Overexpression of NbMAPKKKβ and NbMAPKKKγ induces pathogen-independent cell death in N. benthamiana leaves. N. benthamiana leaves were infiltrated with Agrobacterium strains carrying pEarleyGate203 vector  derivatives harboring the coding sequences for mutant or full-length MAPKKKs. All experiments, except that shown in Figure 4, were repeated at least three times with similar results. A) Symptoms of infiltrated N. benthamiana leaf areas overexpressing the indicated MAPKKK kinase domain (KDs) or ATP-binding site-deficient (K → M) KD mutants. Images of the same leaves after DAB staining are shown in the lower panels. DAB staining to detect hydrogen peroxide production was performed as previously described . Pictures were taken 5 days post-infiltration (dpi). Each protein was transiently co-expressed with silencing suppressor p19. Agrobacterium cultures were grown to a turbidity (OD600) of 0.5 for use in agroinfiltration. B) Ion leakage in the infiltrated leaf areas overexpressing wild-type or K → M mutant NbMAPKKKβ or NbMAPKKKγ or their wild-type or K → M mutant KDs or NbMAPKKKε2 KD. GUS and NbMAPKKKα were used as internal controls. The ion leakage assay was performed as previously described . Data shown represent means ± standard deviation of at least three independent plants. C) Western blot analysis of Myc-tagged NbMAPKKKβ or NbMAPKKKγ KDs or their K → M mutants or NbMAPKKKε2 KD. Myc-tagged GUS was used as an internal control. Total proteins were extracted from each gene-infiltrated area at 5 dpi. Two replicates are shown for each Myc-tagged construct. D) Symptoms of infiltrated leaf areas overexpressing full-length wild-type or K → M mutant NbMAPKKKβ or NbMAPKKKγ. Images of the same leaves after DAB staining are shown in the lower panels.