Project PXD009099

PRIDE Assigned Tags:
Biological Dataset

Summary

Title

Apoplastic proteome upon powdery mildew Bgh challenge

Description

In order to identify the key factors that are crucial for non-host resistance against Bgh and are secreted depending on SYP4-mediated membrane trafficking, we have performed apoplastic fluid preparation of a series of mutants defective in the SYP4 pathway. This project aims to identify proteomic profiles of these apoplastic fluids.

Sample Processing Protocol

Apoplastic protein was extracted as described before with slight modifications (Ruhe et al., 2016, Frontiers in Plant Sci). Briefly, A. thaliana 32-day-old plants growing under short-day condition were inoculated with Bgh K1 isolate and incubated for 0, 24, and 48 hours under the same condition. Shoots were collected from 12-50 plants in a glass beaker and submerged in sodium acetate buffer (pH 4.3) supplemented with 0.2 M CaCl2 and protease inhibitor cocktail (Roche). After 10 minutes of vacuum infiltration and gentle release, excessive buffer on the leaf surface was removed with a paper towel and introduced into a 20-mL syringe. Apoplastic fluid was extracted by centrifuging the syringe in a 50-mL conical tube (Eppendorf) at 1,000 g for 20 minutes and sterilized by centrifuging in a Ultrafree 0.22-m filter tube for 2 minutes at 12,000 g (Millipore). Proteins were purified and concentrated by chloroform/methanol precipitation (Wessel and Flügge, 1984). Proteins were reduced, alkylated and digested in-solution. Protein pellets were dissolved in 8 M urea, 0.1 M Tris-HCl pH 8.0. Cysteins were reduced by adding DTT to a final concentration of 5 mM and incubation for 30 min. Alkylation was subsequently performed by adding chloroacetamide to a final concentration of 14 mM and incubation for 30 min. The reaction was quenched by addition of DTT. Urea concentration was adjusted to 4 M by dilution with 0.1 M Tris-HCl pH 8.0, 1 mM CaCl2, and digested with Lys-C (1:100 enzyme-to-protein ratio) for 4 hours at room temperature. Urea concentration was further adjusted to 1 M, and trypsin digestion (1:100 enzyme-to-protein ratio) was performed over night at 37°C and stopped by addition of 1% formic acid. Digested samples were desalted with StageTips (Empore C18, 3M) as described previously. {Rappsilber, 2007 #32}.

Data Processing Protocol

Raw data were processed using MaxQuant software (version 1.5.7.4, http://www.maxquant.org/) {Cox, 2008 #33} with label-free quantification (LFQ) and iBAQ enabled {Cox, 2014 #34}. MS/MS spectra were searched by the Andromeda search engine against a combined database containing the sequences from A. thaliana (TAIR10_pep_20101214; ftp://ftp.arabidopsis.org/home/tair/Proteins/TAIR10_protein_lists/) and Bgh isolate K1 (6-frame translation of the published genome sequences {Hacquard, 2013 #37;Spanu, 2010 #36} with MaxQuant; only translated polypeptide sequences with a minimum length of 20 amino acids were kept for database search). Sequences of 248 common contaminant proteins and decoy sequences were automatically added during the search. Trypsin specificity was required and a maximum of two missed cleavages allowed. Minimal peptide length was set to seven amino acids. Carbamidomethylation of cysteine residues was set as fixed, oxidation of methionine and protein N-terminal acetylation as variable modifications. Peptide-spectrum-matches and proteins were retained if they were below a false discovery rate of 1%.

Contact

Hirofumi Nakagami, Max Planck Institute for Plant Breeding Research
Hirofumi Nakagami, Protein Mass Spectrometry Max Planck Institute for Plant Breeding Research ( lab head )

Submission Date

02/03/2018

Publication Date

11/01/2019

Tissue

Not available

Cell Type

plant cell

Instrument

Q Exactive

Software

Not available

Experiment Type

Shotgun proteomics

Publication

    Uemura T, Nakano RT, Takagi J, Wang Y, Kramer K, Finkemeier I, Nakagami H, Tsuda K, Ueda T, Schulze-Lefert P, Nakano A. A Golgi-released subpopulation of the trans-Golgi network mediates protein secretion in Arabidopsis. Plant Physiol. 2018 PubMed: 30545905