The receptor-like cytosolic kinase RIPK activates NADP-malic enzyme 2 to generate NADPH for fueling ROS production
The receptor-like cytosolic kinase RIPK activates NADP-malic enzyme 2 to generate NADPH for fueling ROS production
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DOI：
10.1016/j.molp.2022.03.003
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发表时间：
2022-05-02
期刊：
影响因子：
27.5
通讯作者：
Liang, Yan
中科院分区：
文献类型：
--
作者：
Wu, Binyan;Li, Ping;Liang, Yan
活性氧（ROS）的产生是拟南芥中一种保守的免疫反应，主要由呼吸爆发氧化酶同源物D（RBOHD）介导，呼吸爆发氧化酶同源物D是一种与质膜相关的烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶。NADPH的快速增加是RBOHD蛋白质的必需燃料，从而维持ROS的产生。然而，NADPH产生的燃料RBOHD的分子机制仍不清楚。在这项研究中，我们分离出一个新的突变等位基因鞭毛蛋白不敏感4（FIN 4），它编码的第一个酶在从头NAD的生物合成。fin 4突变体显示降低的NADPH水平和受损的ROS产生。然而，FIN 4和其他参与NAD和NADPH生成途径的基因在激发子处理后并没有高度上调，这提高了细胞溶质NADP连接的脱氢酶可能被转录后激活以维持NADPH供应接近RBOHD的可能性。为了验证这种可能性，我们分离了与RPM 1诱导的蛋白激酶（RIPK）相关的蛋白质，RIPK是一种受体样细胞质激酶，在植物免疫中调节广谱ROS信号传导，并鉴定了NADP-苹果酸酶2（NADP-ME 2），一种NADPH生成酶。与野生型植物相比，nadp-me 2突变体显示NADP-ME活性降低，NADPH水平降低，并减少响应于免疫激发子的ROS产生。此外，我们发现RIPK可以直接磷酸化NADP-ME 2，并在体外增强其活性。NADP-ME 2 S371残基的磷酸化有助于免疫诱导剂处理后的ROS产生和对坏死营养细菌胡萝卜果胶杆菌的易感性。总的来说，我们的研究表明，RIPK磷酸化和激活NADP-ME 2，以快速增加胞质NADPH，从而促进RBOHD维持植物免疫中的ROS产生。
Reactive oxygen species (ROS) production is a conserved immune response in Arabidopsis primarily mediated by respiratory burst oxidase homolog D (RBOHD), a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase associated with the plasma membrane. A rapid increase in NADPH is necessary to fuel RBOHD proteins and thus maintain ROS production. However, the molecular mechanism by which NADPH is generated to fuel RBOHD remains unclear. In this study, we isolated a new mutant allele of FLAGELLIN-INSENSITIVE 4 (FIN4), which encodes the first enzyme in de novo NAD biosynthesis. fin4 mutants show reduced NADPH levels and impaired ROS production. However, FIN4 and other genes involved in NAD- and NADPH-generating pathways are not highly upregulated upon elicitor treatment, raising a possibility that a cytosolic NADP-linked dehydrogenase might be post-transcriptionally activated to maintain the NADPH supply close to RBOHD. To verify this possibility, we isolated the proteins associated with RPM1-INDUCED PROTEIN KINASE (RIPK), a receptor-like cytoplasmic kinase that regulates broadspectrum ROS signaling in plant immunity, and identified NADP-malic enzyme 2 (NADP-ME2), an NADPH-generating enzyme. Compared with wild-type plants, nadp-me2 mutants display decreased NADP-ME activity, lower NADPH levels, and reduced ROS production in response to immune elicitors. Furthermore, we found that RIPK can directly phosphorylate NADP-ME2 and enhance its activity in vitro. The phosphorylation of the NADP-ME2 S371 residue contributes to ROS production upon immune elicitor treatment and susceptibility to the necrotrophic bacterium Pectobacterium carotovorum. Collectively, our study suggests that RIPK phosphorylates and activates NADP-ME2 to rapidly increase cytosolic NADPH, thus fueling RBOHD to sustain ROS production in plant immunity.