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k Menke"],"technology_type":["Data-dependent acquisition","Mass Spectrometry","Bottom-up proteomics"],"software":[""],"submitter_keywords":[""],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD063345"],"tissue":["Plant Cell"],"sample_protocol":["For phosphorylation site analysis of His-BIK1* by MIK2 kinase and GRF2 by BIK1, samples were separated on a NuPAGE 4–12% Bis-Tris gel (Invitrogen) and bands excised after InstantBlue staining (Abcam). For AHA1 IP-MS, two-week-old Col-0/AHA1pro::AHA1-GFP and Col-0/GFP-LTI6b seedlings were collected. IP beads were resuspended in 50 µL elution buffer (Invitrogen), incubated at 80 °C for 8 min, and samples run into a NuPAGE 4–12% gel. Lanes were excised post-InstantBlue staining, cut into small pieces, washed thrice with 50% acetonitrile/50 mM ammonium bicarbonate (AcN/ABC) for 30 min, and dehydrated in acetonitrile (10 min). Gel pieces were reduced with 10 mM DTT (30 min, 45 °C), alkylated with 55 mM chloroacetamide (20 min, RT), then washed three times with 50% AcN/ABC (30 min). After dehydration, gels were rehydrated with 40 µL of trypsin (100 ng in 50 mM ABC, 5% AcN). If needed, 50 mM ABC was added to cover gels before overnight incubation at 37 °C. Tryptic peptides were extracted thrice in 50% acetonitrile/5% formic acid (Pierce) for 30 min each, dried, and resuspended in 2% acetonitrile/0.2% TFA (Sigma). Three biological replicates were performed per sample.  Samples were analyzed on either an Orbitrap Fusion Tribrid or Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific), both coupled to a U3000 nano-UPLC system.  For Orbitrap Fusion, ~35% of peptides were injected onto a reverse-phase trap column (NanoEase m/z Symmetry C18, 5 μm, 180 μm × 20 mm; Waters) at 20 μL min⁻¹ in 2% acetonitrile/0.05% TFA, and eluted to the analytical column (NanoEase m/z HSS C18 T3, 1.8 μm, 75 μm × 250 mm; Waters). Elution used a gradient of buffer B (80% acetonitrile/0.05% FA; A: 0.1% FA): 2.5 min 3% B, 5 min 6.3%, 13 min 12.5%, 50 min 42.5%, 58 min 50%, 61 min 65%, 63 min 99%, 66 min 99%, 67 min 3%, 90 min 3%; flow rate 200 nL min⁻¹. The MS was in positive ion mode using a nano-electrospray source (+2.2 kV, 275 °C capillary temp, PicoTip emitter ID 10 μm). It operated in data-dependent mode (full scan m/z 300–1800, resolution 120K, AGC target 1e6), followed by MS/MS on top 40 ions (CE 30%, 1.6 m/z isolation, resolution 120K, AGC target 1e5). Precursors (charge 2–7) were fragmented and dynamically excluded for 30 s. Multistage activation was enabled for neutral losses (-98, -49, -32.7 m/z). Minimum AGC was 5e3, intensity threshold 4.8e4. Peptide match was preferred and isotope exclusion enabled.  For Orbitrap Eclipse with FAIMS, ~20% of peptides were loaded onto a trap (PepMap Neo, C18, 5 μm, 0.3 × 5 mm; Thermo) in 0.1% TFA at 15 μL min⁻¹ for 3 min. The trap was switched in-line with an analytical column (Aurora Frontier TS, 60 cm, 75 μm ID, C18, 1.7 μm, 120 Å; IonOpticks) and separated at 60 °C using buffers A (water, 0.1% FA) and B (80% acetonitrile, 0.1% FA), 0.26 μL min⁻¹: 0–3 min 1% B (trapping), 3–10 min to 8% (curve 4), 10–102 min linear to 48%, ramp to 99% B, then re-equilibrated to 0%, total 140 min. MS data were acquired with FAIMS at three CVs (–35, –50, –65 V) for 1 s each. MS1: OT resolution 120K, profile mode, m/z 300–1600, AGC 100%, max IT 50 ms. MS2: IT Turbo, 1 Da isolation, charge 2–5, threshold 1e4, HCD CE 30, standard AGC, dynamic IT, exclusion 15 s (±10 ppm), one charge state per precursor."],"repository":["Pride"],"quantification_method":[""],"modification":[""],"data_protocol":["Peak lists in the form of Mascot generic files were prepared from raw data files using MS Convert v.3.0 (Proteowizard) and sent to a peptide search on Mascot server v.2.8.2 using Mascot Daemon (Matrix Science) against an in-house constructs and contaminants database and the E. coli K12 protein database. Tryptic peptides with up to two possible mis-cleavages and charge states +2 and +3 were allowed in the search. The following