Rapid Oligo-Galacturonide Induced Changes in Protein Phosphorylation in Arabidopsis
2016; Elsevier BV; Volume: 15; Issue: 4 Linguagem: Inglês
10.1074/mcp.m115.055368
ISSN1535-9484
AutoresBruce D. Kohorn, Divya Hoon, Benjamin B. Minkoff, Michael R. Sussman, Susan L. Kohorn,
Tópico(s)Plant Molecular Biology Research
ResumoThe wall-associated kinases (WAKs) 1The abbreviations used are:WAKswall-associated kinasesOGsoligo-galacturonidesEGFepidermal growth factorPMEpectin methylesterases. are receptor protein kinases that bind to long polymers of cross-linked pectin in the cell wall. These plasma-membrane-associated protein kinases also bind soluble pectin fragments called oligo-galacturonides (OGs) released from the wall after pathogen attack and damage. WAKs are required for cell expansion during development but bind water soluble OGs generated from walls with a higher affinity than the wall-associated polysaccharides. OGs activate a WAK-dependent, distinct stress-like response pathway to help plants resist pathogen attack. In this report, a quantitative mass-spectrometric-based phosphoproteomic analysis was used to identify Arabidopsis cellular events rapidly induced by OGs in planta. Using N14/N15 isotopic in vivo metabolic labeling, we screened 1,000 phosphoproteins for rapid OG-induced changes and found 50 proteins with increased phosphorylation, while there were none that decreased significantly. Seven of the phosphosites within these proteins overlap with those altered by another signaling molecule plants use to indicate the presence of pathogens (the bacterial "elicitor" peptide Flg22), indicating distinct but overlapping pathways activated by these two types of chemicals. Genetic analysis of genes encoding 10 OG-specific and two Flg22/OG-induced phosphoproteins reveals that null mutations in eight proteins compromise the OG response. These phosphorylated proteins with genetic evidence supporting their role in the OG response include two cytoplasmic kinases, two membrane-associated scaffold proteins, a phospholipase C, a CDPK, an unknown cadmium response protein, and a motor protein. Null mutants in two proteins, the putative scaffold protein REM1.3, and a cytoplasmic receptor like kinase ROG2, enhance and suppress, respectively, a dominant WAK allele. Altogether, the results of these chemical and genetic experiments reveal the identity of several phosphorylated proteins involved in the kinase/phosphatase-mediated signaling pathway initiated by cell wall changes. The wall-associated kinases (WAKs) 1The abbreviations used are:WAKswall-associated kinasesOGsoligo-galacturonidesEGFepidermal growth factorPMEpectin methylesterases. are receptor protein kinases that bind to long polymers of cross-linked pectin in the cell wall. These plasma-membrane-associated protein kinases also bind soluble pectin fragments called oligo-galacturonides (OGs) released from the wall after pathogen attack and damage. WAKs are required for cell expansion during development but bind water soluble OGs generated from walls with a higher affinity than the wall-associated polysaccharides. OGs activate a WAK-dependent, distinct stress-like response pathway to help plants resist pathogen attack. In this report, a quantitative mass-spectrometric-based phosphoproteomic analysis was used to identify Arabidopsis cellular events rapidly induced by OGs in planta. Using N14/N15 isotopic in vivo metabolic labeling, we screened 1,000 phosphoproteins for rapid OG-induced changes and found 50 proteins with increased phosphorylation, while there were none that decreased significantly. Seven of the phosphosites within these proteins overlap with those altered by another signaling molecule plants use to indicate the presence of pathogens (the bacterial "elicitor" peptide Flg22), indicating distinct but overlapping pathways activated by these two types of chemicals. Genetic analysis of genes encoding 10 OG-specific and two Flg22/OG-induced phosphoproteins reveals that null mutations in eight proteins compromise the OG response. These