Epistasis analysis of the functional relationships among NbMAPKKKα, NbMAPKKKβ, and NbMAPKKKγ. Epistasis analysis was performed by combining functional activation and suppression of specific combinations of MAPKKKs using transient overexpression and VIGS, respectively. Cell death in infiltrated areas was quantified using ion leakage assays at 4 dpi. Data shown represent means ± standard deviation for at least five plants. Asterisks indicate statistically significant differences from control plants (P < 0.05). All experiments described in this figure were repeated two times with similar results. A) Cell death in the NbMAPKKKβ kinase domain (βkinase)- or NbMAPKKKγ (γORF)-overexpressing areas of non-silenced and NbMAPKKKα-silenced plants. Agrobacterium cultures expressing βkinase and γORF were grown to a turbidity of 1.0 and 0.05, respectively, for infiltration. B) Cell death in the NbMAPKKKα (αORF)- or γORF-overexpressing areas of non-silenced and NbMAPKKKβ-silenced plants. Agrobacterium cultures expressing αORF and γORF were grown to a turbidity of 0.5 or 0.05, respectively, for infiltration. C) Cell death in the αORF- or βkinase-overexpressing areas of non-silenced and NbMAPKKKγ-silenced plants. Agrobacterium cultures expressing αORF and βkinase were grown to a turbidity of 0.5 or 1.0, respectively, for infiltration.