2.1. Plant material and pathogen inoculation
Wheat (Triticum aestivum L) cv Dongxuan 3 seeds were collected from Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing-China. Wheat seeds were sterilized with 30% sodium hypochlorite for 5 min, rinsed 5 times in water, and germinated for 30 days in incubator (AUCMA, Qing Dao, China) to vernalize. After vernalization, seedlings were grown in a 1:2 mixture of organic matter (peat moss, Beijing, China) and soil (Beijing, China) in pots. The pots were kept in incubator (AUCMA, Qing Dao, China) under a 14 h light: 10 h dark (8–10 ˚C, 70% humidity) regime and temperature was increased to 20 ˚C during boot stage. The pathogenic fungus T. controversa Kühn was gifted by Blair Goates (the United States Department of Agriculture (USDA), Agricultural Research Service (ARS), Aberdeen, Idaho, USA). T. controversa was propagated in soil agar medium (20 g agar powder, 75 g soil in one liter distilled water) and autoclaved for pouring the media into sterilized plates. After pouring, the plates were incubated at 5 °C in a 24 h light in incubator (MLR 352H, Panasonic, USA) after covering with parafilm for 60 days incubation. Mycelium production were observed under an automated invert fluorescence microscope (IX83, Olympus, Japan). Hyphae were collected with distilled water and used to inoculate wheat plants. During the boot stage, hyphae was injected into spike with the syringe. The hyphae was inoculated twice a day (5 mL per spike) and consecutively for 5 days, while only ddH2O was used for control plants. The pathogen infected grains (galls) and normal grains was harvested and quickly dipped in liquid nitrogen and stored at -80 ˚ C for further use. Nine replications of each treatment was used for reproducibility.
2. 2. Sample Processing
Metabolites were extracted from a 50 mg of crushed grains using ultrapure (Watsons, China) water. The choice of extraction method is an important factor in any metabolomics study. The grains were crushed by following the developed method of our laboratory. Briefly, two-three grains were dipped into 2 ml Eppendorf tube containing sterilized steel ball (1.5 mm) with appropriate amount of steel balls and grinded in grinder machine (FastPrep 24 5G, MP Biomedicals, USA) for I minute at 70 Hz for crushing. Every sample grinded 3 times for better results. Powdered tissue sample (50 mg) were first mixed with 500 µl of a ultrapure water (1:10 w/V) and sonicated for 10 min. After this time, 50 µl was taken from homogenate mixture and added into 450 µl of precipitant containing internal standard (methanol: acetonitrile = 1:1). The samples were then vortex for 1 min, centrifuging at 13000 rpm for 10 min. The finally resultant product (100 µL) was then transferred to separate new sterilized Eppendorf tubes for LC-MS analysis.
2.3. LC-MS analysis
LC-MS analysis of the precipitant was done in 450 µl of a methanol- acetonitrile mix. Analysis was done using a liquid chromatograph mass spectrometry (Dionex ultimate 3000, Thermo Fisher) with a C18 column (2.1 × 100 mm, 3 µm particle size, Waters). After a 5 µl of sample injection chromatographic analysis was achieved with a liquid phase of 2 mmol / L ammonium acetate and 0.1% formic acid in water (A) and acetonitrile (D). The detailed gradient elution program was shown as followed, 95% (A) and 5% (D) to 0–2 min; 5% (A) and 95% (D) to 42–47 min; 95% (A) and 5% (B) to 47.10–50 min.
Mass spectrometry (MS) was performed in positive electrospray ionization mode (ESI +). The instrument parameters were optimized as follows: ion spray voltage, 3000V; evaporation temperature, 350 ˚C; sheath gas, 35Arb; auxiliary gas, 10Arb; capillary temperature, 320 ˚C. The compound spectrometry parameters were optimized as follows: resolution, 70000; AGC target, 1e6; maximum TT, 100 ms; scanning range, 100-1500m / Z.
2.4. Data processing
All LC-MS dates were further filtered by using the R platform loaded with the xcms tool kit including; peak matching, retention time correction, variable integration and data standardization. The preprocessed data was then analysed by multivariate analysis, including principal components analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) by EZinfo software, and hierarchical cluster analysis (HCA) was performed by MetaboAnalyst 4.0 software. Metabolic pathways were further analyzed using KEGG (http://www.genome.jp/kegg/). The correlation analysis of differential metabolites was calculated by using R software and Cytoscape software was used for network construction.