phosphorylated proteins with genetic evidence supporting their role in the OG response include two cytoplasmic kinases, two membrane-associated scaffold proteins, a phospholipase C, a CDPK, an unknown cadmium response protein, and a motor protein. Null mutants in two proteins, the putative scaffold protein REM1.3, and a cytoplasmic receptor like kinase ROG2, enhance and suppress, respectively, a dominant WAK allele. Altogether, the results of these chemical and genetic experiments reveal the identity of several phosphorylated proteins involved in the kinase/phosphatase-mediated signaling pathway initiated by cell wall changes. wall-associated kinases oligo-galacturonides epidermal growth factor pectin methylesterases. The cell walls of angiosperms are composed of a complex arrangement of cellulose, hemicellulose, and pectin and are assembled through a complex, developmentally regulated coordination of synthesis, turnover, and interactions between protein and carbohydrates (1.Seifert G.J. Blaukopf C. Irritable walls: The plant extracellular matrix and signaling.Plant Physiol. 2010; 153: 467-478Crossref PubMed Scopus (146) Google Scholar). The pectins can be selectively and locally cross-linked into a structural network that is subsequently remodeled and degraded by enzymes, and these events have dramatic effects on cell enlargement (2.Somerville C. Bauer S. Brininstool G. Facette M. Hamann T. Milne J. Osborne E. Paredez A. Persson S. Raab T. Vorwerk S. Youngs H. Toward a systems approach to understanding plant cell walls.Science. 2004; 306: 2206-2211Crossref PubMed Scopus (942) Google Scholar, 3.Anderson C.T. Carroll A. Akhmetova L. Somerville C. 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A peptide hormone and its receptor protein kinase regulate plant cell expansion.Science. 2014; 343: 408-411Crossref PubMed Scopus (467) Google Scholar) and typically have extracellular domains containing leucine-rich regions and a malectin carbohydrate-binding domain, although an experimentally demonstrated role for polysaccharide binding to their extracellular domains is unclear. Of the plant putative "wall sensors" only the wall-associated kinases (WAKs) are known to bind to a cell wall component, pectin, and these are distinguished also by their unique extracellular domain that lacks leucine-rich repeats and contains instead epidermal growth factor (EGF) repeats as well as a pectin-binding region (19.Kohorn B.D. Kohorn S.L. The cell wall-associated kinases, WAKs, as pectin receptors.Frontiers Plant Sci. 2012; 3: 88Crossref PubMed Scopus (208) Google Scholar). Pectins are synthesized in the Golgi apparatus as methyl esterified 1–4 d-galacturonic acids and are secreted into an extracellular plant cell wall matrix composed of cellulose, hemicellulose, and a variety of proteins (1.Seifert G.J. Blaukopf C. Irritable walls: The plant extracellular matrix and signaling.Plant Physiol. 2010; 153: 467-478Crossref PubMed Scopus (146) Google Scholar, 2.Somerville C. Bauer S. Brininstool G. Facette M. Hamann T. Milne J. Osborne E. Paredez A. Persson S. Raab T. Vorwerk S. Youngs H. Toward a systems approach to understanding plant cell walls.Science. 2004; 306: 2206-2211Crossref PubMed Scopus (942) Google Scholar, 3.Anderson C.T. Carroll A. Akhmetova L. Somerville C. Real-time imaging of cellulose reorientation during cell wall expansion in Arabidopsis roots.Plant Physiol. 2010; 152: 787-796Crossref PubMed Scopus (306) Google Scholar, 4.Kohorn B.D. Plasma membrane-cell wall contacts.Plant Physiol. 2000; 124: 31-38Crossref PubMed Scopus (117) Google Scholar, 5.Caffall K.H. Mohnen D. The structure, function, and biosynthesis of plant cell wall pectic polysaccharides.Carbohydrate Res. 2009; 344: 1879-1900Crossref PubMed Scopus (1060) Google Scholar). Pectin methylesterases (PME) in the wall creates negatively charged pectins, leading to calcium-based crosslinking that is hypothesized to provide lateral structure and directionality of growth for a variety of cell types (16.Wolf S. Höfte H. Growth control: A saga of cell walls, ROS, and peptide receptors.Plant Cell. 2014; 26: 1848-1856Crossref PubMed Scopus (76) Google Scholar, 20.Bosch M. Cheung A.Y. Hepler P.K. Pectin methylesterase, a regulator of pollen tube growth.Plant Physiol. 