Experimental treatment of Botrytis cinerea with wuyiencin
The standard strain (B05.10) of B. cinerea was inoculated onto Potato Dextrose Agar (PDA) medium  containing wuyiencin at a final concentration of 0 ppm (CK), 50 ppm, 100 ppm and 200 ppm. Cultures were incubated at 20 ℃ and mycelia morphology was observed under a light microscope after 3 days. Conidia were eluted with 0.2% Tween 20 after 7 days and the number of spores in droplets was quantified using a hemocytometer under a microscope. Three replicates were performed for growth measurements and spore production. The final concentration of wuyiencin is approximately 50–100 ppm in agricultural production ; this study showed that 200 ppm wuyiencin is lethal to B. cinerea, but there is little effect on cell growth at 50 ppm wuyiencin. Thus, we cultivated B05.10 in the dark at 20 °C with 100 ppm and without wuyiencin.
Scanning electron microscopy analysis
The morphology of the hyphae and their behaviour within the contact zone was investigated by scanning electron microscopy (SEM). After 7 days of cultivation on PDA, B05.10 samples (2 × 4 mm) were fixed in 2% (v/v) glutaraldehyde with 0.2 M phosphate buffer (pH 6.8) at 4 °C for 4–6 h and then rinsed with the same buffer for 2 h. The samples were then dehydrated in a graded acetone series (30%, 50%, 70%, 80%, 90%, and 100%), with samples immersed in each grade for 30 min and repeated three times for 100% acetone. Finally, the fully dehydrated samples were dried in a critical point dryer (HCP-2, Hitachi), mounted on stubs, and then coated strain (B05.10) (approximately 200 nm thick) using a sputter coater (S-3400N, Hitachi). The coated specimens were observed with a SEM HV (S-3400N, Hitachi) at 10 kV.
Transmission electron microscopy
For transmission electron microscopy (TEM) evaluations, B05.10 were cultured on PDA medium for 7 days before samples of hyphae were removed using a clean scalpel and washed three times with sterilised distilled water. Samples were then fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at 4 °C overnight. Subsequently, the samples were rinsed three times with phosphate buffer (50 mM, pH 6.8) and post-fixed overnight in 1% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.0) at 4 °C for 2 h. After rinsing with phosphate buffer, the samples were dehydrated in a gradient ethanol series and finally embedded in Epon 812 resin. The ultrathin sections were stained in 2% uranium acetate followed by lead citrate and visualised under a TEM (Hitachi, H-7650) operating at 80 kV.
The standard strain B05.10 of B. cinerea was inoculated onto PDA medium containing cellophane at a final concentration of 0 ppm (CK) and 100 ppm of wuyiencin, and placed in a 20 ℃ incubator. After 7 days, the hyphae were collected. The total proteins of B. cinerea were extracted without and with wuyiencin treatment. Three replicates represented control B. cinerea and three treatments represented wuyiencin-treated B. cinerea.
Hyphae (wet weight 0.5 g) scraped from fresh B. cinerea cultured on PDA plates with cellophane were added to 1 ml cold 10% TCA（Trichloroacetic acid）-0.07% β-me/acetone (β-mercaptoethanol). The sample was chilled at 20℃ for at least 1 h before centrifugation at 13,000 × g for 30 min at 4℃. The supernatant was discarded and cold acetone/0.07% β-mercaptoethanol (6 volumes) was added to the pellet. The solution was vortexed vigorously to mix well and stored at 20 ℃ for 30 min. Sample tubes were inverted several times every 10 min. The lysate was centrifuged twice at 13,000 × g for 30 min at 4 ℃ and each time, the supernatant was discarded. Finally, the B. cinerea pellet was air-dried and at room temperature to remove any residual acetone.
Lysis buffer was added to the B. cinerea pellet and incubated at room temperature for 30 min. The lysed samples were sonicated at 4 ℃ according to the following settings: 200 W, 10 s ON/15 s OFF pulses, (repeat 5 times). Finally, sonicated samples were centrifuged at 13,000 × g for 30 min at 4 ℃. The supernatant was retained and used for further experiments.
