicates no significant difference; error bars represent SEM (n = 10). The experiment was repeated three time with three biological replicates. (e) Fifteen female F. occidentalis of the same age were reared on WT or FoCSP1‐transgenic Arabidopsis pre‐inoculated with TSWV or mock. Leaves were collected at 3 dpf, and the total number of eggs was counted. The mean number of eggs laid per female was calculated. Asterisks indicate significant differences based on one‐way ANOVA with Tukey's test (*p < 0.05 **p < 0.01); error bars represent SEM (n = 15). The experiment was repeated three times with three biological replicates.