CRK2 and C-terminal phosphorylation of NADPH oxidase RBOHD regulate ROS production in Arabidopsis

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 (RLK)-dependent signaling networks. Here we show that the cysteine-rich RLK CRK2 kinase activity is required for plant growth and CRK2 exists in a preformed complex with the NADPH oxidase RBOHD in Arabidopsis. 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 alanine reduced ROS production in Arabidopsis. Phylogenetic analysis suggests that phospho-sites in 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.


Introduction 54
Plants are continuously confronted with stimuli from the surrounding environment, including abiotic cues 55 and invading pathogens. Plant cells also perceive a plethora of signals from neighboring cells and distant 56 tissues. Numerous plasma membrane proteins are involved in the meticulous monitoring and transduction of 57 signals for inter-and intracellular communication. A common early feature of many cellular responses to 58 various environmental changes involves the production of reactive oxygen species (ROS) (Kimura et al.,59 form a monophyletic group, which is separated from human NOX2 and NOX5. The phospho-sites in the C-247 terminal region displayed strong conservation throughout the plant RBOH clade. S703 was conserved in a 248 monophyletic clade containing eight of the ten RBOHs from Arabidopsis but not in the clade containing 249 RBOHH and RBOHJ (Fig. 7). The phospho-sites S611 as well as S862, which may be involved in negative 250 regulation based on experiments in HEK293T cells (Fig. 5b), were strongly conserved in all plant RBOHs. 251 The sequence motifs harboring S611 and S862 are intriguingly conserved even in human NOX2 and NOX5 252 (Fig. 7). The C-terminal region binds FAD and NADPH. Therefore it may underlie strong evolutionary 253 constraints to conserve these binding properties. This may be also reflected in the strong conservation of C-254 terminal phospho-sites and their sequence context not only among plant NADPH oxidases but also animal 255 NOX.

