The versatility of mitogen-activated protein kinases (MAPKs) in translating exogenous and endogenous stimuli into appropriate cellular responses depends on its substrate specificity. In animals, several mechanisms have been proposed about how MAPKs maintain specificity to regulate distinct functional pathways. However, little is known of mechanisms that enable substrate selectivity in plant MAPKs. Small ubiquitin-like modifier (SUMO), a posttranslational modification system, plays an important role in plant development and defense by rapid reprogramming of cellular events. In this study we identified a functional SUMO interaction motif (SIM) in Arabidopsis MPK3 and MPK6 that reveals a mechanism for selective interaction of MPK3/6 with SUMO-conjugated WRKY33, during defense. We show that WRKY33 is rapidly SUMOylated in response to Botrytis cinerea infection and flg22 elicitor treatment. SUMOylation mediates WRKY33 phosphorylation by MPKs and consequent transcription factor activity. Disruption of either WRKY33 SUMO or MPK3/6 SIM sites attenuates their interaction and inactivates WRKY33-mediated defense. However, MPK3/6 SIM mutants show normal interaction with a non-SUMOylated form of another transcription factor, SPEECHLESS, unraveling a role for SUMOylation in differential substrate selectivity by MPKs. We reveal that the SUMO proteases, SUMO PROTEASE RELATED TO FERTILITY1 (SPF1) and SPF2 control WRKY33 SUMOylation and demonstrate a role for these SUMO proteases in defense. Our data reveal a mechanism by which MPK3/6 prioritize molecular pathways by differentially selecting substrates using the SUMO–SIM module during defense responses.

丝裂原活化蛋白激酶 (MAPK) 在将外源性和内源性刺激物转化为适当的细胞反应方面的多功能性取决于其底物特异性。在动物中，已经提出了几种关于 MAPK 如何保持特异性以调节不同功能途径的机制。然而，在植物 MAPK 中实现底物选择性的机制知之甚少。小泛素样修饰剂 (SUMO) 是一种翻译后修饰系统，通过细胞事件的快速重编程在植物发育和防御中发挥重要作用。在这项研究中，我们在拟南芥中鉴定了一个功能性相扑相互作用基序（SIM）MPK3 和 MPK6 揭示了在防御过程中 MPK3/6 与 SUMO 结合的 WRKY33 选择性相互作用的机制。我们表明 WRKY33 响应灰霉病菌感染和 flg22 诱导剂治疗而迅速 SUMO 化。SUMOylation 通过 MPK 和随后的转录因子活性介导 WRKY33 磷酸化。WRKY33 SUMO 或 MPK3/6 SIM 站点的中断会减弱它们的相互作用并使 WRKY33 介导的防御失活。然而，MPK3 / 6 SIM突变体显示与另一个转录因子，SPEECHLESS非SUMO化形式正常相互作用，解开一个ROL ëMPKs 在差异底物选择性中的 SUMOylation。我们揭示了 SUMO 蛋白酶、SUMO PROTEASE RELATED TO FERTILITY1 (SPF1) 和 SPF2 控制 WRKY33 SUMOylation 并证明这些 SUMO 蛋白酶在防御中的作用。我们的数据揭示了 MPK3/6 通过在防御反应期间使用 SUMO-SIM 模块差异选择底物来优先考虑分子途径的机制。