Fig. 9: Perturbation of Nb14-3-3a-MAPKKKα functional module by the CP is a common strategy employed by necroviruses.

a, b BiFC analysis of the interaction between CP of TNV-A^C (a) or TNV-D^H (b) and 14-3-3a. The indicated combinations of different proteins were transiently co-expressed in N. benthamiana leaves. Confocal analysis was performed at 3 dpi. The experiments were repeated three times and representative results are shown. Scale bars = 50 µm. c, d CP inhibits MAPKKKα-induced cell death. Agrobacterium carrying wild-type TNV-A^C (c) or TNV-D^H (d) CP or their variants was co-infiltrated with Agrobacterium containing HA-MAPKKKα controlled by an estradiol-inducible system into different leaf regions. Empty vector (EV) served as the negative control. β-estradiol was applied 48 h after agroinfiltration. Leaves were stained by trypan blue and representative photographs were taken 3 days after estradiol treatment. e, f Quantification of cell death by measuring electrolyte leakage of leaf regions shown in (c) and (d). Error bars indicate ± SD of the mean (n = 6 biologically independent plants). Different letters in the chart denote statistically significant differences among different groups according to the one-way ANOVA analysis with Tukey’s multiple comparison test (P < 0.05). g, h CP affects MAPKKKα protein stability and MAPK activation in non-transgenic N. benthamiana. Agrobacterium carrying CP or CP_Y194A was co-infiltrated with Agrobacterium containing HA-MAPKKKα controlled by an estradiol-inducible system into the leaves. Empty vector (EV) served as the negative control. In all, 30 μM β-estradiol was infiltrated into the leaves 48 h after agroinfiltration. Samples were collected at 0, 12, 24 h after β-estradiol treatment and subjected to western blot with different antibodies indicated on the right. Actin served as the loading control. The experiments were repeated three times with similar results. i, j Phenotype observation of TNV-A^C (i) or TNV-D^H (j) -infected MAPKKKα-KO plants. N. benthamiana leaves were inoculated with 100 ng TNV-A^C virions. Representative photographs were taken at 9 dpi. k Western blot analysis of TNV-A^C CP levels in the plants shown in panel i using an anti-TNV-A^C antibody. Actin served as the loading control. The experiment was repeated three times with similar results. l RT-qPCR analysis of TNV-D^H genomic RNA levels in the plants shown in panel j. Values represent ± SD of the mean (n = 6 biologically independent plants). An asterisk indicates the significant difference based on one-sided Student’s t test (*P = 0.0357).