Summary of Study ST002917

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001813. The data can be accessed directly via it's Project DOI: 10.21228/M8C713 This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002917
Study TitleTransporter-mediated depletion of apoplastic proline directly contributes to plant pattern-triggered immunity against a bacterial pathogen
Study SummaryGC-MS analysis of apoplastic fluid extracted from arabidopsis plants treated with 100 nM flg22 or a mock treatment for 8 hours. Col-0 is wild type arabidopsis plants, QKO is a quadruple knockout mutant in the Col-0 background with knockouts in dde2-2, ein2-1, pad4-1, and sid2-2.
Institute
Oregon State University
DepartmentBotany and Plant Pathology
LaboratoryJeff C Anderson
Last NameRogan
First NameConner
AddressCordley Hall, 2701 SW Campus Way, Corvallis, OR 97331
Emailroganco@oregonstate.edu
Phone3146004945
Submit Date2023-08-28
Raw Data AvailableYes
Raw Data File Type(s)d
Analysis Type DetailGC-MS
Release Date2024-02-28
Release Version1
Conner Rogan Conner Rogan
https://dx.doi.org/10.21228/M8C713
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:


Project:

Project ID:PR001813
Project DOI:doi: 10.21228/M8C713
Project Title:Transporter-mediated depletion of apoplastic proline directly contributes to plant pattern-triggered immunity against a bacterial pathogen
Project Type:Untargeted GC-MS analysis
Project Summary:In plants and animals, detection of pathogen-associated molecular patterns (PAMPs) by membrane-localized receptors initiates pattern-triggered (PTI) immunity against pathogens. In plants, many PAMP-induced signaling pathways and cellular responses are known, yet how PTI limits pathogen growth is poorly understood. Through a combined metabolomics and genetics approach, we discovered that plant-exuded proline is a virulence-inducing signal and nutrient for the bacterial pathogen Pseudomonas syringae, and that PAMP-induced depletion of proline from the extracellular spaces of Arabidopsis leaves directly contributes to PTI against P. syringae. We further show that PAMP-induced depletion of extracellular proline requires the amino acid transporter Lysine Histidine Transporter 1 (LHT1). This study demonstrates that depletion of a single extracellular metabolite is an effective component of plant immunity. Given the important role for amino acids as N and C sources for microbial growth, their depletion at sites of infection may be a broadly effective defense against many pathogens.
Institute:Oregon State University
Department:Botany and Plant Pathology
Laboratory:Jeff C Anderson
Last Name:Rogan
First Name:Conner
Address:2701 SW Campus Way, Corvallis, OR, 97330, USA
Email:roganco@oregonstate.edu
Phone:3146004945

Subject:

Subject ID:SU003030
Subject Type:Plant
Subject Species:Arabidopsis thaliana
Taxonomy ID:3702
Age Or Age Range:4-5 weeks
Species Group:Plants

Factors:

Subject type: Plant; Subject species: Arabidopsis thaliana (Factor headings shown in green)

mb_sample_id local_sample_id Genotype Treatment
SA316606flg22(1-1)Col-0 flg22
SA316607flg22(2-5)Col-0 flg22
SA316608flg22(1-6)Col-0 flg22
SA316609flg22(3-4)Col-0 flg22
SA316610flg22(2-4)Col-0 flg22
SA316611flg22(4-3)Col-0 flg22
SA316612flg22(1-4)Col-0 flg22
SA316613flg22(2-6)Col-0 flg22
SA316614flg22(3-6)Col-0 flg22
SA316615flg22(2-8)Col-0 flg22
SA316616flg22(3-8)Col-0 flg22
SA316617flg22(1-8)Col-0 flg22
SA316618flg22(3-7)Col-0 flg22
SA316619flg22(1-7)Col-0 flg22
SA316620flg22(2-7)Col-0 flg22
SA316621flg22(3-3)Col-0 flg22
SA316622flg22(1-5)Col-0 flg22
SA316623flg22(4-1)Col-0 flg22
SA316624flg22(3-2)Col-0 flg22
SA316625flg22(4-2)Col-0 flg22
SA316626flg22(3-1)Col-0 flg22
SA316627flg22(2-2)Col-0 flg22
SA316628flg22(1-3)Col-0 flg22
SA316629flg22(2-3)Col-0 flg22
SA316630flg22(2-1)Col-0 flg22
SA316631flg22(1-2)Col-0 flg22
SA316632Mock(3-1)Col-0 mock
SA316633Mock(4-1)Col-0 mock
SA316634Mock(2-1)Col-0 mock
SA316635Mock(3-7)Col-0 mock
SA316636Mock(2-8)Col-0 mock
SA316637Mock(3-8)Col-0 mock
SA316638Mock(1-8)Col-0 mock
SA316639Mock(1-1)Col-0 mock
SA316640Mock(2-7)Col-0 mock
SA316641Mock(2-6)Col-0 mock
SA316642Mock(1-7)Col-0 mock
SA316643Mock(3-6)Col-0 mock
SA316644Mock(4-2)Col-0 mock
SA316645Mock(3-2)Col-0 mock
SA316646Mock(1-6)Col-0 mock
SA316647Mock(3-3)Col-0 mock
SA316648Mock(1-3)Col-0 mock
SA316649Mock(2-3)Col-0 mock
SA316650Mock(2-2)Col-0 mock
SA316651Mock(4-3)Col-0 mock
SA316652Mock(2-5)Col-0 mock
SA316653Mock(1-4)Col-0 mock
SA316654Mock(1-2)Col-0 mock
SA316655Mock(1-5)Col-0 mock
SA316656Mock(3-4)Col-0 mock
SA316657Mock(2-4)Col-0 mock
SA316658flg22 QKO-1QKO flg22
SA316659flg22 QKO-3QKO flg22
SA316660flg22 QKO-4QKO flg22
SA316661flg22 QKO-2QKO flg22
SA316662Mock QKO-2QKO mock
SA316663Mock QKO-1QKO mock
SA316664Mock QKO-3QKO mock
SA316665Mock QKO-4QKO mock
Showing results 1 to 60 of 60