2005; 138: 1334-1346Crossref PubMed Scopus (292) Google Scholar, 21.Winship L.J. Obermeyer G. Geitmann A. Hepler P.K. 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Polygalacturonase involved in expansion1 functions in cell elongation and flower development in Arabidopsis.Plant Cell. 2014; 26: 1018-1035Crossref PubMed Scopus (111) Google Scholar, 25.González-Carranza Z.H. Elliott K.A. Roberts J.A. Expression of polygalacturonases and evidence to support their role during cell separation processes in Arabidopsis thaliana.J. Exp. Bot. 2007; 58: 3719-3730Crossref PubMed Scopus (146) Google Scholar). Abscission zones at the root cap, petiole, and sepal base also express pectin-degrading enzymes that are part of the loosening process. In summary, current models predict that pectin digestion is a part of numerous regulated developmental processes, and by nature generates local pools of pectin fragments, or oligo-galacturonides (OGs). Indeed, the biological activity of pectin fragments in developmental processes has been suggested for many years, although the molecular mechanisms remain unknown (6.Harholt J. Suttangkakul A. Vibe Scheller H. Biosynthesis of pectin.Plant Physiol. 2010; 153: 384-395Crossref PubMed Scopus (387) Google Scholar, 7.Ferrari S. Savatin D.V. Sicilia F. Gramegna G. Cervone F. Lorenzo G.D. Oligogalacturonides: Plant damage-associated molecular patterns and regulators of growth and development.Frontiers Plant Sci. 2013; 4: 49Crossref PubMed Scopus (316) Google Scholar, 26.Willats W.G. McCartney L. Mackie W. Knox J.P. Pectin: Cell biology and prospects for functional analysis.Plant Mol. Biol. 2001; 47: 9-27Crossref PubMed Scopus (945) Google Scholar, 27.Yamazaki N. Fry S.C. Darvill A.G. Albersheim P. Host–pathogen interactions : XXIV. Fragments isolated from suspension-cultured sycamore cell walls inhibit the ability of the cells to incorporate [C]leucine into proteins.Plant Physiol. 1983; 72: 864-869Crossref PubMed Google Scholar, 28.Mohnen D. Pectin structure and biosynthesis.Curr. Opin Plant Biol. 2008; 11: 266-277Crossref PubMed Scopus (1470) Google Scholar). Pectins are also the target of numerous pathogens that digest the wall as they approach the plant cell, thereby generating de-esterified pectin fragments or OGs (7.Ferrari S. Savatin D.V. Sicilia F. Gramegna G. Cervone F. Lorenzo G.D. Oligogalacturonides: Plant damage-associated molecular patterns and regulators of growth and development.Frontiers Plant Sci. 2013; 4: 49Crossref PubMed Scopus (316) Google Scholar). These OGs can activate a plant stress response, indicating that OGs signal to the plant that a pathogen is present (7.Ferrari S. Savatin D.V. Sicilia F. Gramegna G. Cervone F. Lorenzo G.D. Oligogalacturonides: Plant damage-associated molecular patterns and regulators of growth and development.Frontiers Plant Sci. 2013; 4: 49Crossref PubMed Scopus (316) Google Scholar, 29.Denoux C. Galletti R. Mammarella N. Gopalan S. Werck D. De Lorenzo G. Ferrari S. Ausubel F.M. Dewdney J. Activation of defense response pathways by OGs and Flg22 elicitors in Arabidopsis seedlings.Mol. Plant. 2008; 1: 423-445Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Physical wounding and herbivory can also trigger the accumulation of OGs and a stress response, presumably through a similar signaling pathway. WAKs are plasma membrane receptors that bind pectin, thereby activating several different possible responses. During seedling growth, WAK2 is required for cell expansion and for the pectin activation of MPK3 and vacuolar invertase, presumably to increase turgor-driven expansion (19.Kohorn B.D. Kohorn S.L. The cell wall-associated kinases, WAKs, as pectin receptors.Frontiers Plant Sci. 2012; 3: 88Crossref PubMed Scopus (208) Google Scholar, 30.Kohorn B.D. Johansen S. Shishido A. Todorova T. Martinez R. Defeo