Discussion 257
CRKs are a large group of RLKs involved in biotic and abiotic stress signaling in Arabidopsis (Bourdais et 258 al., 2015). We have previously shown that flg22-triggered extracellular ROS production is altered in several 259 crk mutants (Bourdais et al., 2015). In particular CRK2, a member of the basal clade of CRKs (Vaattovaara 260 et al., 2019), has been highlighted since crk2 displays striking phenotypes (Bourdais et al., 2015) including 261 reduced rosette size and reduced flg22-induced ROS production. Functional CRK2 restored the reduced 262 rosette size (Fig. 1) as well as the MAMP-induced ROS burst (Fig. 2). In addition to their role in stress 263 responses, extracellular ROS have also been implicated in leaf cell expansion (Schmidt et al., 2016), and the 264 rbohd rbohf double mutant displays reduced rosette size (Torres et al., 2002). Overexpression of CRKs has 265 been associated with increased SA accumulation (Acharya et al., Chen et al., 2004). However, since 266 SA levels were unaltered in the loss-of-function mutant crk2 (Fig. S1b), its smaller size may be a 267 consequence of impaired ROS production (Fig. 2). This is supported by the observation that CRK2 enhanced 268 the activity of RBOHD but also RBOHF in HEK293T cells ( Fig. 3a  Phosphorylation of the C-terminus is critical for the regulation of human NADPH oxidases. Phosphorylation 293 of the NOX2 C-terminus by protein kinase C (PKC) enhances assembly of the multimeric NOX2 complex 294 and its activity, whereas phosphorylation by ataxia telangiectasia-mutated ( Although the C-terminal catalytic domain of RBOHs is highly conserved in plants and animal, the N-299 terminus has been considered as important for activation of the ROS-production activity and multiple 300 phospho-sites (S8, S39, S133, S148, S163, S339, S334 and S347) have been reported. Intriguingly, 301 CRK2cyto interacted with and phosphorylated the RBOHD C-terminal region at S611, S703 and S862 (Fig.  302 3e, 4b and Table S1). Phosphorylation of S703 upon xylanase treatment has been reported (Benschop et al., 303 2007) but not linked with other MAMPs or modulation of ROS production. Treatment of flg22 enhanced 304 phosphorylation of S703 in Arabidopsis (Fig. 6c). Mutation S703A in RBOHD led to reduced CRK2-305 dependent RBOHD activity in HEK293T cells (Fig. 5b) and reduced flg22-induced ROS production in 306 Arabidopsis (Fig. 6d). These results suggest that phosphorylation of the RBOHD C-terminus at S703 likely 307 contributes to the regulation of MAMP-induced ROS production. Phosphorylation sites in the C-terminus 308 were highly conserved among RBOHs (Fig. 7) suggesting that phosphorylation of the C-terminal region 309 could be a general feature of plant NADPH oxidases. Remarkably, two putative RBOHD phospho-sites, 310 S611 and S862, were identified even in the human NADPH oxidases NOX2 and NOX5 (Fig. 7). Substitution 311 of RBOHD S862 to alanine resulted in enhanced ROS-producing activity in HEK293T cells. But substitution 312 of RBOHD S611 to alanine, similarly to RBOHD S39A, did not alter ROS production in HEK293T cells 313 (Fig. 5). RBOHD S39A also did not affect flg22-induced ROS production but phospho-mimic S39D 314 enhanced the ROS production and phosphorylation of S39 is enhanced by MAMP treatment in planta 315 (Kadota et al., 2014) (Fig. 6b, S5c). These results suggest the importance to determine the phosphorylation 316 status of S611 and S862 in planta. RBOHD can also be regulated by cysteine S-nitrosylation in the C-317 terminus (Yun et al., 2011) but it is unclear how this modification is integrated with other regulatory 318 mechanisms. Taken together, our results suggest that phosphorylation of the C-terminal region of plant 319 NADPH oxidases is strongly conserved and important for controlling ROS production. 320 Several protein kinases phosphorylate RBOHD N-terminus and regulate the activity including RLCKs 321 However, reduced flg22-induced ROS production in crk2 was not due to lower BIK1 transcript abundance 326 (Fig. S7). BIK1 homologs, AvrPphB SUSCEPTIBLE1 (PBS1) and AvrPphB SUSCEPTIBLE1-LIKE (PBL) 327 kinases, contribute to the regulation of RBOH activity and ROS production is progressively reduced in double mutants with bik1 (Lin et al., 2015;Zhang et al., 2018). CRK2 and BIK1 could synergistically 329 regulate ROS production but we were unable to obtain a double mutant between bik1 and crk2 (Table S2). 330 Therefore, we propose that at least one of these components is essentially required. BIK1 has previously 331 been shown to interact with other kinases including CRKs (Lee, 2017) but interaction with CRK2 has not 332 been investigated. BIK1 and CRK2 are likely highly coordinated in order to precisely control ROS 333 production in response to environmental stimuli (Fig. 8). Like CRKs, RBOHs are involved in diverse 334 processes in stress responses and also plant development and it is conceivable that different CRKs regulate 335 the diverse set of RBOH proteins in various cellular contexts potentially via phosphorylation of the C-336 terminal region. 337 In summary, we propose that CRK2 is a central element in orchestrating the extracellular ROS burst and in 338 mediating the balance between different defense responses. The full complexity and integration of the 339 regulatory components controlling RBOH activity is still a topic of much speculation (Kimura et al. 2017). 340 The diversity of regulators converging at RBOHs reflects the prominent role of apoplastic ROS in signal 341 transduction while simultaneously strict control is required to circumnavigate oxidative damage. We suggest 342 that RBOHD is regulated by phosphorylation of the C-terminal region to complement regulatory 343 mechanisms targeting the N-terminus (Fig. 8). Based on the conservation of serine and threonine residues in 344 the C-terminus of NADPH oxidases we propose that this mode of regulation could be evolutionarily 345 conserved in plants and animals. In the future it will be interesting to investigate how CRK-mediated 346 phosphorylation of the RBOH C-terminus is integrated in the diverse processes which incorporate 347 extracellular ROS.

