MetaboLightsapplication/xmlftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS1039/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS1039/a_MTBLS1039_LC-MS_positive_reverse-phase_metabolite_profiling.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS1039/s_MTBLS1039.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS1039ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS1039/DERIVED_FILESftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS1039/files-all.jsonprimaryOK200Curt FischerSolanum lycopersicummass spectrometry<p><strong>Metabolite extraction from wild-type tomato and <em>N. benthamiana</em> leaves </strong></p><p>Frozen leaf tissues were lyophilized to dryness. The samples (n = 3, 10 mg of dried tissue/tube) were homogenized on a ball mill (Retsch MM 400) using 5 mm diameter stainless steel beads, shaking at 25 Hz for 2 min.</p><p><br></p><p><strong>Methanol extraction of metabolites from tomato leaves after elicitation</strong></p><p>60mL of an 80:20 MeOH/H2O (v/v) solution was added per milligram of dry tissue, and the mixture was heated at 65 °C for 10 min and filtered through 0.45mm PTFE filters before liquid chromatography-mass spectrometry (LC-MS) analysis. Extraction solvent composition was selected to capture the wide range of polarity of compounds in the tissues.</p><p><br></p><p><strong>Methanol extraction of metabolites from <em>N. benthamiana</em></strong></p><p>80mL of methanol was added per milligram of dry tissue. After incubation at room temperature for 20 min with continuous mixing, samples were filtered through 0.45mm PTFE filters before liquid chromatography-mass spectrometry (LC-MS) analysis.</p><p><br></p><p><strong>Saponification of metabolites from <em>N. benthamiana </em>and CRISPR tomato mutants</strong></p><p>Dried leaf extract from above was mixed with 200 mL of 6% KOH. Tubes were incubated for 2 h at 60 °C with continuous shaking. After cooling to room temperature, the reaction mixture was acidified by the addition of 50 mL of 29% HCl. Tubes were vortexed thoroughly and spun for 30 s using a table centrifuge. The fatty acids were extracted with 500 mL of chloroform/hexane mixture (1:4, v:v).After vortexing and 10 s of centrifugation, the organic phase was collected. Extraction with the chloroform/hexane mixture was repeated twice and the collected fatty acid containing organic phases was combined and dried under N2. The final product was dissolved in 200 mL of 100% MeOH for LC-MS analysis.</p>https://www.ebi.ac.uk/metabolights/MTBLS1039Kimberly Wemmer.Cosima Dufour-Schroif.Elizabeth Sattely.Curt R Fischer.Niraj Mehta.Jung-Gun Kim.Ju Eun Jeon.Mary Beth Mudgett.<p>MS data were analyzed using <strong>MassHunter Qualitative Analysis software</strong> (Agilent) and <strong>XCMS </strong>(Scripps Center for Metabolomics)(Smith et al., 2006). For untargeted metabolomics, <strong>MassHunter </strong>(Agilent) data files were converted to <strong>mzXML</strong> format using <strong>trapper </strong>(Seattle Proteome Center). Grouped <strong>mzXML </strong>files were preprocessed and analyzed by <strong>XCMS.</strong></p>RunPathogentimepoint<p><strong>Treatment of tomato leaves with biotic elicitors</strong></p><p><strong>Leaflets </strong>of tomato VF36 plants were hand-inoculated using a needleless 1 mL syringe with: 1) fungal elicitors: <strong>chitin </strong>(0.5 mg/mL in water),<strong>M. restricta</strong> (OD600= 0.5 in water) or <strong>C. fulvum </strong>(OD600= 1.0 in water); 2) bacterial elicitors: <strong>flg22 </strong>(1mM in water),<strong>S. epidermidis </strong>(OD600= 1.0 in water), <strong>P. acnes </strong>(OD600= 1.0 in water) or <strong>X. euvesicatoria </strong>(OD600= 0.2 in 10mM MgCl2); or 3) <strong>mock </strong>(water, control) (<strong>Figure S1 </strong>in the paper associated with this study). After treatment, <strong>leaves </strong>(one <strong>leaf </strong>from 3 individual plants per treatment, n = 3) were collected at 12, 24, and 48 <strong>hours post-inoculation</strong> (<strong>hpi</strong>) using a razor, flash frozen by liquid nitrogen, and stored at -80 °C for later use in metab-olomics analysis, gene profiling and RNA-Sequencing (RNA-Seq).