Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075759/4597_PG_Report.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075759/4597_Name.tsvftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075759/4597_PG_Report.setup.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075759/checksum.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075759/E2_4597_07.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075759/E2_4597_12.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075759/E2_4597_10.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075759/E2_4597_13.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075759/E2_4597_15.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075759/E2_4597_06.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075759/E2_4597_11.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075759/E2_4597_16.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075759/E2_4597_14.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075759/E2_4597_08.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075759/4597_AXE_GFP_FASTA.fastaftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075759/4597.sneprimaryOK200azuccaro@uni-koeln.deProteomics FacilityMass SpectrometryData-independent acquisitionSymbiosisHemicellulose remodelingHost immune modulationCarbohydrate-active enzymes (cazymes)Acetyl-xylan esterasePlant-microbe interactionshttps://www.ebi.ac.uk/pride/archive/projects/PXD075759Protein SecretionApoplastic fluid (AF) was isolated from barley seedlings as described previously (Saake et al, 2025 https://doi.org/10.1111/nph.70022). Germinated barley seedlings were inoculated with S. indica on 1/10 PNM medium and per replicate, 40 barley seedlings were harvested at 14 dpi, which yielded approximately ~0.5 ml AF per replicate. Harvested roots were surface washed thoroughly in ice-cold water to remove external fungal material, cut into pieces of 2 cm, transferred to ice cold water and vacuum infiltrated (five cycles of15 min 250 mbar, 1.5 min ATM). Then, the roots were gently dried on tissue paper and transferred into a 20 ml syringe in a 50 ml falcon tube, centrifuged for 15 min at 4 °C, 2000 rpm (lowest de- and acceleration) to collect AF in the falcon tube. The isolated AF was stored at -20°C. Proteome analysis was done for the following samples: AF from barley colonized by Serendipita indica expressing GFP (GFP-2), SiAXE (AXE-3). As control, protein was extracted from mycelium of GFP-2 grown in axenic culture by the following protocol: a 100 mL starter culture of CM medium was inoculated with chlamydospores and incubated for one week at 28°C while shaking at 120 rpm. The mycelium was harvested using Miracloth (22-25 µm pore size) and washed with sterile MQ-H2O containing 0.9 % NaCl. The washed mycelium was resuspended in 100 mL fresh CM medium and homogenized with a blender. The resulting mycelial fragments were incubated at 28 °C while shaking at 130 rpm to allow regeneration. After three days, the mycelium was harvested using Miracloth (22-25 µm pore size). The mycelium was washed with 0.9 % NaCl, blotted dry on tissue paper and frozen in liquid nitrogen for protein extraction with the following method: S. indica mycelium was ground with mortar and pestle under liquid N2 to a fine powder and 200 