Regulation of rice NADPH oxidase by binding of Rac GTPase to its N-terminal extension
Regulation of rice NADPH oxidase by binding of Rac GTPase to its N-terminal extension
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DOI：
10.1105/tpc.107.055624
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发表时间：
2007-12-01
期刊：
影响因子：
11.6
通讯作者：
Shimamoto, Ko
中科院分区：
文献类型：
--
作者：
Wong, Hann Ling;Pinontoan, Reinhard;Shimamoto, Ko
NADPH氧化酶产生的活性氧在包括植物天然免疫反应在内的多种细胞活动中起着重要作用。与吞噬细胞的大型多蛋白NADPH氧化酶复合物相反，在植物中，仅发现催化亚基gp 91（phox）和胞质调节剂小GTbac Rac的同源物。gp 91 phox亚基的植物同源物被称为Rboh（呼吸爆发氧化酶同源物）。虽然许多Rboh已在植物中分离，但酶活性的调节仍然未知。迄今为止鉴定的所有rboh基因都具有保守的N末端延伸，其中包含两个Ca 2+结合EF-手基序。在此之前，我们确定了一个小的GTTRac Rac（Os Rac 1）增强病原体相关的分子模式诱导的活性氧产生和水稻（Oryza sativa）对病原体的抗性。在这项研究中，使用酵母双杂交试验，我们发现，Rac GTP酶和N-末端延伸之间的相互作用是普遍存在的，并且Rboh的N-末端区域的相当大的一部分，包括两个EF-手基序，是相互作用所必需的。直接Rac-Rboh相互作用得到了进一步研究的支持，使用体外下拉测定，核磁共振滴定实验，和体内荧光共振能量转移（FRET）显微镜。FRET分析还表明，胞质Ca 2+浓度可以调节Rac-Rboh相互作用的动态方式。此外，Os Rac 1和rbohB的瞬时共表达增强了本氏烟草中ROS的产生，表明Rac-Rboh直接相互作用可能激活植物中NADPH氧化酶的活性。总之，这些结果表明，胞质Ca 2+浓度可能通过调节Rac GT3和Rboh之间的相互作用来调节NADPH氧化酶活性。
Reactive oxygen species (ROS) produced by NADPH oxidase play critical roles in various cellular activities, including plant innate immunity response. In contrast with the large multiprotein NADPH oxidase complex of phagocytes, in plants, only the homologs of the catalytic subunit gp91(phox) and the cytosolic regulator small GTPase Rac are found. Plant homologs of the gp91phox subunit are known as Rboh (for respiratory burst oxidase homolog). Although numerous Rboh have been isolated in plants, the regulation of enzymatic activity remains unknown. All rboh genes identified to date possess a conserved N-terminal extension that contains two Ca2+ binding EF-hand motifs. Previously, we ascertained that a small GTPase Rac (Os Rac1) enhanced pathogen-associated molecular pattern-induced ROS production and resistance to pathogens in rice (Oryza sativa). In this study, using yeast two-hybrid assay, we found that interaction between Rac GTPases and the N-terminal extension is ubiquitous and that a substantial part of the N-terminal region of Rboh, including the two EF-hand motifs, is required for the interaction. The direct Rac-Rboh interaction was supported by further studies using in vitro pull-down assay, a nuclear magnetic resonance titration experiment, and in vivo fluorescence resonance energy transfer (FRET) microscopy. The FRET analysis also suggests that cytosolic Ca2+ concentration may regulate Rac-Rboh interaction in a dynamic manner. Furthermore, transient coexpression of Os Rac1 and rbohB enhanced ROS production in Nicotiana benthamiana, suggesting that direct Rac-Rboh interaction may activate NADPH oxidase activity in plants. Taken together, the results suggest that cytosolic Ca2+ concentration may modulate NADPH oxidase activity by regulating the interaction between Rac GTPase and Rboh.