Fig 1.
Identification of the interaction between V2 and SlGRXC6 proteins.
(a) Y2H confirmed the interaction between V2 and SlGRXC6. SlGRXC6 was fused with a GAL4 activation domain (AD-SlGRXC6) and V2 was fused to a GAL4-binding domain (BD-V2), respectively. Yeast cells expressing the indicated protein pairs were plated onto the selection medium (SD/-His/-Leu/-Trp/-Ade) with X-α-Gal to screen for positive interactions. Yeast cells coexpressing pGADT7-largeT (AD-largeT) and pGBKT7-p53 (BD-p53) or AD-largeT and pGBKT7-LaminC (BD-LaminC) served as a positive and negative control, respectively. (b) The V2-SlGRXC6 interaction was confirmed by using a BiFC assay in N. benthamiana cells. The V2-SlGRXC6 interaction led to a reconstituted fluorescence signal. DAPI stains DNA in the nucleus. Bars: 50 μm. Experiments were repeated three times and 30 cells were observed in each repeat. (c) A co-IP assay to test the interaction between V2 and SlGRXC6, SlGRXC6T53A or SlGRXC6C58A. N. benthamiana leaves were co-infiltrated with FLAG-V2 and SlGRXC6-YFP (Lane 1), FLAG-V2 and SlGRXC6T53A-YFP (Lane 2), FLAG-V2 and SlGRXC6C58A-YFP (Lane 3), or FLAG-V2 and YFP (Lane 4). Cell lysates were incubated with FLAG-Trap beads and proteins pulled down with beads were tested using the indicated antibodies. Samples before (Input) and after (IP) immunoprecipitation were analyzed by using anti-GFP or -FLAG antibody. (d) Schematic representation of the truncated mutants of SlGRXC6 and their interactions with V2 as analyzed using Y2H. (e) Interactions between the V2 and the SlGRXC6 mutants SlGRXC6T53A, SlGRXC6G56A, SlGRXC6C58A, SlGRXC6S69A, SlGRXC6S72A using Y2H. (f) Subcellular localization of SlGRXC6-YFP, SlGRXC6T53A-YFP or SlGRXC6C58A-YFP in H2B transgenic N. benthamiana. The H2B-RFP signal represents the nucleus. Bars: 50 μm. Experiments were repeated three times with similar results.
Fig 2.
The overexpression of SlGRXC6 inhibits TYLCV infection in tomato plants.
(a) The relative levels of SlGRXC6 transcripts in plants, which were treated with PVX or PVX-SlGRXC6, were determined by qRT-PCR at 8 dpai. Total RNA was extracted from newly emerged systemic leaves. Values represent the mean relative to the mock-treated plants (n = 3 biological replicates) and normalized with SlActin as an internal reference. Data are means ± SD (n = 3). Asterisk indicates a statistically significant difference (*p<0.05) according to Student’s t-test. (b) Tomato plants treated with PVX or PVX-SlGRXC6. Plants and newly emerged systemic leaves were photographed at 8 dpai. Bar: 5 cm. (c) The aboveground plant heights of PVX or PVX-SlGRXC6 plants were measured at 16 dpai. (d) Tomato plants treated with PVX and PVX-SlGRXC6 responded differently to TYLCV infection. Plants and newly emerged leaves were photographed 13 days after TYLCV inoculation. Bar: 5 cm. (e) The plant height of TYLCV-inoculated tomato plants treated with PVX or PVX-SlGRXC6. Plants were measured 13 days after TYLCV inoculation. (f) The time course of TYLCV infection in PVX control or PVX-SlGRXC6 plants. Values represent percentages of systemically infected plants at the indicated time points. In each experiment, 15 plants were inoculated and three independent repeats were performed. Experiments were repeated three times with similar results. (g) The viral genomic DNA accumulation in systemic leaves as measured by qPCR. Accumulated levels of viral genomic DNA were tested in PVX control or PVX-SlGRXC6 tomato plants infected with TYLCV at 3, 13, 23, and 33 dpi as in a. Data are means ± SD (n = 3). Experiments were repeated three times with similar results.
Fig 3.
Knocking down the expression of SlGRXC6 promotes TYLCV infection.
(a) The relative levels of SlGRXC6 transcripts in control (TRV) and knockdown (TRV-SlGRXC6) tomato plants were determined by qRT-PCR at 12 dpai with SlActin as an internal control. Data are means ± SD (n = 3). Asterisk indicates a statistically significant difference (*p<0.05) according to Student’s t-test. (b) Tomato plants are smaller when treated with TRV-SlGRXC6 compared to those treated with TRV. Bar: 5 cm. (c) The aboveground height of control and SlGRXC6-silenced plants were tested at 16 dpai. (d) Symptoms caused by TYLCV in control or SlGRXC6-silenced plants. Leaves were photographed at 13 dpi. Bar: 5 cm. (e) The aboveground height of TRV- or TRV-SlGRXC6-treated tomato plants as measured 13 days after TYLCV infection. (f) The time course of TYLCV infection in control or SlGRXC6-silenced plants as shown in Fig 2F. Experiments were repeated three times with similar results. (g) The accumulated viral genomic DNA in systemic leaves as measured by qPCR. Accumulated levels of viral genomic DNA were tested in control or SlGRXC6-silenced tomato plants infected with TYLCV at 3, 13, 23, and 33 dpi as shown in Fig 2A. Data are means ± SD (n = 3). Experiments were repeated three times with similar results.
