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Regulation of Rice NADPH Oxidase by Binding of Rac GTPase to Its N-Terminal Extension^[W],[OA]

^a Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, Ikoma, 630-0192 Nara, Japan
^b Laboratory of Biophysics, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, 630-0192 Nara, Japan
^c Department of Biology, Faculty of Science, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan
^d Laboratory of Defense in Plant–Pathogen Interactions, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan

⁴ Address correspondence to simamoto{at}bs.naist.jp.

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 gp91^phox 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.

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