Plant Rac Proteins Induce Superoxide Production in Mammalian Cells

doi:10.1006/bbrc.2000.2791

Biochemical and Biophysical Research Communications

Volume 272, Issue 3, 16 June 2000, Pages 783-788

https://doi.org/10.1006/bbrc.2000.2791 Get rights and content

Abstract

The small GTP-binding protein family including Rac proteins represents a paradigm for signaling molecules shared by animal and plants. In mammalian cells, Rac induces the activation of NADPH oxidase leading to superoxide production. In plants, evidence suggests that resistance to pathogens depends on superoxide that is generated via NADPH oxidase-like enzymes. We have identified four closely related Rho/Rac genes from Zea mays that exhibit a high degree of homology to the human Rac. We hypothesized that these plant Rac proteins could function as their mammalian counterpart and activate an enzymatic complex that leads to superoxide production. Here, we show that like human Rac1, activated Zea mays Rac genes can induce superoxide production, when expressed in a mammalian system: NIH 3T3 cells. Our results suggest that in plants, Rac proteins can function as activators of oxidative burst and indicate the remarkable functional and structural conservation of Rho/Rac proteins between plant and animal kingdoms during evolution.

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The role of ROPs in plant defense is often associated with H2O2 production. In mammalian cells, over-expression of the dominant active form of maize ZmRac triggers an increase in reactive oxygen species (ROS) production (Hassanain et al., 2000). Rice over-expressing OsRac1 significantly improved its resistance against Magnaporthe grisea via NADPH oxidase mediated H2O2 accumulation (Kawasaki et al., 1999, 2006; Kosami et al., 2014; Oda et al., 2010; Ono et al., 2001; Wang et al., 2018).
ROP GTPases (Rho-related GTPases from plant), a unique subgroup of the Rho family in plants, is a group of key regulators of different signaling pathways controlling plant growth and development, cell polarity and differentiation, and plant response against biotic and abiotic stresses. The present study determined the potential regulatory mechanism of potato ROP GTPase (StRac1) against Phytophthora infestans (P. infestans) infection. Protein secondary structure analysis indicated that StRAC1 is a Rho GTPase. The expression level of StRac1 was variable in different tissues of potato, with the highest expression in young leaves of both Shepody and Hutou potato varieties. After challenging with P. infestans, the expression level of StRac1was higher in resistance varieties Zihuabai and Longshu 7 than in susceptible varieties Shepody and Desiree. StRAC1 fusion with GFP subcellularly localized at the plasma membrane (PM) in tobacco epidermal cells. The potato with transient or stable over-expression of CA-StRac1 (constitutively active form of StRac1)exhibited a dramatic enhancement of its resistance against P. infestans infections. The increased resistance level in transgenic potato was accompanied with elevated H₂O₂ levels. Importantly, silencing StRac1 via virus-induced gene silencing (VIGS) in potato resulted in higher susceptibility to P. infestans infection than in control plants. In summary, our data reveal that StRac1 regulates potato resistance against P. infestans via positively modulating the accumulation of H₂O₂.
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Hyperoxia has been shown to improve wound healing; however, the mechanism for such therapeutic effects of oxygen remains hypothetical. Rac 1 regulates a wide variety of cellular activities, including cell proliferation and migration, and also is a key regulator for the activity of the nicotinamide dinucleotide phosphate oxidase the enzyme complex responsible for the production of a large fraction of cellular superoxide.
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^☆: Abbreviations used: EPR spectroscopy, electron paramagnetic resonance specroscopy; DEPMPO, 5-(diethoxyphosphory)-5-methyl-1-pyrroline N-oxide; CM-DCF-DA, chloromethyl1-2′,7′-dichlorodihydro-fluorescein diacetate; FITC-Phalloidin, fluorescein-5-isothiocyanate-Phalloidin; ROS, reactive oxygen species; NADPH oxidase, β-nicotinamide adenine dinucleotide phosphate oxidase.

¹: These authors have contributed equally to this study.

²: Present address: Institute of Chemical Kinetics & Combustion, Novosibirsk 630090, Russia.

³: To whom correspondence should be addressed. Fax: (614) 688-5778. E-mail: [email protected].

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Regular ArticlePlant Rac Proteins Induce Superoxide Production in Mammalian Cells☆

Abstract

FEBS Lett.

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Rho GTPases and signaling networks

Genes Dev.

Arabidopsis rho-related GTPases: Differential gene expression in pollen and polar localization in fission yeast [In Process Citation]

Plant Physiol.

Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1

Nature

Interaction of Rac with p67phox and regulation of phagocytic NADPH oxidase activity

Science

Regulation of NADPH oxidase by Rac GTPase activity protein(s)

Mol. Biol. Cell

Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac 2

Science

Function and activation of NF-kappa B in the immune system

Annu. Rev. Immunol.

Structure, regulation and function of NF-kappa B

Annu. Rev. Cell Biol.

rac1 regulates a cytokine-stimulated, redox-dependent pathway necessary for NF-kappaB activation

Mol. Cell. Biol.

Regular Article
Plant Rac Proteins Induce Superoxide Production in Mammalian Cells☆