GATA8-Mediated Antiviral Defence Is Countered by Tomato Chlorosis Virus-Encoded Pathogenicity Protein p27

Abstract

Tomato chlorosis virus (ToCV), a phloem-restricted RNA virus within the genus Crinivirus of the family Closteroviridae, exhibits a broad host range and severely impacts the yield and quality of multiple crops. Viral infection directly alters endogenous phytohormone levels, which are intricately associated with viral mobility, replication, symptom development and defence mechanisms. Previous studies have demonstrated that GATA transcription factors regulate several hormone signalling pathways in plants. In this study, we explored the interaction between ToCV p27 and SlGATA8/NbGATA11. Results indicated that ToCV p27 interacts with an 18-amino-acid at the C-terminus of SlGATA8 and NbGATA11 proteins. Silencing and overexpressing of SlGATA8 revealed its positive role in regulating tomato defence against ToCV infection. Additionally, the interaction redirected SlGATA8's subcellular localisation to plasmodesmata. Furthermore, SlGATA8 promoted the transcriptional expression of SlSnRK2 to regulate the abscisic acid (ABA) signalling pathway. In conclusion, this study confirmed that ToCV p27 impaired the transcriptional activation activity of SlGATA8 through direct interaction, thereby inhibiting the ABA pathway and ultimately facilitating viral infection. This study established a link among virus, GATA family transcription factors and phytohormones, elucidating the molecular mechanism by which ToCV-encoded p27 protein interacts with SlGATA8 to disrupt ABA balance and promote virus infection.

Keywords: GATA transcription factors; SlSnRK2; counterdefense; phytohormone; tomato chlorosis virus.

FIGURE 1

Tomato chlorosis virus (ToCV) p27 interacts with SlGATA8. (a) Yeast two‐hybrid (Y2H) assays were performed to validate the ToCV p27 interactions with SlGATA8. pGBKT7‐53 + PGADT7‐T was used as positive control, and pGBKT7‐Lam + PGADT7‐T was used as negative control. (b) Luciferase complementation imaging (LCI) assays were performed to verify the interaction of ToCV p27 with SlGATA8. nLUC, cLUC are empty vector controls. (c) Co‐immunoprecipitation (Co‐IP) assays were performed to verify the interaction of ToCV p27 with SlGATA8. GUS‐3Flag served as negative control. (d) The interaction interface between SlGATA8 and p27 was predicted using AlphaFold 3. (e, f) The major structural domains of SlGATA8 responsible for the interaction with ToCV p27 were verified by Y2H assays (e) and LCI assays (f). (g, h) The major structural domains of p27 responsible for the interaction with SlGATA8 were verified by Y2H assays (g) and LCI assays (h).

FIGURE 2

SlGATA8 binds to the promoter of the SlSnRK2 gene. (a) The transcriptional activation activity of SlGATA8 was verified by yeast assays. (b) Visual model of the promoter cis‐acting element of the SlSnRK2 gene. (b) Schematic of the reporter vector and effector vector for dual luciferase assays. (c) SlGATA8 binds to the promoter sequence of the SlSnRK2 gene as verified by dual‐luciferase assays. LUC/REN ratio was analysed for LUC fluorescence activity. Data were collected in three biological experiments, and error bars represent standard deviations of the mean (n = 3). Statistical analyses were performed by Student's t test (*p < 0.05). (d) The binding affinity of SlGATA8 to the promoter of SlSnRK2 was demonstrated via electrophoretic mobility shift assays in vitro. mProbe, the designed mutant probe.

FIGURE 3

SlGATA8 positively regulates the abscisic acid (ABA) signalling pathway. (a) Tomato symptoms were observed following inoculatiion with PVX‐SlGATA8 or potato virus X (PVX) at 15 days post‐inoculation (dpi). PVX is the negative control. Scale bar, 6 cm. (b) The expression levels of SlGATA8 were assessed by reverse transcription‐quantitative PCR (RT‐qPCR) following PVX‐SlGATA8 or PVX infection. (c) The expression levels of SlSnRK2 were measured by RT‐qPCR in tomato plants heterologously expressing SlGATA8. (d) The phenotypes were observed in SlGATA8‐silenced and non‐silenced tomato. TRV‐GUS is the negative control. Scale bar, 6 cm. (e) Silencing efficiency was assessed by RT‐qPCR. (f) The expression levels of SlSnRK2 were measured by RT‐qPCR in SlGATA8‐silenced tomato plants. (g) The expression levels of genes related to the ABA pathway were measured via RT‐qPCR assays in tomato following PVX‐SlGATA8 infection. (h) The expression levels of genes related to the ABA pathway was measured via RT‐qPCR in SlGATA8‐silenced and non‐silenced tomato. Data were collected from more than three biological experiments, and error bars represent standard deviations of the mean (n = 4). Statistical analyses were performed by Student's t test (**p < 0.01, ***p < 0.001).

