Fig. 7: CP suppresses the MAPKKKα-mediated immune signaling by targeting Nb14-3-3a.

a Interaction with 14-3-3a is required for CP to inhibit MAPKKKα-induced cell death. Agrobacterium carrying wild-type CP or its variants was co-infiltrated with Agrobacterium containing HA-MAPKKKα controlled by an estradiol-inducible system into different leaf regions. β-estradiol was used to induce protein expression 48 h after agroinfiltration. Leaves were stained by trypan blue and representative photographs were taken three days after estradiol treatment. b Quantification of cell death by measuring electrolyte leakage of leaf regions shown in (a). 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). c Western blot analysis to confirm the expression of the CP or its variants with an anti-CP antibody. d CP suppresses MAPKKKα protein accumulation and MAPK activation in WT N. benthamiana. Agrobacterium carrying CP, CP_Y194A, or empty vector (EV) control was co-infiltrated with Agrobacterium containing HA-MAPKKKα controlled by an estradiol-inducible system into leaves. 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 analysis with different antibodies indicated on the right. Actin served as the loading control. e CP suppresses MAPKKKα protein accumulation and MAPK activation in 14-3-3a-OE transgenic N. benthamiana. Agrobacterium carrying CP, CP_Y194A or empty vector was co-infiltrated with Agrobacterium containing HA-MAPKKKα controlled by an estradiol-inducible system into leaves. In total, 30 μM β-estradiol was applied to the leaves 48 h after agroinfiltration. Samples were collected at 0, 12, 24 h after β-estradiol treatment and subjected to western blot analysis with different antibodies indicated on the right. CBB-stained RbcL served as the loading control. f CP-transgenic N. benthamiana exhibited reduced accumulation of HA-MAPKKKα protein and MAPK phosphorylation compared with WT plants. Agrobacterium carrying HA-MAPKKKα controlled by an estradiol-inducible system was infiltrated into leaves. In all, 30 μM β-estradiol was applied to 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. For panels c–f, the experiments were repeated three times with similar results. g Symptom observation of engineered PVX-inoculated N. benthamiana plants. Agrobacterium carrying engineered PVX infection clones were infiltrated into two leaves of each plant. Representative photographs were taken at 6 dpi. h Western blot analysis of viral CP in the systemic leaves shown in panel g by using anti-PVX-CP or anti-BBSV CP antibody. Actin served as the loading control. The experiment was repeated three times with similar results.