Fig 4.
The V2-SlGRXC6 interaction is critical to TYLCV infection.
(a) The aboveground height of PVX-, PVX-SlGRXC6T53A-, or PVX-SlGRXC6C58A-inoculated tomato plants as measured at 16 dpai. (b) Symptoms in control, PVX-SlGRXC6T53A-, PVX-SlGRXC6C58A-inoculated plants. Leaves were photographed 23 days after TYLCV inoculation. Bar: 5 cm. (c) The accumulated TYLCV viral DNA in systemic leaves as measured by qPCR. Accumulated levels of viral genomic DNA were tested in PVX-, PVX-SlGRXC6T53A-, PVX-SlGRXC6C58A-treated tomato plants infected with TYLCV at 3, 13, 23, and 33 dpi as in Fig 2A. Data are means ± SD (n = 3). Experiments were repeated three times with similar results.
Fig 5.
The effect of SlGRXC6 on the nuclear distribution of the V2 and V1 protein.
(a) Localization of V2-YFP in the absence or presence of the SlGRXC6 protein in N. benthamiana cells. V2-YFP expressed alone or coexpressed with SlGRXC6-FLAG was detected by confocal microscopy. Experiments were repeated three times and 30 cells were observed in each repeat. DAPI stains DNA in the nucleus. Bars: 50 μm. (b) The distribution of V2 in the absence or presence of SlGRXC6-FLAG in H2B-RFP transgenic N. benthamiana plants was analyzed using a nuclear-cytoplasmic fractionation assay. Western blotting was conducted with antibodies specific to the indicated proteins. PEPC and H2B-RFP were used as markers for the cytoplasmic and nuclear fraction, respectively. The intensity of the protein signal was measured using ImageQuant TL (GE healthcare), with levels of the cytoplasm and nucleus totaling 100%. Values represent the average of three plants. Experiments were repeated three times. (c) Localization of V2-YFP when expressed alone or in the presence of SlGRXC6, SlGRXC6T53A, or SlGRXC6C58A in H2B transgenic N. benthamiana cells. Bars: 50 μm. Experiments were repeated three times and 30 cells were observed in each repeat. (d) Subcellular localization of V1 was expressed alone or coexpressed with FLAG-V2 and FLAG-tagged SlGRXC6, SlGRXC6T53A, or SlGRXC6C58A in H2B transgenic N. benthamiana cells. The H2B-RFP signal represents the nucleus. The enlarged areas show the nuclear region. Bars: 50 μm. Experiments were repeated three times and 30 cells were observed in each repeat. (e) The number of cells with a nuclear distribution of V1-YFP was counted and the percentage of cells with a nuclear distribution was calculated. Experiments were repeated three times and 30 cells were observed in each repeat. Values represent percentages of cells with nuclear distribution of YFP signal ± SD (standard deviation). Data were analyzed using Student’s t-test and asterisks denote significant differences (*P < 0.05).
Fig 6.
Identification of the interaction between SlGRXC6 and SlNTRC80.
(a) The interaction between SlGRXC6 and SlNTRC80 was tested in the Y2H. Yeast cells expressing the indicated pairs of proteins grew on selection medium (SD/- His/-Leu/-Trp/-Ade) supplied with X-α-Gal. (b) The SlGRXC6-SlNTRC80 interaction was confirmed by co-IP assay. (c) Tomato plants treated with TRV or TRV-SlNTRC80. Plants and newly emerged systemic leaves were photographed at 12 dpai. Bar: 5 cm. Experiments were repeated three times with similar results. (d) Relative expression of SlNTRC80 was tested in control and SlNTRC80-silenced plants using qRT-PCR as shown in Fig 2A. Data are means ± SD (n = 3). (e) Aboveground plant heights of control and SlNTRC80-silenced plants at 12, 16, and 24 dpai. (f) The interaction between V2 and SlNTRC80 was tested in Y2H. Yeast cells expressing the indicated pair of proteins were plated onto the selection medium (SD/-His/-Leu/-Trp) in 10-fold serial dilutions. (g) In vitro competitive pulldown assays. The indicated amounts of His6-V2 or His6 protein were mixed with 2 μg of MBP-SlNTRC80 and pulled down by 2 μg of GST-SlGRXC6. The bound protein was detected by immunoblotting with the indicated antibodies. Experiments were repeated three times with similar results.
Fig 7.
A proposed model for possible roles of the SlGRXC6-V2 interaction in the regulation of plant growth and the restriction of TYLCV infection.
SlGRXC6 and SlNTRC80 physically interact and are cooperatively involved in regulating plant growth. Disruption of the interactions or the balance between them leads to growth defects. The TYLCV V2 protein is a symptom determinant and interacts with SlGRXC6 during viral infection. The V2-SlGRXC6 interaction inhibits or slows down the nuclear export of V2, the V2-mediated nuclear export of V1, and the V1-viral DNA complex, and therefore, restricts viral infection. The V2-SlGRXC6 interaction also sequesters SlGRXC6 from binding to SlNTRC80, interfering the SlNTRC80-SlGRXC6 interaction and leading to growth defects.