mg FW transferred per sample to a 2 ml tube. Then, 500 µl of pre-cooled TKMES extraction buffer (100 mM tricine; pH=7.5, 10 mM KCL, 10 mM MgCl2, 1 mM EDTA, 10% w/v sucrose, freshly added 0.2 % v/v Triton X-100, 1 mM DTT, 100 µg ml-1 PMSF, 1x protease inhibitor cocktail (Sigma Aldrich; #P9599) was added and vortexed vigorously for 20 seconds. The homogenate was centrifuged at 10000 g for 10 minutes at 4°C to pellet cell debris and the supernatant harvested. For AF and mycelium samples, protein amount was determined with Bradford reagent (Bio-Rad) and 5 µg total protein precipitated with acetone, prior to reduction with DTT and alkylation of cysteine residues with chloroacetamide. Then, protein were subjected to on-bead digestion on Sera-Mag beads with trypsin and endoproteinase Lys-C and desalted using SDB-RPS StageTips and prepared for label-free quantitative proteomics. Protein mass spectrometry of digested peptides was done on an Orbitrap Exploris 480 mass spectrometer coupled to a Vanquish neo in trap-and-elute setup (Thermo Scientific). Samples were loaded onto a 2 cm precolumn (Acclaim Pepmap 100, Thermo Scientific) with a flow of 10 µl min-1 of eluent A (0.1% formic acid) before in-line flushed onto an Auroa Ultimate column (25 cm length, 75 µm inner diameter, Ionopticks). Peptides were chromatographically separated with an initial flow rate of 400 nL min-1 and the following gradient: initial 4% B (0.1% formic acid in 80 % acetonitrile), up to 8.5 % in 4 min. Then, flow was reduced to 300 nl/min and B increased to 35% B in 75 min, and up to 98% solvent B within 1.0 min while again increasing the flow to 400 nl/min, followed by column wash and reequilibration to initial condition.MS1 scans were acquired from 399 m/z to 1001 m/z at 30k resolution. Maximum injection time was set to 54 ms and the AGC target to 100%. MS2 scans ranged from 400 m/z to 1000 m/z and were acquired at 30 k resolution with a maximum injection time of 54 ms and an AGC target of 1000%. DIA scans covering the precursor range from 400-1000 m/z and were acquired in 30 x 20 m/z windows with an overlap of 1 m/z. All scans were stored as centroidPrideNot availableSamples were analyzed in Spectronaut 19 (Biognosys) in directDIA mode using the Serendipita indica DSM 11827 protein database (downloaded 12.09.25 at fungi.ensembl.org). Seach parameters were following recommended standard settings with minimum and maximum fragement filters set to six and 12, respectively. Afterwards, analysis of results was performed in Perseus 1.6.15. Low-intensity quantifications were converted to NA, followed by log2 transformation of remaining LFQ values, filtering on data completeness in at least one replicate group and FDR-controlled t-tests between the two groups of interest. Samples were filtered for NA in mycelium protein samples and differential protein abundance visualized in a volcano plot of log2FC against -log10-transformed p-values.ProteomicsProf. Dr. Alga ZuccaroInstitute for Plant Sciences, University of Cologne, 50674 Cologne, GermanyPARTIALSerendipita Indica Dsm 11827proteomics-facility@uni-koeln.deNot availableCECAD Research CenterGermanyfalseHost-adapted enzymatic deconstruction of acetylated xylan enables mutualistic colonization of monocot rootsIntracellular accommodation of