Phytohormone analysis 466
SA was analyzed from liquid-cultured seedlings as described previously (

Protein extraction and Co-immunoprecipitation 483
Co-immunoprecipitation was performed as described previously (Kadota et  In brief, SepPaks were conditioned using methanol (1 mL), buffer B (80 % acetonitrile, 0.1 % TFA; 1 mL) 555 and buffer A (0.1 % TFA; 2 mL). Samples were loaded by gravity flow, washed with buffer A (1 x 1 mL, 1 x 556 2 mL) and eluted with buffer B (2 x 400 µL). 40 µ L of eluates were kept separately to measure non-557 phosphopeptides and the rest were used for further phosphopeptide enrichment. Phosphopeptide enrichment 558 was performed by hydroxy acid-modified metal-oxide chromatography (HAMMOC) using titania as 559 described previously with minor modifications (Nakagami, 2014;Sugiyama et al., 2007). Carbamidomethylation of cysteine residues was set as fixed, phosphorylation of serine, threonine and 583 tyrosine, oxidation of methionine and protein N-terminal acetylation as variable modifications. The match 584 between runs option was disabled. Peptide-spectrum-matches and proteins were retained if they were below 585 a false discovery rate of 1 % in both cases. The "msms.txt" output from MaxQuant was further analyzed 586 using Skyline in PRM mode. Trypsin specificity was required and a maximum of two missed cleavages 587 allowed. Minimal and maximum peptide lengths were set to 7 and 25 amino acids, respectively. 588 Carbamidomethylation of cysteine, phosphorylation of serine, threonine and tyrosine, oxidation of 589 methionine, and protein N-terminal acetylation were set as modifications. Results were filtered for precursor 590 charges of 2 and 3, and b-and y-ions with ion charges of +1 and +2. Product ions were set to "from ion 1 to 591 last ion". All chromatograms were inspected manually and peak integration was corrected for best 592 representation of MS2 signals. Peak area data was exported and further processed.  MAMPs are recognized by MAMP receptor complexes. RBOHD N-terminus is phosphorylated by BIK1 and 922 SIK1 and apoplastic ROS production is induced. Apoplastic ROS production by RBOHD leads to Ca 2+ 923 influx into the cytosol. Ca 2+ -binding to RBOHD N-terminus and to CPKs leads to Ca 2+ -dependent activation 924 of RBOHD. We found that CRK2 also contributes to the activation of RBOHD via phosphorylation of its C-

S1. In vitro phosphorylation sites of 6His-MBP-RBOHDcyto by 6His-GST-CRK2cyto 1046
The 6His-MBP-RBOHD cytosolic regions were incubated with 6His-GST-CRK2cyto. The 6His-MBP-1047 RBOHDcyto bands were excised from a SDS polyacrylamide gel and subsequently digested by trypsin or Lys-C. 1048 The peptides were analyzed by LC-MS/MS. Phosphorylated peptides are designated as pS. 1049 Table S2. Progeny of CRK2/crk2 BIK1/bik1 parent and CRK2/crk2 bik1/bik1 parent  1050 The genotypes of F2 and F3 progenies were determined by PCR. Observed, the number of individuals observed; 1051 Expected, the expected number based on Mendelian inheritance. Chi-square test was used to determine the 1052 probability (P) of which the deviation of the observed value from the expected value was due to chance. 1053 Table S3       cytosol. Ca -binding to RBOHD N-terminus and to CPKs leads to Ca -dependent activation of RBOHD. We found that CRK2 also contributes to the activation of RBOHD via phosphorylation of its C-terminus at S703. CRK2 can also mediates inhibition of MAPK activation and callose deposition via CALS after MAMP perception. MPK, mitogenactivated protein kinase; MP2K, MPKK; MP3K, MPKKK.