P. umbellatus culture
P. umbellatus were derived from wild triennial sclerotia, and were cultured as described previously8. In the current study, P. umbellatus were cultured on plates containing fructose complete medium at room temperature in the dark to obtain sclerotia. P. umbellatus sclerotia collected on day 30, 40 or 90 after inoculation were defined as initial sclerotia (IS), developmental sclerotia (DS) and mature sclerotia (MS), respectively for PRM validation, ELISA analysis and enzyme activity assays.
For PRM analysis, proteins and peptides were prepared as previously reported4. Briefly, proteins were extracted from sclerotia, and then purified with ice cold acetone twice and dissolved with 0.2% RapiGestSFTM (Waters) in 50 mM ammonium bicarbonate. Peptides were digested using sequencing grade modified trypsin digestion (enzyme to protein ratio about 1:40) with final protein concentrations of approximately 1μg/μL. Candidate DEPs in this work were confirmed using parallel reaction monitoring (PRM). Nano LC-MS/MS analysis was carried out using an Orbitrap Fusion (Thermo-Fisher Scientific, San Jose, CA) mass spectrometer equipped with a nanospray Flex Ion Source and the UltiMate3000 RSLC nano (Dionex, Sunnyvale, CA). Peptide samples were injected onto a PepMap C-18 RP nano trap column (3µm, 100 µm×20 mm, thermo-Fisher Scientific) at a 6 uL/min flow rate for on-line desalting, and separated on a PepMap C-18 RP nano column (2 µm, 75 µm×25 cm). Peptides were eluted at a flow rate of 300 nL/min in 0.1% formic acid at 55 ℃ with a linear, segmented gradient of 3-8-28-99% of solvent B (0.1% FA, 80% ACN) over 120 min prior electro spray ionization.
The Orbitrap Fusion was operated in positive ion mode with nano spray voltage set at 2.1 kV and source temperature at 275℃. The instrument was operated in PRM mode.MS survey scanned at a resolving power of 60,000 FWHM (full width half maximum) at m/z 200, for the mass range of m/z 350～2000, and MS/MS scanned at 30,000 with Q isolation window (m/z) at 1.2 for the mass range m/z 105～2000. All data were acquired under Xcalibur 4.3 operation software.
The PRM data were processed using Skyline (v.3.6).Peptide settings: the enzyme was set as trypsin [KR/P], and the maximum number of missed cleavages was set as 2. The peptide length was set as 8-25; the variable modifications were set as carbamidomethyl on Cys and oxidationon Met; and the maximum number of variable modifications was set as 3.
Transition settings: the precursor charges were set as 2+, 3 and 4+; the ion charges were set as 1 and 2; and the ion types were set as b and y. The product ions were set as ranging from ion 3 to the last ion, and the ion match tolerance was set as 0.02 Da.
Enzyme activity assay
Sclerotia of P. umbellatus were ground into a fine powder in liquid nitrogen, then a protein extraction solution of lactase or β-1, 3 GA enzymes in an amount of 1.0mL/0.1g was added, and extracted at 4 ℃ for 20min. Extracts were then centrifuged at 14000×g and 4 ℃ for 10min, and the supernatant was collected as a protein solution.
For enzymatic activity of lactase, 40 μL of the sample protein solution was added to the measurement tube, 40 μL of the boiled sample protein solution was added to the control tube, and then 240 μL of the working solution was added. The solution was mixed and incubated at 60 ℃ in a water bath for 3min and cooled to room temperature. 200 μL of the resulting solution was placed into each well of a 96-well plate, and absorbance was measured 420 nm on an EnSpire® multimode reader (PerkinElmer Co. Ltd.). Three biological samples were taken, and each biological sample was measured twice. The optical density (OD) value of the test tube minus the OD value of the control tube acted as the actual OD value (△A). Enzyme activity (U/g) was calculated according to 65*△A /w in which w was the weight of sample.