beneficial fungi requires host cell wall remodeling that avoids excessive immune activation. The root endophyte Serendipita indica, a generalist mutualist capable of colonizing both monocot and dicot plants, employs a monocot-specific enzymatic module to deconstruct acetyl-xylan, the dominant hemicellulose of grasses. Central to this module are the glycoside hydrolase SiGH11, which releases acetylated xylooligosaccharides, and SiAXE, a previously uncharacterized SGNH-like acetyl-xylan esterase that sequentially removes acetyl groups from soluble XOS. Both enzymes are co-expressed within a monocot-enriched transcriptional program that also includes sugar transporters and metabolic regulators. Their coordinated activity, together with co-expressed exo-enzymes, promotes efficient xylan hydrolysis while limiting the prolonged accumulation of immunogenic damage-associated molecular patterns (DAMPs). Functional genetics demonstrated that SiAXE is required for sustained intracellular growth in monocot roots: its deletion impaired colonization, whereas overexpression transiently accelerated entry but provoked immune responses, underscoring the importance of temporal regulation and enzyme coordination for immune-compatible colonization. These findings provide mechanistic insight into an immune-compatible fungal strategy for host cell wall remodeling and reveal how a broadly colonizing mutualist has repurposed ancestral saprotrophic enzymes into specialized host-adapted modules that balance nutrient acquisition with immune modulation.2026-04-202026-03-17PXD075759NEWT:3555NEWT:71647NEWT:35554NEWT:32046NEWT:544496NEWT:2042546NEWT:32049NEWT:1392696NEWT:259447NEWT:45351NEWT:445974NEWT:43179NEWT:180454NEWT:5722NCBITaxon:1280NEWT:1129NEWT:309807NEWT:55153NEWT:309800NEWT:281395NEWT:1211601NEWT:876138NEWT:44271NEWT:10036NEWT:1590NEWT:498019NEWT:1351NEWT:1438992NEWT:1352NEWT:2649997NEWT:1147161NEWT:224326NEWT:1333499NCBITaxon:79857NEWT:1096976NEWT:95648NEWT:1589NEWT:135622NEWT:1349NEWT:96731NEWT:383379NEWT:10029NEWT:913645NEWT:44491NEWT:556484NEWT:317447NEWT:4688NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:3112NEWT:4442NEWT:192875NEWT:1214915NEWT:12637NEWT:59729NEWT:2164133NEWT:108061NEWT:1316931NEWT:224308NEWT:3347NEWT:511145NEWT:931281NEWT:658457NEWT:1310161NEWT:77133NEWT:295358NEWT:145481NCBITaxon:79824NCBITaxon:4563NEWT:5755NEWT:44689NEWT:44447NEWT:5759NEWT:1736231NEWT:1392NEWT:498217NEWT:2242NEWT:11320NEWT:286NEWT:391619NEWT:246196NEWT:287NEWT:246197NEWT:633149NEWT:10239NEWT:44685NEWT:161934NEWT:1148NEWT:5508NEWT:5507NEWT:410661NEWT:55571NEWT:35500NEWT:1140NCBITaxon:2157NEWT:1143NEWT:4896NEWT:1390NEWT:11557NEWT:1094343NEWT:1336795NEWT:644042NEWT:294NEWT:1773NEWT:1182590NEWT:3712NEWT:82380NEWT:935293NEWT:375146NEWT:118698NEWT:263NEWT:118696NEWT:52283NEWT:284812NEWT:8175NEWT:43330NEWT:1603293NEWT:408169NEWT:44664NEWT:47946NEWT:3702NEWT:243277NEWT:7067NEWT:118694NEWT:2850NEWT:118691NEWT:33548NEWT:274NEWT:408172NEWT:408170NEWT:3708NEWT:332648NEWT:44670NEWT:536231NEWT:376219NEWT:1436733NEWT:460519NEWT:572307NEWT:1432138NEWT:1424507NEWT:1194599NEWT:272844NEWT:9483NEWT:1513458NCBITaxon:40559NEWT:84588NEWT:1679718NEWT:480NEWT:67767NEWT:46835NEWT:109757NEWT:582580NEWT:300852NEWT:1502NEWT:376686NEWT:95486NEWT:508771NEWT:1883446NEWT:253NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