peptide modifications were included in the search: carbamidomethylated cysteine (fixed), oxidized methionine (variable), with phosphorylated serine, threonine and tyrosine (variable). Data were searched with a monoisotopic precursor and fragment ion mass tolerance 10 ppm and 0.8 Da respectively. Decoy database was used to validate peptide sequence matches. Mascot results were combined in Scaffold v.5.0.0 (Proteome Software) and filtered to show only phospho-peptides. For His-BIK1* phosphopeptide analysis, peptide probability and protein threshold were set at 50.0 % and 99.0 % respectively and spectra were manually curated to verify validity of identification and position of the phosphorylated residue. For His-MBP-GRF2 analysis, peptide probability and protein threshold were each set at 1 % false discovery rate (FDR). Spectral counts for each identified phospho-peptide from three biological replicates were summed. Spectral counts from mis-cleaved peptides identifying the same phosphorylation site were then summed to give final spectral counts for each site. For identification of GRFs potentially interacting with AHA1, peptide probability and protein threshold were each set at 1 % FDR. Mean spectral counts from three biological replicates each for AHA1-GFP and GFP-LTI6b controls were then compared."],"omics_type":["Proteomics"],"labhead":["Frank Menke"],"instrument_platform":[""],"labhead_affiliation":["The Sainsbury Laboratory"],"submission_type":["PARTIAL"],"species":["Arabidopsis Thaliana (mouse-ear Cress)"],"publication":["Not available"],"submitter_mail":["frank.menke@tsl.ac.uk"],"submitter_affiliation":["the Sainsbury laboratory"],"submitter_country":["United Kingdom"],"additional_accession":[]},"is_claimable":false,"name":"A phosphorelay circuit drives extracellular alkalinization in receptor kinase-mediatedimmune and cell wall damage signaling","description":"Extracellular alkalinization has long been recognized as a hallmark of plant cell-surfacereceptor activation, including during pattern-triggered immunity (PTI); yet themechanisms driving elicitor-induced alkalinization and its role in plant signaling remainunclear. Here, we demonstrate that inhibition of autoinhibited H+-ATPases (AHAs) isrequired for elicitor-induced extracellular alkalinization. This alkalinization is essentialfor immune and cell wall damage signaling mediated by diverse plasma membrane-localised receptor kinases (RKs) likely through modulation of ligand-receptorinteractions. Mechanistically, RKs transduce elicitor-triggered signaling via thereceptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1), whichinhibits AHA activity by disrupting AHA-GENERAL REGULATORY FACTOR (GRF)interactions through a conserved phosphorylation event. This phosphorylation-drivenextracellular alkalinization module is required for disease resistance and cell walldamage responses initiated by ligand-RK pairs. Our findings uncover a conservedphosphorelay circuit that broadly regulates extracellular alkalinization to coordinate RK signaling, illuminating a general mechanism for RK activation and stress resilience.","dates":{"publication":"2026-04-14","submission":"2025-04-25"},"accession":"PXD063345","cross_references":{"TAXONOMY":["NEWT:6945","NEWT:3555","NEWT:2","NEWT:157546","NEWT:35554","NEWT:38942","NEWT:307972","NEWT:32046","NEWT:544496","NEWT:2042546","NEWT:45351","NEWT:43179","NEWT:4513","NEWT:5722","NEWT:55153","NCBITaxon:10407","NEWT:1736309","NEWT:309800","NEWT:1211601","NEWT:876138","NEWT:237561","NEWT:5833","NEWT:6928","NEWT:10036","NEWT:36745","NEWT:1351","NEWT:1438992","NEWT:2649997","NEWT:272563","NEWT:224326","NCBITaxon:79857","NEWT:1096976","NEWT:95648","NEWT:3885","NEWT:3888","NEWT:1589","NEWT:135622","NCBITaxon:4896","NEWT:6915","NEWT:3649","NEWT:101510","NEWT:3880","NEWT:272559","NEWT:3641","NEWT:383379","NEWT:466585","NEWT:10029","NEWT:1000589","NEWT:85963","NEWT:85962","NEWT:317447","NEWT:7955","NEWT:7959","NEWT:2261","NEWT:31156","NEWT:398580","NEWT:4565","NEWT:1264690","NEWT:515619","NEWT:192875","NEWT:34305","NEWT:59729","NCBITaxon:183674","NEWT:224308","NEWT:84645","NEWT:626528","NEWT:139927","NEWT:4558","NEWT:209285","NEWT:1283","NEWT:931281","NEWT:4550","NEWT:1000561","NEWT:197","NCBITaxon:79824","NEWT:4787","NCBITaxon:4563","NEWT:5755","NEWT:44689","NEWT:3218","NEWT:5759","NEWT:1736231","NEWT:1270","NEWT:2242","NEWT:4784","NEWT:11320","NEWT:360106","NEWT:286","NEWT:287","NEWT:10117","NEWT:10239","NEWT:10116","NEWT:1280","NEWT:1735272","NEWT:83334","NEWT:83332","NEWT:44685","NEWT:317513","NEWT:1148","NEWT:580240","NEWT:294128","NEWT:11676","NEWT:55571","NEWT:100226","NEWT:4530","NEWT:4896","NEWT:75058","NEWT:13616","NEWT:1094343","NEWT:296543","NEWT:1773","NEWT:1895","NEWT:1182590","NEWT:3712","NEWT:935293","NEWT:64152","NEWT:4924","NEWT:749200","NEWT:990346","NEWT:145953","NEWT:257309","NEWT:100816","NEWT:263","NEWT:230741","NEWT:52283","NEWT:284812","NCBITaxon:1313","NEWT:43330","NEWT:1603293","NEWT:408169","NEWT:44544","NEWT:4911","NEWT:645463","NEWT:3702","NEWT:129249","NEWT:243277","NEWT:990119","NEWT:408172","NEWT:408170","NEWT:493760","NEWT:260710","NEWT:257313","NEWT:400772","NEWT:3708","NEWT:128161","NEWT:332648","NEWT:106592","NEWT:536231","NEWT:460519","NEWT:1187947","NEWT:1432138","NEWT:10312","NEWT:1424507","NCBITaxon:1773","NEWT:9598","NEWT:8030","NEWT:1639","NEWT:188229","NEWT:3818","NEWT:480","NEWT:4909","NEWT:67767","NEWT:432359","NEWT:46835","NEWT:1182263","NEWT:2711","NEWT:376686","NEWT:95486","NEWT:9103","NEWT:29159","NEWT:253","NEWT:10306","NCBITaxon:2759","NEWT:1233435","NEWT:8022","NEWT:145943","NCBITaxon:4932","NEWT:595536","NEWT:240906","NEWT:593117","NEWT:3635","NEWT:5811","NEWT:235443","NEWT:272624","NEWT:411483","NEWT:884019","NEWT:198215","NEWT:411490","NEWT:983964","NEWT:169963","NEWT:32644","NEWT:499175","NEWT:109779","NEWT:476272","NEWT:3747","NEWT:195051","NEWT:367830","NEWT:1255228","NEWT:178616","NEWT:410289","NEWT:373153","NEWT:352472","NEWT:357","NEWT:360094","NEWT:470","NEWT:1313","NEWT:411469","NEWT:84023","NEWT:559292","NEWT:39491","NCBITaxon:5811","NEWT:411464","NEWT:411460","NEWT:2014887","NEWT:2762","NEWT:1174673","NEWT:562","NEWT:411470","NEWT:33952","NEWT:2094720","NCBITaxon:2697049","NEWT:571256","NEWT:28038","NEWT:1663","NEWT:1423","NEWT:4932","NEWT:3603","NEWT:2759","NEWT:3847","NEWT:327159","NEWT:178876","NEWT:327160","NEWT:573","NEWT:9031","NEWT:7091","NEWT:241368","NEWT:42528","NEWT:190802","NEWT:9778","NEWT:150475","NEWT:303","NEWT:9417","NEWT:7111","NEWT:347515","NEWT:1216979","NEWT:5180","NEWT:256737","NEWT:115104","NEWT:1121114","NEWT:663","NEWT:1081927","NEWT:1238993","NEWT:67825","NEWT:185579","NEWT:941442","NEWT:220668","NEWT:13076","NEWT:1249668","NEWT:7108","NEWT:317","NEWT:7227","NEWT:7469","NEWT:885318","NEWT:9402","NEWT:415540","NEWT:550","NEWT:675060","NEWT:4081","NEWT:334542","NEWT:554","NEWT:98334","NEWT:426428","NEWT:7574","NEWT:1715256","NEWT:7215","NEWT:575412","NEWT:29204","NEWT:2172103","NEWT:507601","NEWT:643680","NCBITaxon:6157","NEWT:746360","NEWT:6239","NEWT:470150","NEWT:216257","NEWT:102169","NEWT:9986","NEWT:4054","NEWT:73239","NEWT:226186","NEWT:1268063","NEWT:8782","NEWT:1263854","NEWT:435590","NEWT:1902","NEWT:160488","NEWT:28104","NEWT:1908","NCBITaxon:2","NEWT:985076","NEWT:1215323","NEWT:52641","NEWT:7038","NEWT:6192","NEWT:28532","NCBITaxon:38727","NEWT:353152","NEWT:2829","NEWT:366581","NEWT:216599","NEWT:216595","NEWT:243230","NEWT:8355","NEWT:9685","NEWT:7029","NEWT:1080772","NEWT:1283300","NEWT:6183","NEWT:6063","NEWT:334747","NEWT:61235","NEWT:6289","NEWT:436486","NEWT:6287","NEWT:300641","NEWT:727","NEWT:9796","NEWT:725","NEWT:170187","NEWT:260707","NCBITaxon:6191","NEWT:1836","NEWT:185431","NEWT:29760","NEWT:260704","NEWT:703612","NEWT:260705","NEWT:80863","NEWT:2697049","NEWT:105231","NEWT:1216981","NCBITaxon:6073","NEWT:884204","NEWT:6279","NEWT:1123869","NEWT:9544","NEWT:7370","NEWT:83906","NEWT:607699","NEWT:6282","NEWT:208964","NEWT:1134506","NEWT:575584","NEWT:38783","NEWT:8727","NEWT:4006","NEWT:8726","NEWT:6426","NEWT:6669","NEWT:10090","NEWT:4120","NEWT:51515","NEWT:5693","NEWT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