E. Obregon P. Pectin activation of MAP kinase and gene expression is WAK2 dependent.Plant J. 2009; 60: 974-982Crossref PubMed Scopus (138) Google Scholar, 31.Kohorn B.D. Kobayashi M. Johansen S. Riese J. Huang L.F. Koch K. Fu S. Dotson A. Byers N. An Arabidopsis cell wall-associated kinase required for invertase activity and cell growth.Plant J. 2006; 46: 307-316Crossref PubMed Scopus (133) Google Scholar). Pectin also causes the induction and repression of hundreds of genes involved in cell wall biogenesis and stress responses, and this response is WAK2 dependent (30.Kohorn B.D. Johansen S. Shishido A. Todorova T. Martinez R. Defeo E. Obregon P. Pectin activation of MAP kinase and gene expression is WAK2 dependent.Plant J. 2009; 60: 974-982Crossref PubMed Scopus (138) Google Scholar, 31.Kohorn B.D. Kobayashi M. Johansen S. Riese J. Huang L.F. Koch K. Fu S. Dotson A. Byers N. An Arabidopsis cell wall-associated kinase required for invertase activity and cell growth.Plant J. 2006; 46: 307-316Crossref PubMed Scopus (133) Google Scholar). Overall, this work suggested that WAKs serve as pectin receptors, a conclusion that has received additional support from the results of experiments in which the WAK1 extracellular domain was fused to the kinase domain of the EGFR receptor (32.Brutus A. Sicilia F. Macone A. Cervone F. De Lorenzo G. A domain swap approach reveals a role of the plant wall-associated kinase 1 (WAK1) as a receptor of oligogalacturonides.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 9452-9457Crossref PubMed Scopus (492) Google Scholar). OGs activated a pathway downstream of this hybrid kinase when it was transiently expressed in tobacco leaves. A dominant WAK2 allele WAK2cTAP, whose encoded protein requires a functional pectin-binding domain and an active kinase, induces a constitutive stress response (33.Kohorn B.D. Kohorn S.L. Saba N.J. Martinez V.M. Requirement for pectin methyl esterase and preference for fragmented over native pectins for wall-associated kinase-activated, EDS1/PAD4-dependent stress response in Arabidopsis.J. Biol. Chem. 2014; 289: 18978-18986Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 34.Kohorn B.D. Kohorn S.L. Todorova T. Baptiste G. Stansky K. McCullough M. A dominant allele of Arabidopsis pectin-binding wall-associated kinase induces a stress response suppressed by MPK6 but not MPK3 mutations.Mol. Plant. 2012; 5: 841-851Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The response is dependent upon MPK6 and the pathogen response transcription factors EDS1 and PAD4. Importantly, the WAK2cTAP allele is also suppressed by a null allele of a pectin methyl esterase, pme3 (33.Kohorn B.D. Kohorn S.L. Saba N.J. Martinez V.M. Requirement for pectin methyl esterase and preference for fragmented over native pectins for wall-associated kinase-activated, EDS1/PAD4-dependent stress response in Arabidopsis.J. Biol. Chem. 2014; 289: 18978-18986Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). This provides genetic evidence that WAKs are sensing the de-esterified form of pectin, consistent with the higher affinity in vitro of WAKs for de-esterified over esterified pectin and for pectin fragments of limited degrees of polymerization, 9–15 sugars (30.Kohorn B.D. Johansen S. Shishido A. Todorova T. Martinez R. Defeo E. Obregon P. Pectin activation of MAP kinase and gene expression is WAK2 dependent.Plant J. 2009; 60: 974-982Crossref PubMed Scopus (138) Google Scholar, 31.Kohorn B.D. Kobayashi M. Johansen S. Riese J. Huang L.F. Koch K. Fu S. Dotson A. Byers N. An Arabidopsis cell wall-associated kinase required for invertase activity and cell growth.Plant J. 2006; 46: 307-316Crossref PubMed Scopus (133) Google Scholar, 35.Decreux A. Messiaen J. Wall-associated kinase WAK1 interacts with cell wall pectins in a calcium-induced conformation.Plant Cell Physiol. 2005; 46: 268-278Crossref PubMed Scopus (222) Google Scholar, 36.Decreux A. Thomas A. Spies B. Brasseur R. Van Cutsem P. Messiaen J. In vitro characterization of the homogalacturonan-binding domain of the wall-associated kinase WAK1 using site-directed mutagenesis.Phytochemistry. 