Host GRXC6 restricts Tomato yellow leaf curl virus infection by inhibiting the nuclear export of the V2 protein
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).