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

Donghyuk Lee,Savithramma P Dinesh-Kumar,Jun Liu,Bardo Castro,Neeraj K Lal,Shisong Ma,Tania Toruño,Zuh-Jyh Daniel Lin,Gitta Coaker

doi:10.1038/s41467-020-15601-5

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.

Highlights

Production of reactive oxygen species (ROS) is critical for successful activation of immune responses against pathogen infection
Following FLAGELLIN-SENSING 2 (FLS2)−BAK1 complex formation, a series of trans-phosphorylation events between various intracellular kinases associated with the pattern recognition receptors (PRRs) complex leads to activation of downstream signaling including a rapid burst of reactive oxygen species (ROS), activation of mitogen-activated protein kinase (MAPK) cascades, calcium influx, regulation of calciumdependent protein kinases (CPKs), transcriptional reprogramming and phytohormone regulation[6]
Since the majority of pattern-triggered immunity (PTI)-induced ROS in plants is produced by RBOHD and its homologs[13,14,15], we investigated if RBOHD can associate with PBS1-like kinase 13 (PBL13)

Summary

Introduction

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. PIRE and PBL13 mutants display higher RBOHD protein accumulation, increased ROS production, and are more resistant to bacterial infection. Plant innate immunity relies on plasma membrane localized pattern recognition receptors (PRRs) to provide resistance against a broad spectrum of pathogens[1]. Following FLS2−BAK1 complex formation, a series of trans-phosphorylation events between various intracellular kinases associated with the PRR complex leads to activation of downstream signaling including a rapid burst of reactive oxygen species (ROS), activation of mitogen-activated protein kinase (MAPK) cascades, calcium influx, regulation of calciumdependent protein kinases (CPKs), transcriptional reprogramming and phytohormone regulation[6]

Methods

Results

Conclusion