Reactive oxygen species (ROS) are important messengers in eukaryotic organisms. Extracellular ROS production by NADPH oxidases in plants is triggered by receptor-like protein kinase (RLK)-dependent signaling networks. This ROS production is tightly controlled by multiple mechanisms including phosphorylation by different kinases. Here we show that the cysteine-rich RLK CRK2 exists in a preformed complex with the NADPH oxidase RBOHD at the plasma membrane. Functional CRK2 is required for the full pathogen-induced ROS burst and consequently the crk2 mutant is impaired in defense against the bacterial pathogen Pseudomonas syringae. We identified phosphorylation sites in the C-terminal region of RBOHD and mutations of the phosphorylation sites modulate ROS production in response to biotic stimuli. Our work demonstrates that CRK2 occupies a central role in controlling ROS production and highlights that regulation of NADPH oxidase activity by phosphorylation of the C-terminal region is an ancient mechanism which is conserved between animals and plants.

Targeted (phospho)peptide analysis Plant treatment and phosphopeptide enrichment. Arabidopsis seeds were sterilized by incubating with 1.5% NaClO 0.02% Triton X-100 solution for 5 min and vernalized at 4 °C for 2 days. Sterilized seeds were germinated and grown in liquid culture on 6 well plates (30 seeds/well) in MGRL medium with 0.1% (w/v) sucrose (2 mL/well) [1] at 23 °C under continuous light (100 µmol m-2 s-1) in a Percival growth chamber. Plates with 11-day old seedlings were transferred from the growth chamber to a workbench and kept o/n for acclimatization before treatments. Seedlings were treated with either 1 µM flg22 or sterile water for 5 minutes after which seedlings were immediately collected and flash-frozen in liquid nitrogen and stored at -80 °C. Frozen seedlings were disrupted using a Retsch mill (5 min, 30 Hz), and 500 µL urea extraction buffer (8M urea in 100mM Tris, pH 8.5, 20µL/mL Phosphatase Inhibitor Cocktail 3 (Sigma, P0044), 20µL/mL Phosphatase Inhibitor Cocktail 2 (Sigma, P5726), 5 mM DTT) was added to the disrupted frozen powders, mixed briefly and incubated at RT for 30 min. After centrifugation at 15,000 x g for 10 min, supernatants were transferred to fresh tubes. Protein concentrations were determined using Pierce 660 nm protein assay (Thermo Scientific). Extracts with 500 µg of protein were alkylated with 14 mM chloroacetamide (CAA) at RT for 30 min in the dark, CAA was quenched by addition of 1/200 sample volume 1M DTT. Samples were diluted 1:8 with 0.1 M Tris, pH 8.5, 1 mM CaCl2 and were digested o/n at RT either with 5 µg trypsin or 5 µg LysC . Digestion reaction was terminated by addition of TFA (0.1% final concentration), and peptides were desalted using C18 SepPaks (1cc cartridge, 100 mg (WAT023590)). In brief, SepPaks were conditioned using methanol (1 mL), buffer B (80% acetonitrile, 0.1% TFA) (1 mL) and buffer A (0.1% TFA) (2 mL). Samples were loaded by gravity flow, washed with buffer A (1 x 1 mL, 1 x 2 mL) and eluted with buffer B (2 x 400 µL). 40 µL of eluates were kept separately to measure non-phosphopeptides and the rest were used for further phosphopeptide enrichment. Phosphopeptide enrichment was performed by hydroxy acid-modified metal-oxide chromatography (HAMMOC) using titania as described previously with minor modifications [2, 3]. LC-MS/MS data acquisition. Samples were analyzed using an EASY-nLC 1200 (Thermo Fisher) coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher). Peptides were separated on 16 cm frit-less silica emitters (New Objective, 0.75 µm inner diameter), packed in-house with reversed-phase ReproSil-Pur C18 AQ 1.9 µm resin (Dr. Maisch). Peptides were loaded on the column and eluted for 115 min using a segmented