Trypsin digestion of proteins
The total protein was quantified by Bradford method . Samples of protein (200 μg) were placed in a centrifuge tube, mixed evenly with 5 ml DTT (1 M) and incubated for 1 h at 37 ℃. After the addition of 20 μl iodoacetamide ( IAA, 1 M), the solution was mixed at room temperature for 1 h. Samples were drawn into an ultrafiltration tube and the supernatant was discarded after centrifugation at 14,000 × g for 15 min at 4 ℃. Subsequently, 100 ml UA (8 M urea, 100 mm Tris-HCL, pH 8.0) were added to the ultrafiltration tube and the supernatant was discarded after two further rounds of centrifugation. TEAB buffer (0.5 M) was added (100 μl) and the supernatant was discarded following another three rounds of centrifugation. Finally, trypsin was added to the ultrafiltration tube at a protein to enzyme ratio of 50:1 and incubated for 12–16 h at 37 ℃.
Tandem mass tag (TMT) labelling
First, 45 μl of TEAB buffer (100 nM) was added to 100 μg re-labelled protein sample, and the total volume made up to 100 μl using distilled water. Next, 5 μl iodoacetamide (175 mM) was added and the sample incubated in the dark for 30 min before the addition of 1 ml pre-chilled acetone and precipitation at -20 ℃ overnight. After centrifugation at 12,000 ´g, the protein precipitate was air-dried and dissolved in 100 μl TEAB buffer (100 mM). The tryptic digests were then desalted using Pierce C18 spin columns (Thermo Scientific) according to manufacturer’s instructions. The digested peptides were labelled using a TMT kit according to the manufacturer’s instructions. After incubation for 1 h at room temperature, the labeling reaction was terminated by incubation with 5% hydroxylamine for 15 min. All samples were then combined and stored at -80 ℃.
Ion exchange chromatography
Off-line grading of labeled peptides was performed using the Waters HPLC system (series 2695) and the strong cation exchange column PolyLC Polysulfoethyl aspartamide column (100 mm ´ 2.1 mm, 5 μm particle size, 300 A pore size; PolyLC, Columbia, MD, USA). For gradient elution, 100% Buffer A (10 mM potassium dihydrogen phosphate, 15% acetonitrile, pH 2.7) was switched to 100% Buffer B (10 mM potassium dihydrogen phosphate, 15% acetonitrile, 500 mM potassium chloride, pH 2.7) over a period of 40 min. Gradient elution was performed at a flow rate of 200 μl/min, and elution peak monitoring and collection was performed at 220 nm using a Waters 2998 PDA module.
LTQ Orbitrap mass spectrometry
The fractionated peptides were separated by reverse phase liquid chromatography (RP-LC) using an Easy-nLC 1000 nanoflow high performance liquid chromatography system (Thermo Fisher Scientific, Odense, Denmark) with a capillary C18 reverse phase column (Acclaim PepMap, NanoViper C18, 50 μm × 15 cm). For linear gradient elution (5%–40%) elution, Buffer A (0.1% formic acid in water) was switched to Buffer B (0.1% formic acid, 98% acetonitrile) over a period of 60 min, at a flow rate of 300 nl/min. The separated components entered the LTQ Orbitrap combined mass spectrometer (Thermo Scientific) directly for tandem mass spectrometry identification using the default optimization parameters of the instrument, which were automatically adjusted according to the specific operating conditions. Level 1 scan range was m/z 350–1,800, and the 10 ions with the highest intensity were selected for secondary ion trap analysis. Mass spectrometry data were downloaded to a local computer for analysis using Proteome DiscovererTM 1.3 software (Thermo Fisher Scientific).
