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  • Regulation of reactive oxygen species during plant immunity through phosphorylation and ubiquitination of RBOHD.

Regulation of reactive oxygen species during plant immunity through phosphorylation and ubiquitination of RBOHD.

Nature communications (2020-04-17)
DongHyuk Lee, Neeraj K Lal, Zuh-Jyh Daniel Lin, Shisong Ma, Jun Liu, Bardo Castro, Tania Toruño, Savithramma P Dinesh-Kumar, Gitta Coaker
ABSTRACT

Production of reactive oxygen species (ROS) is critical for successful activation of immune responses against pathogen infection. The plant NADPH oxidase RBOHD is a primary player in ROS production during innate immunity. However, how RBOHD is negatively regulated remains elusive. Here we show that RBOHD is regulated by C-terminal phosphorylation and ubiquitination. Genetic and biochemical analyses reveal that the PBL13 receptor-like cytoplasmic kinase phosphorylates RBOHD's C-terminus and two phosphorylated residues (S862 and T912) affect RBOHD activity and stability, respectively. Using protein array technology, we identified an E3 ubiquitin ligase PIRE (PBL13 interacting RING domain E3 ligase) that interacts with both PBL13 and RBOHD. Mimicking phosphorylation of RBOHD (T912D) results in enhanced ubiquitination and decreased protein abundance. PIRE and PBL13 mutants display higher RBOHD protein accumulation, increased ROS production, and are more resistant to bacterial infection. Thus, our study reveals an intricate post-translational network that negatively regulates the abundance of a conserved NADPH oxidase.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Monoclonal Anti-FLAG-Peroxidase antibody produced in rat, 2-4 mg/mL, clone 6F7, purified immunoglobulin
Roche
Anti-HA-Peroxidase, High Affinity, from rat IgG1
Sigma-Aldrich
Anti-Ubiquitin Antibody, clone P4D1-A11, clone P4D1-A11, Upstate®, from mouse