Abstract
MAPK cascades play pivotal roles in plant immunity. We have identified Nicotiana benthamiana WRKY8 as a protein phosphorylated by pathogen-responsive tobacco MAPKs, SIPK, NTF4 and WIPK. WRKY8 contains a deduced D domain, which is a conserved MAPK-interacting sites identified in yeast and animal MAPK substrates. D domain-mediated high-affinity interaction with MAPK ascertains the specific and high-efficiency phosphorylation by MAPK. In this study, we analyzed contribution of the D domain-like sequence of WRKY8 to the phosphorylation by MAPKs. We examined the interaction between WRKY8 and MAPKs by in vitro pull-down and BiFC assays. WRKY8 was interacted with SIPK, NTF4 and WIPK, but not NTF6. Mutations within the D domain remarkably disrupted the interaction between WRKY8 and SIPK. Phosphorylation intensities by SIPK in vitro and in vivo phosphorylation in response to MEK2DD were reduced markedly in D domain-mutated WRKY8. These results indicate that WRKY8 directly interacts with MAPKs in a D domain-dependent manner and the D domain-dependent interaction is necessary for MAPK-mediated phosphorylation of WRKY8.