linear gradient of 5% to 95% solvent B (0 min : 5%B; 0-5 min -> 5%B; 5-65 min -> 20%B; 65-90 min ->35%B; 90-100 min -> 55%; 100-105 min ->95%, 105-115 min ->95%) (solvent A 0% ACN, 0.1% FA; solvent B 80% ACN, 0.1%FA) at a flow rate of 300 nL/min. Mass spectra were acquired using a targeted (parallel reaction monitoring, PRM) approach. The acquisition method consisted of a full scan method combined with a non-scheduled PRM method. The 16 targeted precursor ions were selected based on the results of a DDA peptide search of phospho-enriched samples in Skyline (Version 4.2.0.x, https://skyline.ms) ({McLean et al., Bioinformatics, 2010, 26, 966. https://academic.oup.com/bioinformatics/article/26/7/966/212410}). MS spectra were acquired in the Orbitrap analyzer with a mass range of 300–1750 m/z at a resolution of 70,000 FWHM and a target value of 3×106 ions, followed by MS/MS acquisition for the 16 targeted precursors. Precursors were selected with an isolation window of 2.0 m/z. HCD fragmentation was performed at a normalized collision energy of 27. MS/MS spectra were acquired with a target value of 2x105 ions at a resolution of 17,500 FWHM, a maximum injection time of 120 ms and a fixed first mass of m/z 100. MS data analysis. Raw data from PRM acquisition were processed using MaxQuant software (version 1.5.7.4, http://www.maxquant.org/) {Cox et al., Nat. Biotechnol. 2008, 26, 1367. https://www.nature.com/articles/nbt.1511}. MS/MS spectra were searched by the Andromeda search engine against a combined database containing the sequences from A. thaliana (TAIR10_pep_20101214; ftp://ftp.arabidopsis.org/home/tair/Proteins/TAIR10_protein_lists/) and sequences of 248 common contaminant proteins and decoy sequences. Trypsin specificity was required and a maximum of two missed cleavages allowed. Minimal peptide length was set to seven amino acids. Carbamidomethylation of cysteine residues was set as fixed, phosphorylation of serine, threonine and tyrosine, oxidation of methionine and protein N-terminal acetylation as variable modifications. The match between runs option was disabled. Peptide-spectrum-matches and proteins were retained if they were below a false discovery rate of 1% in both cases. The “msms.txt” output from MaxQuant was further analyzed using Skyline in PRM mode. Trypsin specificity was required and a maximum of two missed cleavages allowed. Minimal and maximum peptide lengths were set to 7 and 25 amino acids, respectively. Carbamidomethylation of cysteine, phosphorylation of serine, threonine and tyrosine, oxidation of methionine, and protein N-terminal acetylation were set as modifications. Results were filtered for precursor 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 last ion”. All chromatograms were inspected manually and peak integration was corrected for best representation of MS2 signals. Peak area data was exported and further processed. Results of the Skyline analysis have been uploaded to Panorama (https://panoramaweb.org). [1] Fujiwara T, Hirai MY, Chino M, Komeda Y, Naito S. Effects of sulfur nutrition on expression of the soybean seed storage protein genes in transgenic petunia. Plant Physiol. 1992;99: 263–268. pmid:16668860 [2] Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano-LC-MS/MS in proteomics applications. Sugiyama N, Masuda T, Shinoda K, Nakamura A, Tomita M, Ishihama Y. Mol Cell Proteomics. 2007 Jun;6(6):1103-9. Epub 2007 Feb 23. PMID:17322306 [3] StageTip-based HAMMOC, an efficient and inexpensive phosphopeptide enrichment method for plant shotgun phosphoproteomics. Nakagami H. Methods Mol Biol. 2014;1072:595-607. doi: 10.1007/978-1-62703-631-3_40. PMID:24136549

phospho-enriched peptides from Arabidopsis thaliana seedlings (Col-0) treated with 1 uM flg22 peptide or sterile water for 5 min.