Collection:

Collection ID:CO003023
Collection Summary:Apoplastic wash fluid (AWF) was isolated from the leaves of 5- to 6-week-old Arabidopsis plants treated with flg22 or a mock control. To initiate PAMP responses, a needle-less syringe was used to infiltrate the leaves with a solution of 100 nM flg22 in water, or with DMSO in water as a negative control. Six to eight leaves were infiltrated on each plant, and a total of six plants were infiltrated for each treatment condition. Individual plants were infiltrated with flg22 or the DMSO only control, never both. After eight hours, AWF was isolated by syringe-infiltrating the mock- and flg22-treated leaves with sterile H2O containing 164 µM ribitol as an internal standard. Immediately after infiltration, the aerial portion of the plant was removed by cutting the primary stem and briefly washed with H2O to remove surface contaminants. The infiltrated leaves were detached from the rosette and stacked between layers of parafilm, with 2 to 4 leaves in each layer. The parafilm booklet of leaves was wrapped with tape and suspended inside a 15 mL conical centrifuge tube. The tube was centrifuged at 750 x g for seven minutes. The AWF that collected at the bottom of the conical tube was transferred to a clean 1.7 mL microcentrifuge tube, then centrifuged at 21,000 x g for 10 minutes at 4°C. The resulting supernatant was transferred to a clean 1.7 mL microcentrifuge tube. After addition of 50 µL of chloroform, the samples were vortexed for 10 seconds and centrifuged at 21,000 x g for 10 minutes at 4°C. The upper aqueous phase was transferred to a clean 1.7 mL microcentrifuge tube, and the volume recovered was measured with a pipette. The AWF samples were then lyophilized to dryness and stored at -80°C until further use.
Sample Type:Apoplastic washing fluid

Treatment:

Treatment ID:TR003039
Treatment Summary:Plants were treated with 100 nM flg22 dissolved in H2O or a mock treatment for eight hours.

Sample Preparation:

Sampleprep ID:SP003036
Sampleprep Summary:Samples were derivatized with 10 μL of 20 mg/mL methoxyamine hcl in pyridine for 90 minutes at 37 °C. Then 20 μL of MSTFA + 1% TMCS was added and the samples were incubated for 30 minutes at 37 °C. The samples were then randomly injected into the GC-MS.

Combined analysis:

Analysis ID AN004787
Analysis type MS
Chromatography type GC
Chromatography system Agilent 7890B
Column Agilent DB5-MS (30m x 0.25mm, 0.25um)
MS Type EI
MS instrument type Single quadrupole
MS instrument name Agilent 5977B
Ion Mode POSITIVE
Units Peak area

Chromatography:

Chromatography ID:CH003618
Chromatography Summary:For each sample, a 20 µL aliquot of AWF was lyophilized to dryness and resuspended in 10 µL of 30 mg/mL methoxyamine hydrochloride (Sigma-Aldrich) in pyridine (Sigma-Aldrich). The resuspended sample was incubated at 37°C and shaken at 1800 rpm for 90 minutes. After adding 20 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (CovaChem), the sample was incubated at 37°C and shaken at 1800 rpm for 30 minutes. Each derivatized sample was injected with a 10:1 split into an Agilent 7890B GC system with a 30 m +10 m Duraguard x 0.25 mm x 0.25 μm DB-5MS+DG Agilent column. The oven temperature was kept at 60°C for 1 minute, then ramped to 300°C at a rate of 10°C/min and held at 300°C for 10 minutes. Analytes were detected with an Agilent 5977B MSD in EI mode scanning from 50 m/z to 600 m/z. Mass spectrum analysis, component identification and peak area quantification were performed with AMDIS (http://www.amdis.net/). To reduce erroneous identifications during automated feature identification, the FiehnLib library was used to create a custom library that included both identified compounds along with unidentified prominent peaks that were recorded as unknowns and designated by their respective retention times72. Statistics were performed with MetaboAnalyst73 and MetaboAnalystR74. An in house perl script was used to collate AMDIS-generated peak areas into an Excel spreadsheet. Peaks that were present in blank samples containing derivatization chemicals only were removed from the data set. Two to three technical replicates were analyzed for each sample and averaged to produce a single sample value. MetaboAnalyst settings were selected to replace missing values by 1/5 of the minimum positive value for each feature and normalize the peak areas of the features by the peak area of the internal ribitol standard in each respective sample.
Instrument Name:Agilent 7890B
Column Name:Agilent DB5-MS (30m x 0.25mm, 0.25um)
Column Temperature:60 ramped to 300
Flow Gradient:NA
Flow Rate:.52 mL/min helium gas
Injection Temperature:250
Internal Standard:ribitol
Solvent A:NA
Solvent B:NA
Chromatography Type:GC
Solvent C:NA

MS:

MS ID:MS004533
Analysis ID:AN004787
Instrument Name:Agilent 5977B
Instrument Type:Single quadrupole
MS Type:EI
MS Comments:Data were collect with scans from 50 m/z to 600 m/z. Data files were processed with AMDIS using automated feature identification with a library adapted from FiehnLib mass spectral and retention index library.
Ion Mode:POSITIVE
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