Proteome DiscovererTM 1.3 software (Thermo Fisher Scientific) was used in combination with the grey mould database uniprot-botrytis_organism_Acinerea. For relative quantitation of identified proteins, the proteins required a “high” peptide confidence interval and a maximum of two missed trypsin cleavages. N-term acetylation and oxidation (M) were set as variable modifications and carbamidomethylation and oxidation (C) as a fixed modification. The search was performed with a peptide mass tolerance of 15 ppm and a product ion tolerance of 0.02 Da with a 1% false discover rate (FDR). Differential protein screening (determined by the ratio in the treated samples and their corresponding untreated controls) was performed under the condition of 1.5 difference multiples (fc = fold change) and P < 0.05 threshold and under the condition of 2.0 difference multiples.
Protein hierarchical cluster analysis
In this study, we used hierarchical clustering analysis, in which each sample is first considered individually as a class, and the distances between the different classes are merged, before recalculation of the distance between classes. This process continues until all samples are grouped into one category. The standard for calculating the cluster distance indicator d (distance) uses the Euclidean distance, which represents the distance between classes using the full join method, also known as the longest distance method. The pair-wise ‘distance’ of proteins between the control (CK) and wuyiencin-treated (100 ppm) samples was subjected to Log2 normalisation and heatmaps and dendrograms were subsequently generated by pHEATMAP (R package) and g-plots, respectively. All statistical analyses were performed in the R environment.
GO enrichment analysis, functional classification, and expression profile analysisFor GO annotation (http://www.geneontology.org), the GO database (http://www.geneontology.org/page/download-go-annotations) is used to classify proteins into cellular components, molecular function, and biological process categories. Functional annotation of proteins was performed in this study by BLAST2GO, a comprehensive suite for functional analysis, in conjunction with the Perl computer programming language. Annotation was based on sequence similarity identified through the hypergeometric test, where P < 0.05 was considered to be significant. Significant differences in the expression of proteins in the treatment group compared with those in the control group were identified by t-tests. Blast results were mapped to uniprot-botrytis_organism_Acinerea and functional classification of differentially expressed proteins was performed using the online GO classification software QuickGo (http://www.ebi.ac.uk/QuickGO/). In-depth analysis of the main metabolic pathways was performed by KEGG pathway annotations generated from the mapping of GO terms to their enzyme code equivalents, according to the KEGG database (http://www.kegg.jp/kegg/pathway.html).
Target analysis by parallel reaction monitoring (PRM)
PRM analysis was performed with the Q-Exactive HF mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) using the same 90 min LC gradient settings as described previously which carried out at the Beijing Bangfei Bioscience Co., Ltd. (Beijing, China). The MS acquisition mode comprised a combination of two scan events: a full scan and a time-scheduled scan. The full scan was taken at a resolution of 120,000 at m/z 200 with a scan mass range of 300 to 1300 m/z, a target AGC of 3e6 and maximum injection fill time of 80 ms. The scheduled scan was employed at a resolution of 15,000 at m/z 120, a target AGC of 2e5, and maximum injection fill time of 45 ms. The precursor ion of each target peptide was isolated with a 2 Da window. The elution time window was set as ±2.5 min.
PRM MS analysis
MS raw data acquired from the target proteomics analysis were searched with SEQUEST integrated in Proteome Discoverer (version 1.4, Thermo Fisher Scientific, San Jose, CA, USA) against the uniprot-botrytis_organism_Acinerea database. Peptide probabilities were calculated by the Percolator algorithm in Proteome Discoverer, with a FDR set to 0.01. The generated .msf file was used to create a library in Skyline (version 3.1.0) and the cut-off score was set as 0.90. The target precursors list was then uploaded to Skyline and the six most intense product ions matching the library were selected as transitions. Peak extraction and manual inspection of fragment ion mass was performed and corrected according to the transitions, retention time, mass accuracy and MS/MS spectra. Each precursor was assigned the six most intense transitions and only transitions shared by all of the samples were used for quantification. The peak area of each transition was extracted and the area under curve (AUC) was extracted and exported to MSstats for further data analysis.