Re-annotated role of beta-Galactosidase in Flagellin deglycosylation
Most Eukaryotes recognise flagellin as a signature of bacterial invasion. In contrast to animals, plants do not recognise flagellin proteins, but conserved peptides released from flagellin (Felix et al., 1999). However, these peptides (e.g. flg22) are folded and buried deeply inside the flagellin polymer and would need to be released before they can interact with cell surface receptors, such as FLS2 (Fliegman & Felix, 2016). Here we discovered that the hydrolytic pathway releasing the flagellin elicitor in plants is initiated by a host-secreted beta-galactosidase (BGAL), which removes the terminal modified viosamine (mVio) from the O-glycan that cloaks the flagellin polymer. BGAL contributes to flagellin-dependent immunity but only against bacterial Pseudomonas syringae strains that carry mVio. Signatures of arms races at this new level of antagonistic interactions are that BGAL is suppressed during infection by a heat stable metabolite secreted by bacteria, and that other P. syringae strains carry BGAL-insensitive O-glycans.
Sample Processing Protocol
Sample clean-up for LC-MS. Acidified tryptic digests were desalted on home-made C18 StageTips as described (Rappsilber et al 2007 ). On each 2 disc StageTip around 15 µg peptides were loaded (based on the initial protein concentration). After elution from the StageTips, samples were dried using a vacuum concentrator (Eppendorf) and the peptides were taken up in 10 µL 0.1 % formic acid solution. LC-MS/MS. Experiments were performed on an Orbitrap Elite instrument (Thermo,[3 ]) that was coupled to an EASY-nLC 1000 liquid chromatography (LC) system (Thermo). The LC was operated in the one-column mode. The analytical column was a fused silica capillary (75 µm × 25 cm) with an integrated PicoFrit emitter (New Objective) packed in-house with Reprosil-Pur 120 C18-AQ 3 µm resin (Dr. Maisch). The analytical column was encased by a column oven (Sonation) and attached to a nanospray flex ion source (Thermo). The column oven temperature was adjusted to 45 °C during data acquisition. The LC was equipped with two mobile phases: solvent A (0.1% formic acid, FA, in water) and solvent B (0.1% FA in acetonitrile, ACN). All solvents were of UHPLC (ultra high performance liquid chromatography) grade (Sigma). Peptides were directly loaded onto the analytical column with a maximum flow rate that would not exceed the set pressure limit of 980 bar (usually around 0.5 – 0.8 µL/min). Peptides were subsequently separated on the analytical column by running a 70 min gradient of solvent A and solvent B (start with 7% B; gradient 7% to 35% B for 60 min; gradient 35% to 100% B for 5 min and 100% B for 5 min) at a flow rate of 300 nl/min. The mass spectrometer was operated using Xcalibur software (version 2.2 SP1.48). The mass spectrometer was set in the positive ion mode. Precursor ion scanning was performed in the Orbitrap analyzer (FTMS; Fourier Transform Mass Spectrometry) in the scan range of m/z 350-1800 and at a resolution of 60000 with the internal lock mass option turned on (lock mass was 445.120025 m/z, polysiloxane)[4 ]. Product ion spectra were recorded in a data dependent fashion in the ion trap (ITMS; Ion Trap Mass Spectrometry) in a variable scan range and at a rapid scan rate. The ionization potential (spray voltage) was set to 1.8 kV. Peptides were analyzed using a repeating cycle consisting of a full precursor ion scan (1.0 × 106 ions or 30 ms) followed by 10 product ion scans (1.0 × 104 ions or 50 ms) where peptides are isolated based on their intensity in the full survey scan (threshold of 500 counts) for tandem mass spectrum (MS2) generation that permits peptide sequencing and identification. CID (collision-induced dissociation) collision energy was set to 35% for the generation of MS2 spectra. During MS2 data acquisition dynamic ion exclusion was set to 60 seconds with a maximum list of excluded ions consisting of 500 members and a repeat count of one. Ion injection time prediction, preview mode for the FTMS, monoisotopic precursor selection and charge state screening were enabled. Only charge states higher than 1 were considered for fragmentation.
Data Processing Protocol
Peptide and Protein Identification using MaxQuant. RAW spectra were submitted to an Andromeda search in MaxQuant (version 126.96.36.199) using the default settings.[6 ] Label-free quantification and match-between-runs was activated.[7 ] MS/MS spectra data were searched against the ACE_0319_Niben_Final.fasta _(N. benthamiana, 42853 entries) and Uniprot reference database UP000002515_223283.fasta (Pseudomonas syringae pv. tomato (strain ATCC BAA-871 / DC3000), 5431 entries) databases. All searches included also a contaminants database (as implemented in MaxQuant, 267 sequences). The contaminants database contains known MS contaminants and was included to estimate the level of contamination. Andromeda searches allowed oxidation of methionine residues (16 Da), acetylation of protein N-terminus (42 Da as dynamic modification and the static modification of cysteine (57 Da, alkylation with IAM). Enzyme specificity was set to “Trypsin/P” with 2 missed cleavages allowed. The instrument type in Andromeda searches was set to Orbitrap and the precursor mass tolerance was set to ±20 ppm (first search) and ±4.5 ppm (main search). The MS/MS match tolerance was set to ±0.5 Da. The peptide spectrum match FDR and the protein FDR were set to 0.01 (based on target-decoy approach). Minimum peptide length was 7 amino acids. For protein quantification unique and razor peptides were allowed. Modified peptides were allowed for quantification. The minimum score for modified peptides was 40. Data Analysis. Initial data analysis was performed by using the PERSEUS computational platform (version 188.8.131.52.). [8 ]
Buscaill P, Chandrasekar B, Sanguankiattichai N, Kourelis J, Kaschani F, Thomas EL, Morimoto K, Kaiser M, Preston GM, Ichinose Y, van der Hoorn RAL. Glycosidase and glycan polymorphism control hydrolytic release of immunogenic flagellin peptides. Science. 2019 364(6436) PubMed: 30975858