Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter EX4 4QD, UK.
Plant Physiol. 2023 Apr 3;191(4):2551-2569. doi: 10.1093/plphys/kiac603.
Reactive oxygen species are produced in response to pathogens and pathogen-associated molecular patterns, as exemplified by the rapid extracellular oxidative burst dependent on the NADPH oxidase isoform RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) in Arabidopsis (Arabidopsis thaliana). We used the H2O2 biosensor roGFP2-Orp1 and the glutathione redox state biosensor GRX1-roGFP2 targeted to various organelles to reveal unsuspected oxidative events during the pattern-triggered immune response to flagellin (flg22) and after inoculation with Pseudomonas syringae. roGFP2-Orp1 was oxidized in a biphasic manner 1 and 6 h after treatment, with a more intense and faster response in the cytosol compared to chloroplasts, mitochondria, and peroxisomes. Peroxisomal and cytosolic GRX1-roGFP2 were also oxidized in a biphasic manner. Interestingly, our results suggested that bacterial effectors partially suppress the second phase of roGFP2-Orp1 oxidation in the cytosol. Pharmacological and genetic analyses indicated that the pathogen-associated molecular pattern-induced cytosolic oxidation required the BRI1-ASSOCIATED RECEPTOR KINASE (BAK1) and BOTRYTIS-INDUCED KINASE 1 (BIK1) signaling components involved in the immune response but was largely independent of NADPH oxidases RBOHD and RESPIRATORY BURST OXIDASE HOMOLOG F (RBOHF) and apoplastic peroxidases peroxidase 33 (PRX33) and peroxidase 34 (PRX34). The initial apoplastic oxidative burst measured with luminol was followed by a second oxidation burst, both of which preceded the two waves of cytosolic oxidation. In contrast to the cytosolic oxidation, these bursts were RBOHD-dependent. Our results reveal complex oxidative sources and dynamics during the pattern-triggered immune response, including that cytosolic oxidation is largely independent of the preceding extracellular oxidation events.
活性氧物种是针对病原体和病原体相关分子模式产生的，例如拟南芥（Arabidopsis thaliana）中依赖 NADPH 氧化酶同工型 RESPIRATORY BURST OXIDASE HOMOLOG D（RBOHD）的快速细胞外氧化爆发。我们使用 H2O2 生物传感器 roGFP2-Orp1 和靶向各种细胞器的谷胱甘肽氧化还原状态生物传感器 GRX1-roGFP2 来揭示在 flagellin（flg22）引发的模式触发免疫反应期间和接种 Pseudomonas syringae 后未被发现的氧化事件。roGFP2-Orp1 以双相方式被氧化，在处理后 1 和 6 小时，与叶绿体、线粒体和过氧化物酶体相比，细胞质中的反应更强烈、更快。过氧化物酶体和细胞质中的 GRX1-roGFP2 也以双相方式被氧化。有趣的是，我们的结果表明，细菌效应子部分抑制了细胞质中 roGFP2-Orp1 氧化的第二阶段。药理学和遗传学分析表明，病原体相关分子模式诱导的细胞质氧化需要参与免疫反应的 BRI1-ASSOCIATED RECEPTOR KINASE（BAK1）和 BOTRYTIS-INDUCED KINASE 1（BIK1）信号成分，但在很大程度上独立于 NADPH 氧化酶 RBOHD 和 RESPIRATORY BURST OXIDASE HOMOLOG F（RBOHF）和质外体过氧化物酶 peroxidase 33（PRX33）和 peroxidase 34（PRX34）。用发光氨测量的初始质外体氧化爆发后紧接着是第二次氧化爆发，这两次爆发都发生在细胞质氧化的两个波之前。与细胞质氧化相反，这些爆发是 RBOHD 依赖性的。我们的研究结果揭示了模式触发免疫反应期间复杂的氧化来源和动态，包括细胞质氧化在很大程度上独立于先前的细胞外氧化事件。
