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190719_PSL_YoJi_flg22_1 05:42:37111320
Detection of flg22 peptide upon treatment with different Arabidopsis root-derived bacterial supernatant
Data License: CC BY 4.0 | ProteomeXchange: PXD020452
  • Organism: Arabidopsis thaliana
  • Instrument: Q Exactive
  • SpikeIn: No
  • Keywords: Arabidopsis thaliana, root-growth inhibition, root commensals, flg22
  • Lab head: Hirofumi Nakagami Submitter: Sara Stolze
Asymptomatic plants grown in natural soil are colonized by phylogenetically structured communities of microbes known as the microbiota. Individual microbiota members can activate host innate immunity, which limits pathogen proliferation and curtails plant growth, a phenomenon known as the growth-defense trade-off. We report that in mono-associations, 41% (62/151) of taxonomically diverse root commensals suppress Arabidopsis root growth inhibition (RGI) triggered by immune-stimulating microbe-/damage-associated molecular patterns. 16S rRNA gene amplicon sequencing data reveal that immune activation alters the profile of synthetic communities (SynComs) comprised of RGI non-suppressive strains, while the presence of RGI-suppressive strains attenuates this effect. Chronic root transcriptional outputs in response to colonization with RGI-suppressive or non-suppressive SynComs share a core of genes with a stereotyped expression pattern. However, RGI-suppressive SynComs specifically downregulate a subset of immune-related genes. Such SynCom-specific modulation of defense is physiologically relevant as mutation of one commensal-downregulated transcription factor, MYB15, or pre-colonization with an RGI-suppressive SynCom render plants more susceptible to opportunistic Pseudomonas pathogens. Our results suggest that commensals with contrasting MTI modulating capacities interact with the plant host and together buffer the system against pathogen challenge, defense-associated plant growth inhibition and community shift via a crosstalk with the immune system, leading to commensal-host homeostasis.
Experiment Description
For in vitro detection of flg22, 1 µM flg22 was co-incubated with 1ml supernatant for 1 hour at room temperature. Half strength MS medium without sugar was used as a control. 100 µL aliquots sample were mixed with 200 µL UA (8M urea in 100 mM Tris-HCl pH 8.5) and adjusted to 10 mM DTT using 1M stock. Samples were loaded onto 30 kD spin filters (Vivacon 500, Sartorius) and centrifuged at 14k g for 15 min. The filtrate was collected and loaded onto 2 kD spin filters (Vivacon 500, Sartorius) and centrifuged at 14k g for 30 min, after which 300 µL UA were added and samples were centrifuged again (14k g, 45 min, or until most liquid had passed through the filter). Next, 100 µL 55 mM chloroacetamide were added to the filter and samples were incubated for 30 min in the dark, after which they were centrifuged at 14k g for 20 min. 300 µL UA were added and samples were centrifuged at 14k g for 45 min. Samples were washed twice with 300 µL 100mM Tris-HCl, pH 8.5, by centrifugation (14k g, 45 min). For elution, 200 µL Tris-HCl were added, and the inverted spin filters were centrifuged at 2k g for 2 min to collect eluate into a fresh tube. The eluates were desalted using StageTips with C18 Empore disk membranes (3 M)21, final elution was performed using 40% acetonitrile, 0.1% TFA. Samples were dried in a vacuum evaporator, and dissolved in 10 µL 2% ACN, 0.1% TFA for analysis. LC-MS/MS data acquisition. Samples were analyzed using an EASY-nLC 1000 (Thermo Fisher) coupled to a QExactive mass spectrometer (Thermo Fisher). Peptides were separated on 16 cm frit-less silica emitters (New Objective, 0.75 µm inner diameter), packed in-house with reversed-phase ReproSil-Pur C18 AQ 3 µm resin (Dr. Maisch). Peptides were loaded on the column and eluted for 50 min using a segmented linear gradient of 5% to 95% solvent B (0 min : 5%B; 0-5 min -> 5%B; 5-25 min -> 20%B; 25-35 min ->35%B; 35-40 min -> 95%B; 40-50 min ->95%B) (solvent A 0% ACN, 0.1% FA; solvent B 80% ACN, 0.1%FA) at a flow rate of 300 nL/min. Mass spectra were acquired in data-dependent acquisition mode with a TOP10 method. MS spectra were acquired in the Orbitrap analyzer with a mass range of 300–1500 m/z at a resolution of 70,000 FWHM and a target value of 3×106 ions. Precursors were selected with an isolation window of 2.0 m/z. HCD fragmentation was performed at a normalized collision energy of 25. MS/MS spectra were acquired with a target value of 5x105 ions at a resolution of 17,500 FWHM, a maximum injection time of 85 ms and a fixed first mass of m/z 100. Peptides with a charge of 1, greater than 6, or with unassigned charge state were excluded from fragmentation for MS2, dynamic exclusion for 20s prevented repeated selection of precursors. Data analysis Raw data was directly analyzed on MS1 level using Skyline ( {McLean et al., Bioinformatics, 2010, 26, 966.} against the sequence of the flg22 peptide. LysC specificity was required and a maximum of two missed cleavages allowed. Minimal peptide length was set to seven maximum length to 25 amino acids. Carbamidomethylation of cysteine, oxidation of methionine and protein N-terminal acetylation were set as modifications. Results were filtered for precursor charges of 2, 3. Peaks of the intact flg22 peptide precursor were integrated manually, peak areas were exported for further processing.
Sample Description
Preparation of cell-free culture of bacteria 15 to 20 Col-0 were pregerminated on half strength MS agar plate supplemented with 5g/L sucrose. After two weeks, 20ml washed bacterial suspension (OD600=0.0005) in half strength MS medium without sucrose was added to each plate. After another seven days of coincubation, fresh half strength MS medium was added to obtain a final 20ml supernatant after 1 hour of gentle shaking. The supernatant was filter-sterilized by passing through 0.22um PES filter (Millipore). The filtrates were separated into two fractions by passing through the 3kDa ultracentrifugal filter (4000rpm, 60min, Amicon Millipore). The filtrates were filter-sterilized by passing through the 0.22um filter again if necessary. Finally, the fraction larger than 3kDa was heat inactivated by boiling for 5 minutes. To test for RGI suppressive activity, surface sterilized seeds were germinated in 1 ml supernatant supplemented with 5g/L sucrose and 1 µM Atpep1 or flg22 in a 12-well plate for two weeks.
Created on 7/20/20, 5:42 AM