The crystal structure of the plant small GTPase OsRac1 reveals its mode of binding to NADPH oxidase

J Biol Chem. 2014 Oct 10;289(41):28569-78. doi: 10.1074/jbc.M114.603282. Epub 2014 Aug 15.


Rac/Rop proteins are Rho-type small GTPases that act as molecular switches in plants. Recent studies have identified these proteins as key components in many major plant signaling pathways, such as innate immunity, pollen tube growth, and root hair formation. In rice, the Rac/Rop protein OsRac1 plays an important role in regulating the production of reactive oxygen species (ROS) by the NADPH oxidase OsRbohB during innate immunity. However, the molecular mechanism by which OsRac1 regulates OsRbohB remains unknown. Here, we report the crystal structure of OsRac1 complexed with the non-hydrolyzable GTP analog guanosine 5'-(β,γ-imido)triphosphate at 1.9 Å resolution; this represents the first active-form structure of a plant small GTPase. To elucidate the ROS production in rice cells, structural information was used to design OsRac1 mutants that displayed reduced binding to OsRbohB. Only mutations in the OsRac1 Switch I region showed attenuated interactions with OsRbohB in vitro. In particular, Tyr(39) and Asp(45) substitutions suppressed ROS production in rice cells, indicating that these residues are critical for interaction with and activation of OsRbohB. Structural comparison of active-form OsRac1 with AtRop9 in its GDP-bound inactive form showed a large conformational difference in the vicinity of these residues. Our results provide new insights into the molecular mechanism of the immune response through OsRac1 and the various cellular responses associated with plant Rac/Rop proteins.

Keywords: Crystal Structure; NADPH Oxidase; Plant Defense; Rac (Rac GTPase); Rice Immunity; Small GTPase.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Arabidopsis / enzymology
  • Arabidopsis / genetics
  • Arabidopsis Proteins / chemistry
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism
  • Binding Sites
  • Crystallography, X-Ray
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression Regulation, Plant
  • Guanylyl Imidodiphosphate / chemistry*
  • Guanylyl Imidodiphosphate / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Monomeric GTP-Binding Proteins / chemistry
  • Monomeric GTP-Binding Proteins / genetics
  • Monomeric GTP-Binding Proteins / metabolism
  • Mutation
  • NADPH Oxidases / chemistry*
  • NADPH Oxidases / genetics
  • NADPH Oxidases / metabolism
  • Oryza / chemistry*
  • Oryza / enzymology
  • Oryza / genetics
  • Oryza / immunology
  • Oxidation-Reduction
  • Phosphatidylinositol Phosphates / chemistry*
  • Phosphatidylinositol Phosphates / metabolism
  • Plant Immunity
  • Plant Proteins / chemistry*
  • Plant Proteins / genetics
  • Plant Proteins / metabolism
  • Protein Binding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Reactive Oxygen Species / chemistry
  • Reactive Oxygen Species / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Signal Transduction
  • rac1 GTP-Binding Protein / chemistry*
  • rac1 GTP-Binding Protein / genetics
  • rac1 GTP-Binding Protein / metabolism


  • Arabidopsis Proteins
  • Phosphatidylinositol Phosphates
  • Plant Proteins
  • Reactive Oxygen Species
  • Recombinant Proteins
  • phosphatidylinositol 3-phosphate
  • Guanylyl Imidodiphosphate
  • NADPH Oxidases
  • Monomeric GTP-Binding Proteins
  • ROP9 protein, Arabidopsis
  • rac1 GTP-Binding Protein

Associated data

  • PDB/4U5X