For assessing enzymatic activity of β-1, 3GA, 35μL of the sample protein solution was added to the measurement tube and the control tube, followed by 35μlwater which was added to the control tube, and 35 μL of the working solution was added to the measurement tube. The resulted mixture was mixed and incubated at 37 ℃ in a water bath for 60 min. next, the solution was cooled to room temperature and 230μL working solution was added to each tube, and then mixed and incubated at 95 ℃ water bath for 3 min. Next, 200 μL solution was placed into a 96-well plate, and absorbance was measured at 550 nm. Three biological samples were taken and each biological sample was measured twice. The OD value of the test tube minus the OD value of the control tube acted as the actual OD value (△A).Enzyme activity (U/g) was calculated according to 20.876*(△A+0.0192) /w in which w was the weight of sample.
ELISA tests of β-mannosidase and α-L fucose
A gradient of β-mannosidase standard solutions were prepared (5 pg/mL, 10 pg/mL, 20 pg/mL, 40 pg/mL or 60 pg/mL) and a gradient of α-L fucose enzyme standard solutions (20 ng/L, 40 ng/L, 80 ng/L, 160 ng/L or 320 ng/L). Each solution was repeated twice.
Sample concentration determination: a 40μL sample of dilution solution and 10μL of sample were added to the well in sequence. The plate was sealed and incubated at 37 ℃ for 30 minutes. Waste solution was discarded and the plate was dried. Next, 200μL washing solution was added and the well was left to rest for 30s then the solution was discarded, this was repeated 5 times. Next, 50μL of enzyme-labeled reagent was added to each well and incubate at 37 ℃ for 30min before being washed five times again. Then 50μL developer A and B was added to each well in sequence, mixed gently by shaking, and developed at 37 ℃ in the dark for 15min. Next, Added 50μL stop solution was added. Wells without samples and enzyme-labeled reagents acted as blank wells. The OD values of above samples were measured at 450 nmon an EnSpire® multimode reader.
Localization of target protein Puctg 112
Immunocolloidal gold localization technology was introduced to locate the target Puctg 112 (hydrophobin B-like protein) in initial sclerotia of P. umbellatus. The experimental process refer to the previous article8 and the general steps were as follows: Sclerotia was cut into small pieces with a diameter of about 0.3 cm, and were fixed with 4% glutaraldehyde solution (4°C) followed washing with PBS buffer (pH6.8) and then dehydrated with 15%-100% ethanol solution (4°C) for 30 min each time. The dehydrated samples were treated with ethanol-LR White epoxy resin mixture (ethanol: LR White=3:1, 1:1 and 1:3) for 24h each time, and then imbedded in the LR white resin. The pellets were cut into 50~80 nm ultra-thin sections and washed three times with distilled water, PBS-glycine solution and PBS-T for 5 min each time in proper order. The slices were incubated with anti-Puctg 112 antibody (prepared by Beijing Protein Innovation Co., Ltd.) diluted to 6000 times, and incubated at room temperature for 60 min and then incubated at 4 °C overnight. After cleaned them with 0.1% BSA Tris-phosphate buffer (PBS-T), the slices were incubated with goat anti-mouse secondary antibody diluted to 20 times with PBS-T solution for 90 min at room temperature. Washed three times with PBS-T, PBS-glycine solution and distilled water for 5 min each time. Sample sections were treated with sensitization solution and uranyl acetate for 15 minutes each time, and finally were visualized by using JEM-1230 transmission electron microscope (Tokyo, Japan).
Data processing and biological information analysis
Quantified proteins of sclerotia by SWATH were selected, normalized and compared via t-test to identify differentially expressed proteins (DEPs) of MS vs. IS, MS vs. DS and DS vs. IS. Union of the DEPs was done via Venn diagram, and then GO annotation and KEGG pathway enrichment analysis were performed by omicsbeanTM (Shanghai Gene for health Co.). Those DEPs involved in defensive structures of sclerotia were selected for further analyses to investigate morphological changes in sclerotia. Calculated results are presented as M±SD (n=3) followed Grubbs test to remove suspicious data.