Abstract
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.
Highlights
Plants are continuously confronted with stimuli from the surrounding environment, including abiotic cues and invading pathogens
The results showed that the C1 and C3 fragments of 6His-maltose binding protein (MBP)-RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) were preferentially phosphorylated by 6His-glutathione S-transferase (GST)-CRK2cyto, while C2 displayed considerably weaker phosphorylation (Figure 4B)
cysteine-rich RLKs (CRKs) are a large group of receptor-like protein kinase (RLK) involved in biotic and abiotic stress signaling in Arabidopsis (Bourdais et al, 2015)
Summary
Plants are continuously confronted with stimuli from the surrounding environment, including abiotic cues and invading pathogens. While ROS are an inevitable byproduct of aerobic metabolism and their unrestricted accumulation can have deleterious consequences (Waszczak et al, 2018), ROS are ubiquitous signaling molecules in plants and animals (Suzuki et al, 2011; Waszczak et al, 2018). Eukaryotic cells produce ROS in several subcellular compartments as well as the extracellular space, in plants referred to as the apoplast (Kimura et al, 2017; Waszczak et al, 2018). NOX-dependent ROS production is involved in regulation of immune functions, cell growth, and apoptosis in animals and plants (Jiménez-Quesada et al, 2016; Waszczak et al, 2018)
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