2006; 67: 1068-1079Crossref PubMed Scopus (82) Google Scholar). The pme3/pme3 mutant plant is more responsive to OGs than WT plants, as measured by the induction of the FADLox gene, a robust marker for OG induction of transcription (29.Denoux C. Galletti R. Mammarella N. Gopalan S. Werck D. De Lorenzo G. Ferrari S. Ausubel F.M. Dewdney J. Activation of defense response pathways by OGs and Flg22 elicitors in Arabidopsis seedlings.Mol. Plant. 2008; 1: 423-445Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 33.Kohorn B.D. Kohorn S.L. Saba N.J. Martinez V.M. Requirement for pectin methyl esterase and preference for fragmented over native pectins for wall-associated kinase-activated, EDS1/PAD4-dependent stress response in Arabidopsis.J. Biol. Chem. 2014; 289: 18978-18986Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 34.Kohorn B.D. Kohorn S.L. Todorova T. Baptiste G. Stansky K. McCullough M. A dominant allele of Arabidopsis pectin-binding wall-associated kinase induces a stress response suppressed by MPK6 but not MPK3 mutations.Mol. Plant. 2012; 5: 841-851Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The data support a model in which OGs are competing with native pectin to bind WAKs, thus providing a mechanism for WAKs to distinguish changes in the pectin network and activate alternate pathways. There are at least 21 other Arabidopsis genes encoding proteins with receptor kinase sequences similar to WAK that also contain EGF repeats, and these have been coined WAK- like or WAKL genes (37.Verica J.A. Chae L. Tong H. Ingmire P. He Z.H. Tissue-specific and developmentally regulated expression of a cluster of tandemly arrayed cell wall-associated kinase-like kinase genes in Arabidopsis.Plant Physiol. 2003; 133: 1732-1746Crossref PubMed Scopus (97) Google Scholar, 38.Verica J.A. He Z.H. The cell wall-associated kinase (WAK) and WAK-like kinase gene family.Plant Physiol. 2002; 129: 455-459Crossref PubMed Scopus (143) Google Scholar). 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To understand how plant cells respond to pectin fragments that are induced by pathogens, biotic stresses, or during developmental patterns, a phosphoproteomic analysis was carried out to identify proteins phosphorylated as a result of OG treatment. The results reveal a subset of proteins that likely play a role in the pectin-induced signal transduction pathway and provide a new window into how changes to the plant cell wall are perceived. Growth conditions were as previously described (44.Kline K.G. Barrett-Wilt G.A. Sussman M.R. In planta changes in protein phosphorylation induced by the plant hormone abscisic acid.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 15986-15991Crossref PubMed Scopus (186) Google Scholar). Treatment was with 50 μg/ml concentration of OG for 5 min. Three A (14N OG-treated, 15N mock-treated) replicates were used, and three B (14N mock-treated, 15N OG-treated) replicates were used. Each A or B experiment consisted of material from two flasks, combined into 85 ml homogenization buffer (44.Kline K.G. Barrett-Wilt G.A. Sussman M.R. In planta changes in protein phosphorylation induced by the plant hormone abscisic acid.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 15986-15991Crossref PubMed Scopus (186) Google Scholar) with 1 mm DTT, 1 mm PMSF, 21.5 mm leupeptin, 1.5 mm pepstatin, 2 mm bestatin, 50 mm 1,10-phenanthroline, 100 mm vanadate, and 2.8 mm E64 added immediately prior to use. After combination, samples were immediately homogenized using a tissue tearer for 10 s at 10,000 rpm. Following filtration through one layer of miracloth, samples were spun at 6000 × g at 4°C for 7 min to pellet debris. Microsomal fractionation was performed by further spinning the supernatant from the 6,000 × g spin for 1 h at 4C and 100,000 × g. Supernatant from this spin yielded soluble protein-enriched fractions, and the pellet was resuspended into 1 ml of resuspension buffer using a Teflon homogenizer to