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

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.

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

“…Given the importance of NADPH oxidases for defense responses and the conservation of RBOHD's C-terminal residues, it is plausible that multiple kinases and E3 ligases will target RBOHs. Recently, the cysteine-rich receptor like kinase CRK2 was found to target RBOHD-C for phosphorylation, including S703, where mimicking phosphorylation enhanced RBOHD activity 38 . CRK2 also phosphorylates RBOHD at position S862 in vitro, and an S862A variant exhibited increased NADPH activity in HEK293 cells, a result that is consistent with our in planta data 38 .…”

“…Recently, the cysteine-rich receptor like kinase CRK2 was found to target RBOHD-C for phosphorylation, including S703, where mimicking phosphorylation enhanced RBOHD activity 38 . CRK2 also phosphorylates RBOHD at position S862 in vitro, and an S862A variant exhibited increased NADPH activity in HEK293 cells, a result that is consistent with our in planta data 38 . Thus, it is likely that the C-terminus of RBOHD will be subjected to convergent regulation by multiple kinases to ensure appropriate ROS production.…”

Comprehensive Analysis of Respiratory Burst Oxidase Homologs (Rboh) Gene Family and Function of GbRboh5/18 on Verticillium Wilt Resistance in Gossypium barbadense

“…AtRbohD showed the highest expression of the 10 AtRbohs : it functions via ROS production in abscisic acid-induced stomatal closure, flagellin-induced immune responses ( Pogany et al, 2009 ), resistance to Botrytis cinereal ( Galletti et al, 2008 ), as well as phosphorylation mediated by CRK2 in plant defense against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 ( Kimura et al, 2020 ). AtRbohD-dependent H 2 O 2 production was positively regulated by H2Bub1 in the responses against Vd-toxins in Arabidopsis ( Zhao et al, 2020 ).…”

NADPH oxidases and the evolution of plant salinity tolerance

“…At the same time, many other reports questioned the validity of this approach, reporting no or a negative correlation between activity of antioxidant enzymes and plant salinity stress tolerance (Tanveer & Shabala, 2018). The reason for this discrepancy may be related to the fact that ROS also play an important signalling role in plant adaptive and developmental responses Kimura et al, 2020;Mittler, 2017). The ROS signalling network is an evolutionary conserved signal transduction network found in all aerobic organisms that controls a large array of biological processes ranging from the regulation of development and growth to responses to biotic and/or abiotic stimuli (Mittler et al, 2011;Suzuki, Koussevitzky, Mittler, & Miller, 2012 Gilroy et al, 2014;Hu et al, 2020;Suzuki et al, 2012).…”

“…The C-terminus is highly conserved, which contains NADPH-and FAD-binding sites. In this region, S703 is phosphorylated by CRK2, while C890 undergoes S-nitrosylation (Kimura et al, 2020;Yun et al, 2011). (b), (c) and (d) The unique predicted phosphorylation sites in four halophytes.…”