CRK2 and C-terminal Phosphorylation of NADPH Oxidase RBOHD Regulate Reactive Oxygen Species Production in Arabidopsis

Plant Cell. 2020 Apr;32(4):1063-1080. doi: 10.1105/tpc.19.00525. Epub 2020 Feb 7.


Reactive oxygen species (ROS) are important messengers in eukaryotic organisms, and their production is tightly controlled. Active extracellular ROS production by NADPH oxidases in plants is triggered by receptor-like protein kinase-dependent signaling networks. Here, we show that CYSTEINE-RICH RLK2 (CRK2) kinase activity is required for plant growth and CRK2 exists in a preformed complex with the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) in Arabidopsis (Arabidopsis thaliana). Functional CRK2 is required for the full elicitor-induced ROS burst, and consequently the crk2 mutant is impaired in defense against the bacterial pathogen Pseudomonas syringae pv tomato DC3000. Our work demonstrates that CRK2 regulates plant innate immunity. We identified in vitro CRK2-dependent phosphorylation sites in the C-terminal region of RBOHD. Phosphorylation of S703 RBOHD is enhanced upon flg22 treatment, and substitution of S703 with Ala reduced ROS production in Arabidopsis. Phylogenetic analysis suggests that phospho-sites in the C-terminal region of RBOHD are conserved throughout the plant lineage and between animals and plants. We propose that regulation of NADPH oxidase activity by phosphorylation of the C-terminal region might be an ancient mechanism and that CRK2 is an important element in regulating microbe-associated molecular pattern-triggered ROS production.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Arabidopsis / drug effects
  • Arabidopsis / metabolism*
  • Arabidopsis / microbiology
  • Arabidopsis Proteins / chemistry
  • Arabidopsis Proteins / metabolism*
  • Conserved Sequence
  • Cytosol / drug effects
  • Cytosol / metabolism
  • Disease Resistance
  • Flagellin / pharmacology
  • HEK293 Cells
  • Humans
  • Models, Biological
  • NADPH Oxidases / chemistry*
  • NADPH Oxidases / metabolism*
  • Pathogen-Associated Molecular Pattern Molecules / metabolism
  • Phosphorylation / drug effects
  • Phosphoserine / metabolism
  • Plant Development / drug effects
  • Plant Diseases / microbiology
  • Protein Binding / drug effects
  • Protein-Serine-Threonine Kinases / chemistry
  • Protein-Serine-Threonine Kinases / metabolism*
  • Pseudomonas syringae / pathogenicity
  • Pseudomonas syringae / physiology
  • Reactive Oxygen Species / metabolism*
  • Virulence / drug effects


  • Arabidopsis Proteins
  • Pathogen-Associated Molecular Pattern Molecules
  • Reactive Oxygen Species
  • Flagellin
  • Phosphoserine
  • respiratory burst oxidase homolog D, Arabidopsis
  • NADPH Oxidases
  • AT1G70520 protein, Arabidopsis
  • Protein-Serine-Threonine Kinases