• The ethylene‐responsive element binding factor (ERF) family is a large family of transcription factors involved in plant development and environmental stress responses. We previously reported the identification of 29 putative substrates of Mitogen‐activated Protein Kinase3 (AtMPK3), AtMPK4 and AtMPK6, based on a solid‐phase phosphorylation screening using a lambda phage expression library in Arabidopsis thaliana.
• In this study, a putative MPK substrate, AtERF72 (At3g16770), was strongly phosphorylated by AtMPK6 on the serine residue at position 151 (Ser151). AtERF72 binds to the GCC box (AGCCGCC) in the promoters of several pathogenesis‐related (PR) genes and activates their transcription. We also show that the DNA‐binding activity of AtERF72 is enhanced upon phosphorylation by AtMPK6 in vitro.
• In addition, transient co‐expression experiments in Arabidopsis protoplasts revealed that effector constructs expressing a mutant variant of AtERF72, AtERF72^S151D (carrying a Ser to aspartic acid [Asp] substitution at amino acid position 151) showed higher expression of the β‐glucuronidase (GUS) reporter gene driven by the GCC box element than effector constructs expressing the wild‐type AtERF72. Furthermore, yeast two‐hybrid assays revealed that the interaction between AtERF72^S151D and TGA4/OBF4 was stronger than that between wild‐type AtERF72 and TGA4/OBF4.
• Since AtERF72^S151D is equivalent to AtERF72 phosphorylated by AtMPK6 at Ser151, these results suggest that the phosphorylation of AtERF72 by AtMPK6 triggers an event of transcriptional regulation from defence signalling in Arabidopsis.

中文翻译：

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AtMPK6诱导的AtERF72磷酸化增强其DNA结合活性以及与拟南芥中TGA4 / OBF4的相互作用

• 乙烯反应元件结合因子（ERF）家族是涉及植物发育和环境胁迫响应的大量转录因子家族。我们先前报道了在拟南芥中使用lambda噬菌体表达库进行固相磷酸化筛选的基础上，鉴定了29种推定的丝裂素活化蛋白激酶3（AtMPK3），AtMPK4和AtMPK6底物。
• 在这项研究中，推定的MPK底物AtERF72（At3g16770）被AtMPK6在位置151（Ser151）的丝氨酸残基上强烈磷酸化。AtERF72在几个发病相关（PR）基因的启动子中与GCC框（AGCCGCC）结合并激活其转录。我们还显示AtAtF72的DNA结合活性在体外被AtMPK6磷酸化后得到增强。
• 此外，在拟南芥原生质体中的瞬时共表达实验表明，表达AtERF72突变体，AtERF72 ^S151D（在151位氨基酸上进行Ser到天冬氨酸[Asp]取代的表达）的效应子构建体显示β-葡萄糖醛酸苷酶（由GCC框元件驱动的报道基因（GUS）比表达野生型AtERF72的效应子构建体驱动。此外，酵母双杂交检测显示，AtERF72 ^S151D与TGA4 / OBF4之间的相互作用强于野生型AtERF72与TGA4 / OBF4之间的相互作用。
• 由于AtERF72 ^S151D等同于AtMPK6在Ser151处磷酸化的AtERF72，因此这些结果表明AtMPK6对AtERF72的磷酸化触发了拟南芥防御信号转导转录的事件。