yield an insoluble protein-enriched fraction. Methanol/chloroform precipitation was performed on fractions as previously described. Following protein precipitation, tryptic digestion, and desalting, phosphopeptide enrichment was performed using 200 μl spin-enrichment TiO2-packed tips (GL Sciences, Inc. Torrance Ca) using supplier protocol. Dried phosphopeptides were solubilized into Optima LC/MS-grade 0.1% formic acid in water (Fisher Scientific Pittsburg PA) for injection onto an LTQ Orbitrap XL using an Agilent 1100 LC system. Solvent A was 0.1% formic acid, and solvent B was 95% acetonitrile, 0.1% formic acid. Sample was loaded directly onto an analytical column of inner diameter 75 μm and outer diameter 360 μm house-packed with ∼13 cm C18 resin (Magic-C18, 200Å, 3 μm, Michrom Biosources, Inc. Auburn Ca) at 0% B and a flow rate of 0.5 μl/min from 0–45 min, then eluted from 45–235 min at a flow rate of 0.2 μl/min and a gradient to 40% B, then from 235–255 min to 60% B, then from 255–260 min to 100% B. The column was then re-equilibrated by flowing 100% A from 260–278 min, stepping flow rate to 0.5 μl/min at minute 265. MS1 spectra were collected using the Orbitrap at resolution 100,000 with preview mode enabled. The top five MS2 per MS1 were collected in the LTQ, rejecting +1 and unassigned charge states, using CID with an isolation window of 2.5 m/z, normalized CE of 35, activation Q of 0.25, activation at 30ms, and a minimum MS1 signal threshold of 500. Raw data were converted to .mgf files using default settings in the Trans-Proteomic Pipeline and searched using Mascot v2.2.2 and the Arabidopsis Information Resource (TAIR) protein database (version 9, June 19, 2009) with reverse sequences and common contaminants manually appended using BioEdit (Ibis Biosciences, 62,522 sequences in total). The enzyme trypsin was specified as the protease and one missed cleavage was allowed. A precursor and fragment ion tolerance of 20 ppm and 0.6 Da, respectively, were used. Cysteine carbamidomethylation was set as a fixed modification, and methionine oxidation, serine/threonine/tyrosine phosphorylation, and asparagine/glutamine deamidation were set as variable modifications. Mascot output was filtered to 1.0% false discovery rate using an in-house written script, and Census (45.Park S.K. Venable J.D. Xu T. Yates 3rd., J.R. A quantitative analysis software tool for mass spectrometry-based proteomics.Nat. Methods. 2008; 5: 319-322Crossref PubMed Scopus (319) Google Scholar, 46.Park S.K. Yates 3rd., J.R. Census for proteome quantification.Curr. Protoc. Bioinformatics. 2010; 13 (Unit 13.12.1–11)PubMed Google Scholar) was used to extract ion chromatograms and quantify 14/15N area ratios. Census output was reformatted using an in-house written script, and data were median-normalized per injection. 15N Mascot searches were performed for all B experiment .raw files. Identical filtering and processing was applied, and data were added to the list of phosphochanges. Phosphopeptides of interest with Mascot score ≤ 30 or potentially ambiguous phosphorylation localization (i.e. multiple phosphoisoforms were identified) were manually validated by raw MS2 spectrum analysis when possible. Ambiguity, even after manual examination, is noted in the supplemental table. All data have been provided as processed Excel files and raw data, which have been uploaded to and is publicly available via Chorus (https://chorusproject.org), and individual spectra are viewable using Scaffold (Proteome Software) and Supplemental File 2. All scripts are available on the Sussman laboratory website (http://www.biotech.wisc.edu/sussmanlab). To obtain the values highlighted in this manuscript, OG-responsive phosphorylation events had their ratios collapsed into a single data point by averaging all the values for the 14Ntreat/15Ncontrol experiment (A) and the inverse values of the 14Ncontrol/15Ntreat experiment (B). The requirement set for this manual calculation was that the peptides had at leas
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