Co-immunoprecipitation and mass-spectrometry analysis

Total protein extracts from 100 mg of 35S::FW2.2-YFP fruit pericarp tissue were prepared using the following buffer: 1× PBS, cOmplete Protease Inhibitor Cocktail tablets (Roche, Mannheim, Germany), and 1% (v/v) Triton X-100. Samples were incubated in the extraction buffer at 4°C for 30 min with agitation, and then centrifuged (16,000 g, 10 min, 4°C). Prior to co-immunoprecipitation, western-blotting was used to check the presence of the expressed tagged-FW2.2 protein in the supernatant (Supplementary Fig. S12). The supernatant containing the resuspended proteins was then used for immunoprecipitation assay using anti-GFP microbeads provided in the μMACS Epitope Tag Protein Isolation Kit according to the manufacturer’ protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). Approximately 500 μg of soluble proteins was loaded for each co-IP assay.

Fifty µL of the resulting eluate was loaded on a 10% (w/v) SDS-PAGE acrylamide gel; gel bands were manually cut and transferred to 1.5 mL Eppendorf tubes. Bands were first washed with 500 µL of water and then 500 µL of 25 mm NH₄HCO₃. Destaining was performed twice in the presence of 500 µL of 50% (v/v) acetonitrile (ACN) in 25 mm NH₄HCO₃. Gel bands were dehydrated twice by 500 µL of 100% (v/v) ACN, and finally dried at room temperature. Following destaining, proteins were reduced with 500 µL of 10 mm DTT at 56 °C for 45 min. The supernatant was then removed and proteins were alkylated with 500 µL of 55 mm iodoacetamide for 30 min. Gel bands were washed twice with 500 µL of 50% (v/v) ACN in 25 mm NH₄HCO₃, then dehydrated by 500 µL of 100% (v/v) CH₃CN, and finally dried at room temperature. Twenty µL of a trypsin solution (Sequencing Grade Modified Trypsin, Promega, Madison, USA), at a concentration of 0.0125 µg/µL in 25 mm NH₄HCO₃, was added to every gel region and gel bands were kept for 10 min on ice. Fifty µL of 25 mm NH₄HCO₃ was added, and the samples were kept for another 10 min at room temperature. The digestion was performed overnight at 37 °C; then peptides were extracted by addition 100 µL of 2% (v/v) formic acid (FA). Gel bands were extracted twice by addition of 200 µL of 80% (v/v) ACN and 2% FA. After solvent evaporation in a Speed-vac, peptides were resuspended in 10 µL of 2% (v/v) FA, then purified with a micro tip C18 (Zip-Tip C18 Millipore Corporation Billerica MA, USA). Peptides were eluted with a solution containing 2% (v/v) FA and 80% (v/v) ACN and dried until total evaporation. Peptides were resuspended in 7 µL 2% (v/v) FA before LC-MS/MS analysis.

The LC-MS/MS were performed using the Ultimate 3000 RSLC nano system (Thermo Fisher Scientific Inc., Waltham, MA, USA) interfaced online with a nano easy ion source and the Exploris 240 Plus Orbitrap mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The samples were analyzed in Data Dependent Acquisition (DDA). The raw files were analyzed with MaxQuant version 2.0.3 using default settings. The files were searched against the S. lycopersicum genome (ITAG4.1_release January 2022 https://solgenomics.net/organism/solanum_lycopersicum/genome 34,689 entries) added with the FW2.2-YFP. Identified proteins were filtered according to the following criteria: at least 2 different trypsin peptides with at least 1 unique peptide, an E-value below 0.01, and a protein E-value smaller than 0.01 were required. Using the above criteria, the rate of false peptide sequence assignment and false protein identification was lower than 1%. Proteins were quantified by label-free method with MaxQuant software using unique and razor peptides intensities (Cox et al. 2014). Statistical analyses were carried out using RStudio package software. The protein intensity ratio and statistical tests were applied to identify the significant differences in the protein abundance. Hits were retained if they were quantified in at least 4 of the 5 replicates in at least 1 experiment. Proteins with a significant quantitative ratio (P < 0.05 or 0.01 with or without Benjamini–Hochberg correction) were considered as significantly upregulated and downregulated, respectively.

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al. 2022) partner repository with the data set identifier PXD045350.