Phosphorylation of the Nicotiana benthamiana WRKY8 Transcription Factor by MAPK Functions in the Defense Response    

Abstract

Mitogen-activated protein kinase (MAPK) cascades have pivotal roles in plant innate immunity. However, downstream signaling of plant defense-related MAPKs is not well understood. Here, we provide evidence that the Nicotiana benthamiana WRKY8 transcription factor is a physiological substrate of SIPK, NTF4, and WIPK. Clustered Pro-directed Ser residues (SP cluster), which are conserved in group I WRKY proteins, in the N-terminal region of WRKY8 were phosphorylated by these MAPKs in vitro. Antiphosphopeptide antibodies indicated that Ser residues in the SP cluster of WRKY8 are phosphorylated by SIPK, NTF4, and WIPK in vivo. The interaction of WRKY8 with MAPKs depended on its D domain, which is a MAPKinteracting motif, and this interaction was required for effective phosphorylation of WRKY8 in plants. Phosphorylation of WRKY8 increased its DNA binding activity to the cognate W-box sequence. The phospho-mimicking mutant of WRKY8 showed higher transactivation activity, and its ectopic expression induced defense-related genes, such as 3-hydroxy-3-methylglutaryl CoA reductase 2 and NADP-malic enzyme. By contrast, silencing of WRKY8 decreased the expression of defense-related genes and increased disease susceptibility to the pathogens Phytophthora infestans and Colletotrichum orbiculare. Thus, MAPK-mediated phosphorylation of WRKY8 has an important role in the defense response through activation of downstream genes.

Regulation of Specialized Metabolism by WRKY Transcription Factors

“…Overexpression of OsWRKY13 up-regulates genes in the phenylpropanoid pathway while downregulating those for terpenoid biosynthesis, illustrating the potential of WRKY TFs to differentially modulate discrete classes of metabolites (Qiu et al, 2008a). Both OsWRKY53 and Nicotiana benthamiana WRKY8 (Ishihama et al, 2011) are phosphorylated by the MPK3/MPK6 cascade. Rice OsWRKY76 activates cold stress tolerance but represses terpene synthesis (Yokotani et al, 2013).…”

“…Importantly, six WRKY TFs predicted as orthologs (AtWRKY33, NbWRKY8, OsWRKY53, PsWRKY1, VvWRKY2, and WRKY108715) have independently been found to regulate specialized metabolism (Table I; Supplemental Table S1). Duplicate WRKYs of this group function to regulate the biosynthesis of alkaloids, terpenes, and phenylpropanoids (Naoumkina et al, 2008;Guillaumie et al, 2010;Ishihama et al, 2011;Mao et al, 2011;Mishra et al, 2013;Chujo et al, 2014). Therefore, orthologs of the MPK3/ MPK6-AtWRKY33 signaling cascade may be key factors in the regulation of specialized metabolism.…”

A systems biology perspective on the role of WRKY transcription factors in drought responses in plants

“…Also, interaction studies show that group I wRKY proteins are among the targets of MAP kinase signaling cascades, suggesting that phosphorylation plays a major role in wRKY activity. In Nicotiana benthamiana, NbwRKY8 phosphorylation by MAP kinases enhances its DNA-binding and transcriptionactivating activities (Ishihama et al 2011). There are many other examples of wRKY-MAP kinase interactions (Chi et al 2013).…”

Alternative splicing of rice WRKY62 and WRKY76 transcription factor genes in pathogen defense

“…For example, Arabidopsis (Arabidopsis thaliana) WRKY33 is phosphorylated by MPK3/MPK6 in response to infection by Botrytis cinerea and is required for MPK3/MPK6-induced camalexin biosynthesis (Mao et al, 2011). In Nicotiana benthamiana, phosphorylated WRKY8 and its phosphor-mimicking mutants are found to have increased binding to the cognate W box and enhanced transactivation activity, respectively (Ishihama et al, 2011). Panicle blast1 (Pb1) is a panicle blast resistance gene, conferring durable resistance to Magnaporthe oryzae (Hayashi et al, 2010).…”