:93062NCBITaxon:4932NEWT:644223NEWT:235443NEWT:108458NEWT:272623NEWT:272624NEWT:320637NEWT:983964NEWT:118499NEWT:32644NEWT:527796NEWT:499175NEWT:109779NEWT:3745NEWT:1715989NCBITaxon:4751NEWT:476272NEWT:3747NEWT:195051NEWT:3988NEWT:1255228NEWT:649908NEWT:410289NEWT:373153NEWT:352472NEWT:1071661NEWT:360094NEWT:470NEWT:41364NEWT:1313NEWT:39491NCBITaxon:5811NEWT:29491NEWT:2014887NEWT:33952NEWT:261756NEWT:571256NEWT:40483NEWT:272634NEWT:9031NEWT:1872122NEWT:7091NEWT:141262NEWT:108931NEWT:1055524NEWT:4087NEWT:9778NEWT:306NEWT:150475NEWT:303NEWT:267872NEWT:7111NEWT:347515NEWT:9534NEWT:5180NEWT:256737NEWT:4090NEWT:4092NEWT:9541NEWT:4093NEWT:4096NEWT:185579NEWT:941442NEWT:13076NEWT:7108NEWT:40479NEWT:4076NEWT:317NEWT:1006581NEWT:885318NEWT:39488NEWT:550NEWT:4081NEWT:273068NEWT:554NEWT:98334NEWT:451516NEWT:36185NEWT:1225786NEWT:7574NEWT:1715256NEWT:30640NEWT:575412NEWT:929793NEWT:29204NEWT:28112NEWT:114155NEWT:6494NEWT:6491NEWT:507601NEWT:643680NEWT:214092NCBITaxon:6157NEWT:6239NEWT:162425NEWT:216257NEWT:102169NEWT:9986NEWT:4054NEWT:1268063NEWT:1263854NEWT:118503NEWT:2059687NEWT:160488NEWT:28104NEWT:407821NCBITaxon:2NEWT:985076NEWT:1215323NEWT:986NEWT:52641NEWT:7159NEWT:28532NEWT:353152NEWT:8496NEWT:27448NEWT:1194669NEWT:51329NEWT:243230NEWT:1080772NEWT:519NEWT:8479NEWT:8485NEWT:6063NEWT:49240NEWT:104395NEWT:6289NEWT:6287NEWT:300641NEWT:727NEWT:9796NEWT:725NEWT:469008NEWT:256318NEWT:9321NEWT:206411NCBITaxon:6191NEWT:596153NEWT:229533NEWT:1836NEWT:80863NEWT:105231NEWT:52638NEWT:57075NEWT:4097NEWT:884204NEWT:9785NEWT:6279NEWT:1123869NEWT:9544NEWT:7370NEWT:83906NCBITaxon:780NCBITaxon:1502NEWT:6282NEWT:1134506NEWT:38783NEWT:4006NEWT:6426NCBITaxon:5476NEWT:6669NEWT:9935NEWT:89453NEWT:287889NEWT:51515NEWT:588596NEWT:51511NEWT:8845NEWT:92867NEWT:92866NEWT:5334NEWT:51750NEWT:202950NEWT:382352NEWT:413071NEWT:632957NEWT:9925NEWT:89462NCBITaxon:9606NEWT:8839NEWT:90690NEWT:4232NEWT:2758385NEWT:11298NEWT:171101NEWT:714NEWT:421932NEWT:312017NEWT:196627NEWT:200308NEWT:200302NEWT:870435NEWT:284593NEWT:100392NEWT:5315NEWT:9913NEWT:265872NEWT:105841NEWT:337330NEWT:2666255NEWT:2285NEWT:999810NCBITaxon:5693NEWT:1392998NEWT:380394NEWT:981087NEWT:180066NEWT:226900NEWT:231490NEWT:430615NEWT:563041NEWT:326423NEWT:452467NEWT:198822NEWT:36111NEWT:326424NEWT:1416333NEWT:1678078NEWT:749906NEWT:418985NEWT:1817808NEWT:749907NEWT:145263NEWT:694569NEWT:27606NEWT:9739NEWT:59202NEWT:9975NEWT:51953NEWT:3197NEWT:860688NEWT:13443NEWT:36329NEWT:62615NCBITaxon:3044782NEWT:160235NEWT:72407NEWT:349741NEWT:157295NEWT:2096NEWT:192222NEWT:9721NEWT:137221NEWT:9963NEWT:1617910NEWT:565050NEWT:2785785NEWT:411901NEWT:105884NCBITaxon:9615NEWT:58334NEWT:1193501NEWT:58331NEWT:6689NEWT:298315NEWT:70448NEWT:698936NEWT:9940NEWT:3168NEWT:521001NEWT:192011NEWT:1813887NEWT:118702NEWT:118700NEWT:224911NEWT:118705NCBITaxon:11552NEWT:6945NEWT:184922NEWT:6703NEWT:2104NEWT:2NEWT:157546NEWT:474186NEWT:38942NEWT:307972NEWT:1496NEWT:2102NEWT:45474NEWT:1590841NEWT:4513NEWT:1247NEWT:6938NEWT:376741NCBITaxon:10407NEWT:1736309NEWT:4751NEWT:10360NEWT:47664NEWT:317548NEWT:15819NEWT:3654NEWT:237561NEWT:5833NEWT:6928NEWT:1239NEWT:36745NEWT:272563NEWT:82688NEWT:3885NEWT:3888NEWT:5821NEWT:44139NCBITaxon:4896NEWT:6915NEWT:3649NEWT:101510NEWT:28903NEWT:3880NE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