Department of Biochemistry, Molecular Biology, Entomology, and Plant Pathology, Mississippi State University, Mississippi State, Mississippi, USA.
Department of Chemistry, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
J Biol Chem. 2021 Jan-Jun;296:100695. doi: 10.1016/j.jbc.2021.100695. Epub 2021 Apr 22.
Upon pathogen infection, receptors in plants will activate a localized immune response, the effector-triggered immunity (ETI), and a systemic immune response, the systemic acquired response (SAR). Infection also induces oscillations in the redox environment of plant cells, triggering response mechanisms involving sensitive cysteine residues that subsequently alter protein function. Arabidopsis thaliana thimet oligopeptidases TOP1 and TOP2 are required for plant defense against pathogens and the oxidative stress response. Herein, we evaluated the biochemical attributes of TOP isoforms to determine their redox sensitivity using ex vivo Escherichia coli cultures and recombinant proteins. Moreover, we explored the link between their redox regulation and plant immunity in wild-type and mutant Arabidopsis lines. These analyses revealed that redox regulation of TOPs occurs through two mechanisms: (1) oxidative dimerization of full-length TOP1 via intermolecular disulfides engaging cysteines in the N-terminal signal peptide, and (2) oxidative activation of all TOPs via cysteines that are unique and conserved. Further, we detected increased TOP activity in wild-type plants undergoing ETI or SAR following inoculation with Pseudomonas syringae strains. Mutants unable to express the chloroplast NADPH-dependent thioredoxin reductase C (NTRC) showed elevated TOP activity under unstressed conditions and were SAR-incompetent. A top1top2 knockout mutant challenged with P. syringae exhibited misregulation of ROS-induced gene expression in pathogen-inoculated and distal tissues. Furthermore, TOP1 and TOP2 could cleave a peptide derived from the immune component ROC1 with distinct efficiencies at common and specific sites. We propose that Arabidopsis TOPs are thiol-regulated peptidases active in redox-mediated signaling of local and systemic immunity.
在病原体感染后，植物中的受体将激活局部免疫反应，即效应触发免疫（ETI），以及系统免疫反应，即系统获得性反应（SAR）。感染还会引起植物细胞氧化还原环境的振荡，触发涉及敏感半胱氨酸残基的反应机制，随后改变蛋白质功能。拟南芥硫肽寡肽酶 TOP1 和 TOP2 是植物抵御病原体和氧化应激反应所必需的。在此，我们评估了 TOP 同工型的生化特性，以使用体外大肠杆菌培养物和重组蛋白来确定它们的氧化还原敏感性。此外，我们探讨了它们的氧化还原调节与野生型和突变拟南芥系植物免疫之间的联系。这些分析表明，TOP 的氧化还原调节通过两种机制发生：（1）全长 TOP1 通过分子间二硫键的氧化二聚化，涉及 N 端信号肽中的半胱氨酸，以及（2）所有 TOP 的氧化激活通过独特且保守的半胱氨酸。此外，我们在接种丁香假单胞菌菌株后，在经历 ETI 或 SAR 的野生型植物中检测到 TOP 活性增加。不能表达叶绿体 NADPH 依赖性硫氧还蛋白还原酶 C（NTRC）的突变体在非胁迫条件下表现出升高的 TOP 活性，并且 SAR 能力丧失。用 P. syringae 挑战 top1top2 敲除突变体时，在受病原体感染和远端组织中观察到 ROS 诱导基因表达的失调。此外，TOP1 和 TOP2 可以以不同的效率在常见和特定位点切割来自免疫成分 ROC1 的肽。我们提出，拟南芥 TOP 是在局部和系统免疫的氧化还原介导信号转导中活跃的硫醇调节肽酶。
