Mitogen‐activated protein kinase (MAPK) cascades are highly conserved signaling modules that regulate plant immune responses. The Arabidopsis thaliana Raf‐like MAPK kinase kinase ENHANCED DISEASE RESISTANCE1 (EDR1) is a key negative regulator of plant immunity that affects the protein levels of MKK4 and MKK5, two important MAPK cascade members, but the underlying mechanism is poorly understood. Here, genome‐wide phosphorylation analysis demonstrated that the E3 ligase KEEP ON GOING (KEG) is phosphorylated in the edr1 mutant but not the wild type, suggesting that EDR1 negatively affects KEG phosphorylation. The identified phosphorylation sites in KEG appear to be important for its accumulation. The keg‐4 mutant, a previously identified edr1 suppressor, enhances susceptibility to the powdery mildew pathogen Golovinomyces cichoracearum. In addition, MKK4 and MKK5 protein levels are reduced in the keg‐4 mutant. Furthermore, we demonstrate that MKK4 and MKK5 associate with full‐length KEG, but not with truncated KEG‐RK or KEG‐RKA, and that KEG ubiquitinates and mediates the degradation of MKK4 and MKK5. Taken together, these results indicate that MKK4 and MKK5 protein levels are regulated by KEG via ubiquitination, uncovering a mechanism by which plants fine‐tune immune responses by regulating the homeostasis of key MAPK cascade members via ubiquitination and degradation.

中文翻译：

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拟南芥E3连接酶KEG与MKK4和MKK5结合并泛素化以调节植物免疫力

丝裂原激活的蛋白激酶（MAPK）级联反应是高度保守的信号传导模块，可调节植物的免疫反应。在拟南芥的Raf状MAPK激酶激酶增强的抗病RESISTANCE1（EDR1）是植物免疫力影响MKK4和MKK5，两个重要的MAPK级联成员的蛋白水平的关键负调节物，但其机制知之甚少。在这里，全基因组磷酸化分析表明E3连接酶KEEP ON GOING（KEG）在edr1突变体中被磷酸化，而在野生型中不被磷酸化，表明EDR1对KEG磷酸化有负面影响。在KEG中确定的磷酸化位点似乎对其积累很重要。所述小桶-4突变体，先前识别EDR1抑制剂，增强了对白粉病病原菌Golovinomyces cichoracearum的敏感性。此外，keg-4突变体中的MKK4和MKK5蛋白水平降低。此外，我们证明MKK4和MKK5与全长KEG相关，但与截短的KEG-RK或KEG-RKA不相关，并且KEG泛素化并介导MKK4和MKK5的降解。综上所述，这些结果表明MKK4和MKK5蛋白水平受KEG通过泛素化调节，揭示了一种机制，植物可通过泛素化和降解调节关键MAPK级联成员的稳态来微调免疫反应。