FIGURE 4

SlGATA8 negatively regulates tomato chlorosis virus (ToCV) infection. (a) Phenotypes of tomato plants following ToCV inoculation after heterologous overexpression of SlGATA8. Scale bar, 6 cm. (b) ToCV genomic RNA accumulation levels were analysed by reverse transcription‐quantitative PCR (RT‐qPCR) assays in PVX‐SlGATA8 and PVX‐infected tomato. (c) Phenotypes of tomato plants following ToCV inoculation after SlGATA8 silencing. Scale bar, 6 cm. (d) ToCV genomic RNA accumulation levels were analysed by RT‐qPCR assays in SlGATA8‐silenced and non‐silenced tomato plants. (e) ToCV genomic RNA accumulation levels were analysed by RT‐qPCR assays following abscisic acid (ABA) treatment. (f) ToCV genomic RNA accumulation levels were analysed by RT‐qPCR assays in SlGATA8‐silenced tomato plants following ABA treatment. Data were collected in more than three biological experiments, and error bars represent standard deviations of the mean. In (b), n = 4 biologically independent plants. In (d), n = 5 biologically independent plants. In (e) and (f), n = 3 biologically independent plants. Statistical analyses were performed by Student's t test (*p < 0.05, ***p < 0.001, ns, no significance).

FIGURE 5

Tomato chlorosis virus (ToCV)‐encoded p27 protein inhibits the nuclear localisation of SlGATA8. (a) The subcellular localisation of SlGATA8, ToCV p27 and co‐localisation were observed by laser confocal microscopy. NbH2B‐RFP fluorescence indicates nucleus positions. (b) Bimolecular fluorescence complementation (BiFC) assay was conducted to verify the interaction between ToCV p27 and SlGATA8. AtPDLP1‐dsRed severed as plasmodesmata (PD) localisation marker.

FIGURE 6

Tomato chlorosis virus (ToCV)‐encoded p27 protein inhibits abscisic acid (ABA) signalling pathway. (a) Tomato symptoms were observed following inoculation with PVX‐p27 and potato virus X (PVX) at 15 days post‐inoculation (dpi). PVX served as the negative control. Scale bar, 6 cm. (b) PVX genomic RNA accumulation levels were analysed by reverse transcription‐quantitative PCR (RT‐qPCR) assays in PVX‐p27‐ and PVX‐infected tomato plants. (c) The expression levels of SlSnRK2 were analysed by RT‐qPCR assays in tomato following PVX‐p27 or PVX infection. (d) The expression of genes related to the ABA pathway was measured via RT‐qPCR assay in tomato following PVX‐p27 or PVX infection. Data were collected in more than three biological experiments, and error bars represent standard deviations of the mean (n = 4). Statistical analyses were performed by Student's t test (**p < 0.01, ***p < 0.001).

FIGURE 7

NbGATA11 interacts with p27 and inhibits tomato chlorosis virus (ToCV) infection. (a) Yeast two‐hybrid (Y2H) assays were conducted to validate the ToCV p27 interaction with NbGATA11. BD‐53 + AD‐T was used as positive control and BD‐Lam + AD‐T was used as negative control. (b) Bimolecular fluorescence complementation (BiFC) assays were performed to verify the interaction of ToCV p27 with NbGATA11. (c) Luciferase complementation imaging (LCI) assays were performed to verify the interaction of ToCV p27 with NbGATA11. nLUC, cLUC are empty vector controls. (d) Schematic of the reporter vector and effector vector for dual luciferase assays. (e) NbGATA11 binds to the promoter sequence of the NbSnRK2 gene as verified by dual‐luciferase assays. The LUC/REN ratio was analysed for LUC fluorescence activity. (f) The binding affinity of NbGATA11 to the promoter of NbSnRK2 was demonstrated by electrophoretic mobility shift assays in vitro. mProbe, the designed mutant probe. (g) ToCV‐CP accumulation levels were analysed by RT‐qPCR assays in NbGATA11‐silenced and non‐silenced Nicotiana benthamiana. Data were collected from more than three biological experiments, and error bars represent standard deviations of the mean. In (e), n = 3 biologically independent plants. In (g), n = 4 biologically independent plants. Statistical analyses were performed by Student's t test (*p < 0.05, **p < 0.01).