</p><p><br></p><p><br></p>MetaboLightsPublicMetabolomicsLiquid Chromatography MS - positive - reverse phase<p>A coupled Agilent 6520 Accurate-Mass Q-TOF ESI mass spectrometer was used to collect MS data in positive ion mode (parameters: mass range: 100-1700 m/z; drying gas: 300°C, 1 L/min; nebulizer: 25 psig; capillary: 3500 V; fragmentor: 150 V; skimmer: 65 V; OCT 1 RF Vpp: 750 V; 1000 ms per spectrum). The first minute of each run was discarded to avoid salt contamination of the MS apparatus. For tandem mass spectrometry (MS/MS) analysis, 5, 10, 20 and 40 V collision energies were used with an m/z window of 1.3 centered on the m/z analyzed. </p>ultra-performance liquid chromatography-mass spectrometryGenomicsfatty acidpathogenuntargeted metabolitesfalcarindiol<p>Samples were analyzed by reversed-phase chromatography on an Agilent 1260 HPLC, using a 5 μm, 2 x 100 mm Gemini NX-C18 column (Phenomenex). Water with 0.1% formic acid (A) and Acetonitirile with 0.1% formic acid (B) were used as the mobile phase components at a flow rate of 0.4 mL/min with the following 55 min gradient; i) for methanol extracts: 0-30 min, 3-50% B; 30-45 min, 50-97% B; 45-50 min, 97% B; 50-51 min, 97-3% B; 51-55 min, 3% B, ii) for saponified extracts: 0-45 min, 40-97% B; 45-50 min, 97% B; 50-51 min, 97-40% B; 51-55 min, 40% B. </p>A Pathogen-Responsive Gene Cluster for Highly Modified Fatty Acids in Tomato. 10.1016/j.cell.2019.11.037. PMID:31923394ultra-performance liquid chromatography-mass spectrometryGenomicsfatty acidpathogenuntargeted metabolitesfalcarindiolStanford UniversityleafWe provide no identification here. Only raw data is provided.In response to biotic stress, plants produce suites of highly modified fatty acids that bear unusual chemical functionalities. Despite their chemical complexity and proposed roles in pathogen defense, little is known about the biosynthesis of decorated fatty acids in plants. Falcarindiol is a prototypical acetylenic lipid present in carrot, tomato, and celery that inhibits growth of fungi and human cancer cell lines. Using a combination of untargeted metabolomics and RNA sequencing, we discovered a biosynthetic gene cluster in tomato (Solanum lycopersicum) required for falcarindiol production. By reconstituting initial biosynthetic steps in a heterologous host and generating transgenic pathway mutants in tomato, we demonstrate a direct role of the cluster in falcarindiol biosynthesis and resistance to fungal and bacterial pathogens in tomato leaves. This work reveals a mechanism by which plants sculpt their lipid pool in response to pathogens and provides critical insight into the complex biochemistry of alkynyl lipid production.A Pathogen-Responsive Gene Cluster for Highly Modified Fatty Acids in Tomato.Jeon Ju Eun JE, Kim Jung-Gun JG, Fischer Curt R CR, Mehta Niraj N, Dufour-Schroif Cosima C, Wemmer Kimberly K, Mudgett Mary Beth MB, Sattely Elizabeth ESaturated Fatty, acido graso, malignant Growth, lipids, host organism, Ghrfr, Esterified Fatty Acids, human being, acidos grasos, bioformation, RNA Sequence Determination, Sequence Determination, Pflanze, cell type cancer, postnatal development, RNA Sequence, Metabonomic, growth and development, Metabonomics, biosynthesis, Tomatoes, 9-dien-4, viridiplantae, acides gras, Roles, responsivity, resistance, Line, Concepts, Saturated Fatty Acids, Esterified Fatty Acid, Analysis, Metabolomic, heptadeca-1, 9-diene-4, neoplasm, malignant tumour, multicellular organismal biosynthetic process, Esterified, plantae, Pilz, reactivity, Fatty Acids, Filamentous Fungi, single-organism biosynthetic process, Analyses, formation, tomato, Determination, anabolism, Esterified Fatty, neoplasm (disease), Aliphatic, plants, present in organism, Lycopersicon esculentum, Filamentous Fungus, W, Lycopersicon esculentum var. esculentum, man, Sequence Determinations, Sequencing, CA, synthesis, Aliphatic Acids, 6-diyne-3, RNA Sequence Analyses, malignant neoplasm, Lycopersicon lycopersicum, Role Concepts, RNA Sequencing, TG, fungi, Sequence Analyses, malignancy, RNA, Saturated, Molds, Solanum esculentum Dunal, Tomato, RNA Sequence Determinations, Lipid., malignant, Lycopersicum esculentum, land plants, Fungi, Fungus, Cell Lines, Fatty acid, lit, Fettsaeuren, Mycota, falcalindiol, Cell, Solanum esculentum, Mold, Determinations, Concept, development, Fettsaeure, Solanum lycopersicum var. humboldtii, MT, Role Concept, malignant neoplasm (disease), Lipid, 8-diol, organ system cancer, Role, little, Lines, Acid, Fatty Acid, primary cancer, Saturated Fatty Acid, Solanum lycopersicum L., growth pattern, non-developmental growth, postnatal growth, Plant, Aliphatic Acid, fungus, acide gras, (9E)-heptadeca-1, malignant tumor, human, fatty acids, higher plants, Filamentous, malignant neoplastic disease, RNA Sequence Analysis, response to biotic stress, response to abiotic stress, response, Solanum