Maxquant

Plant materials: one-year-old hybrid bamboo plants were planted in the bamboo-growing areas of a reclaimed farmland (103°01′N, 29°54′E) in Sichuan, China. The study area was at an altitude of 515.98 m with an annual temperature of 6.8 to 26.1 °C, and annual precipitation of 1300–1700 mm. Hybrid rice was planted in the rice-growing areas of Chengdu Plain (103°01′N, 29°54′E) in Sichuan, China. The area has an altitude of 530 meters, average annual temperature of 15.9 °C, and average annual precipitation of 1010 mm. All samples were healthy varieties.
Microorganism: A. phaeospermum was isolated from diseased hybrid bamboo²³ . The isolate was maintained on a PDA slant medium containing potato dextrose agar at 4 °C until used.
Instruments and reagents: UA buffer (8 M urea, 150 mM Tris–HCl pH 8.0), NH₄HCO₃ (Sigma, A6141), acetonitrile (Merck, 1499230-935), TMT 10plex kit (Thermo Fisher), Pierce High PH Reversed-Phase Peptide Fractionation Kit (Thermo Fisher), Q-Exactive Plus (Thermo Scientific), Easy-nLC1200 (Thermo Scientific), Trap column (Reverse-phase), 100 μm × 20 mm (5 μm, C18), Thermo Scientific EASY column (Reverse-phase), 75 μm × 120 mm (3 μm, C18), MaxQuant (version 1.6.0.16).

Identification of SUMO modification lysine sites was carried out as described before (42 , 43 , 44 (link)). Briefly, HEK293 cells were transfected with mSUMO3 and HA-GβL, followed by denaturating Ni–NTA pulldown as described previously. The Ni–NTA resin was extensively washed with 50 mM ammonium bicarbonate to remove traces of Triton, and the proteins were digested with trypsin directly on the Ni–NTA solid support for 16 h at 37 °C. The mSUMO3-modified peptides were immunoprecipitated with a custom anti-NQTGG antibody that recognizes the tryptic remnant created on the SUMO-modified lysine side chain, as described before (42 , 43 , 44 (link)). Samples were analyzed on the Q-Exactive HF instrument (ThermoFisher Scientific), and raw files were processed using MaxQuant and Perseus, as described previously (42 , 43 , 44 (link)).

Specific proteins were analyzed using Proteome Discoverer 2.4.1.15. Trypsin was employed as the enzyme, which cleaved after all lysine and arginine residues, with up to two missed cleavages allowed. Carbamidomethylating of cysteine was specified as fixed modification, and protein N-terminal acetylation, oxidation of methionine, and pyro-glutamate formation from glutamine were considered as variable modifications for all groups. The raw file of the mass spectrum was identified and analyzed using the commercial software, Max Quant (Thermo Fisher Scientific, Waltham, MA, USA). The precursor ion mass tolerance was set to 15 ppm and the fragment ion mass tolerance was set to 0.02 Da. All data were searched as a single batch with PSM and protein FDR set to 1% using a target decoy approach. The search parameters were as follows: species, C. sinensis; dynamic modification, oxidation; mass tolerance of the precursor ion, ± 15 ppm; fragment ion mass tolerance, ± 0.5 Da; and protein false discovery rate (FDR), 0.01. The maximum number of missed cleavages was two. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the proteins of C. sinensis was performed to select the most significant pathway and to analyze the relationship between the abundance of specific proteins of different pathways and the different periods of infection.

Specific proteins were analyzed according to the Proteome Discoverer 2.4.1.15. The raw file of the mass spectrum was identified and analyzed using the commercial software, MaxQuant (Thermo Fisher Scientific, Waltham, MA, USA). The search parameters were as follows: value of the enzyme was trypsin, static modification was C-carboxyamidomethylation (57.021 Da), dynamic modification was oxidation (M), species was F. hepatica, mass tolerance of the precursor ion was ±15 ppm, fragment ion mass tolerance was ±0.5 Da, and the protein false discovery rate (FDR) was set at 0.01. The maximum number of missed cleavages was 2. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the specific proteins of F. hepatica was performed to select the most significant pathway and to analyze the relationship between the abundance of specific proteins of different pathways and the different periods of infection.

The raw MS2 data were searched using Maxquant version 1.6.0.1 (Thermo Scientific) against the human UniProt database. Carbamidomethyl (Cys) was set as a fixed modification. The variable modifications included oxidation (Met) and acetyl (protein N-term). A positive identification of peptide length was required to contain a minimum of seven amino acids and a maximum of one peptide PEP. The tolerances of MS/MS were set as 20 ppm. The false discovery rates (FDRs) of the peptide-spectral match and protein identification were set as 0.01. The protein intensity is derived from label-free quantification intensity generated by Maxquant. Proteins with a P-value of < 0.05 were considered DEPs. Finally, Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were applied on DEPs, and DEPs were corrected using principal component analysis (PCA) via the pipeline process. The spectral data were deposited to the Figshare website (https://figshare.com/account/login) with the dataset https://doi.org/10.6084/m9.figshare.12335288.