lycopersicon, growth, cancer, Lycopersicon esculentum Mill.Saturated Fatty, acido graso, Acid, Fatty Acids, Solanum esculentum., Fatty Acid, Esterified Fatty Acids, Saturated, Saturated Fatty Acid, acidos grasos, Solanum lycopersicum L., tomato, Solanum esculentum Dunal, Tomato, Lycopersicum esculentum, Esterified Fatty, Aliphatic, Aliphatic Acid, Fatty acid, acide gras, Lycopersicon esculentum, Fettsaeuren, Lycopersicon esculentum var. esculentum, Tomatoes, Fettsaeure, Aliphatic Acids, fatty acids, Solanum lycopersicum var. humboldtii, acides gras, Lycopersicon lycopersicum, Saturated Fatty Acids, Esterified Fatty Acid, responsive, Solanum lycopersicon, Lycopersicon esculentum Mill., EsterifiedSaturated Fatty, acido graso, malignant Growth, lipids, host organism, Ghrfr, Esterified Fatty Acids, human being, acidos grasos, bioformation, RNA Sequence Determination, Sequence Determination, Pflanze, cell type cancer, postnatal development, RNA Sequence, Metabonomic, growth and development, Metabonomics, biosynthesis, Tomatoes, 9-dien-4, viridiplantae, acides gras, Roles, responsivity, resistance, Line, Concepts, Saturated Fatty Acids, Esterified Fatty Acid, Analysis, Metabolomic, heptadeca-1, 9-diene-4, neoplasm, malignant tumour, multicellular organismal biosynthetic process, Esterified, plantae, Pilz, reactivity, Fatty Acids, Filamentous Fungi, single-organism biosynthetic process, Analyses, formation, tomato, Determination, anabolism, Esterified Fatty, neoplasm (disease), Aliphatic, plants, present in organism, Lycopersicon esculentum, Filamentous Fungus, W, Lycopersicon esculentum var. esculentum, man, Sequence Determinations, Sequencing, CA, synthesis, Aliphatic Acids, 6-diyne-3, RNA Sequence Analyses, malignant neoplasm, Lycopersicon lycopersicum, Role Concepts, RNA Sequencing, TG, fungi, Sequence Analyses, malignancy, RNA, Saturated, Molds, Solanum esculentum Dunal, Tomato, RNA Sequence Determinations, Lipid., malignant, Lycopersicum esculentum, land plants, Fungi, Fungus, Cell Lines, Fatty acid, lit, Fettsaeuren, Mycota, falcalindiol, Cell, Solanum esculentum, Mold, Determinations, Concept, development, Fettsaeure, Solanum lycopersicum var. humboldtii, MT, Role Concept, malignant neoplasm (disease), Lipid, 8-diol, organ system cancer, Role, little, Lines, Acid, Fatty Acid, primary cancer, Saturated Fatty Acid, Solanum lycopersicum L., growth pattern, non-developmental growth, postnatal growth, Plant, Aliphatic Acid, fungus, acide gras, (9E)-heptadeca-1, malignant tumor, human, fatty acids, higher plants, Filamentous, malignant neoplastic disease, RNA Sequence Analysis, response to biotic stress, response to abiotic stress, response, Solanum lycopersicon, growth, cancer, Lycopersicon esculentum Mill.Saturated Fatty, acido graso, Acid, Fatty Acids, Solanum esculentum., Fatty Acid, Esterified Fatty Acids, Saturated, Saturated Fatty Acid, acidos grasos, Solanum lycopersicum L., tomato, Solanum esculentum Dunal, Tomato, Lycopersicum esculentum, Esterified Fatty, Aliphatic, Aliphatic Acid, Fatty acid, acide gras, Lycopersicon esculentum, Fettsaeuren, Lycopersicon esculentum var. esculentum, Tomatoes, Fettsaeure, Aliphatic Acids, fatty acids, Solanum lycopersicum var. humboldtii, acides gras, Lycopersicon lycopersicum, Saturated Fatty Acids, Esterified Fatty Acid, responsive, Solanum lycopersicon, Lycopersicon esculentum Mill., Esterified0.00.00.00.00.0falseA Pathogen-Responsive Gene Cluster for Highly Modified Fatty Acids in TomatoIn response to biotic stress, plants produce suites of highly modified fatty acids that bear unusual chemical functionalities. Despite their chemical complexity and proposed roles in pathogen defense, little is known about the biosynthesis of decorated fatty acids in plants. Falcarindiol is a prototypical acetylenic lipid present in carrot, tomato, and celery that inhibits growth of fungi and human cancer cell lines. Using a combination of untargeted metabolomics and RNA sequencing, we discovered a biosynthetic gene cluster in tomato (Solanum lycopersicum) required for falcarindiol production. By reconstituting initial biosynthetic steps in a heterologous host and generating transgenic pathway mutants in tomato, we demonstrate a direct role of the cluster in falcarindiol biosynthesis and resistance to fungal and bacterial pathogens in tomato leaves. This work reveals a mechanism by which plants sculpt their lipid pool in response to pathogens and provides critical insight into the complex biochemistry of alkynyl lipid production.2022-01-172019-06-05MTBLS103931923394