AA). The resulting plasmid was transformed into E. coli BL 21 (DE3) competent cells. For recombinant protein expression, E. coli was cultured overnight and then diluted 1:100 in Luria Broth medium. The bacteria were incubated at 37 until A600 reached approximately 0.5, then isopropyl-1thio–D-galactopyranoside (IPTG) was added to a final concentration of 1 mM to induce recombinant protein expression. The bacterial cells were incubated for an additional 3 h before the cells were collected by centrifugation for recombinant protein isolation. The recombinant protein was purified using Ni-NTA His Bind Resin according to the manufacturer’s manual (Merck KGaA), followed by further purification by 12 SDS-PAGE. The protein band corresponding to the predicted size of the recombinant SlVPE3 was excised from the gel and used to immunize rabbits at the Beijing Protein Institute Co., Ltd. Polyclonal antibodies that recognized SlVPE3 was affinity-purified from antisera using the AminoLink Plus Coupling Resin following the purification protocol (Thermo Scientific).Wang et al. Genome Biology (2017) 18:Page 18 ofPolyclonal KTI4 antibodies were raised by injecting a rabbit with the synthetic peptide TYASVVDSDGNPVKAGAKYF followed by affinity-purification using the synthetic peptide.Immunoprecipitation of SlVPE3-interacting Biotin-VAD-FMK chemical information proteins for SWATH-MS analysisLeu/-Trp/-His/-Ade medium (SD/-4) containing X-Gal. As controls, AD and BD, KTI4-BD and AD, or SlVPE3-AD and BD were co-transformed. The experiments were repeated three times.Subcellular colocalizationProteins were isolated from 41 dpa tomato fruit using IP buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 NP-40, 50 M MG132, 1 mM PMSF, and the protease inhibitor cocktail tablet (Roche). After centrifugation at 12,000 ?g for 10 min, the supernatant containing the proteins was immunoprecipitated overnight at 4 with 50 g of anti-SlVPE3 or pre-immune serum IgG (negative control) that was coupled to an agarose support, as described in the Pierce?Co-Immunoprecipitation (CoIP) Kit (Pierce Biotechnology). The agarose beads were collected in spin columns and washed twice with IP buffer. The proteins were then eluted from the beads with 0.1 M glycine-HCl (pH 2.2), followed by reduction, alkylation, and digestion using the filter-aided sample preparation (FASP) method [79]. The resulting peptides were collected, dried under vacuum, and redissolved in 0.1 formic acid for NanoLC-MS/MS analysis. Quantitative analysis of SlVPE3-interacting proteins was performed using the SWATH-MS method [28]. Mass spectra were generated on a TripleTOF 5600 plus instrument (AB SCIEX) operating in the SWATH mode. Each sample contained three technical replicates and a t-test was used for statistical analysis. A P value <0.01 was considered to be significant.Y2H analysisFor subcellular localization analysis, the full-length SlVPE3 and KTI4 cDNAs were amplified by PCR (primer sequences in Additional file 13: Table S12) and individually cloned into the pCambia 2300-MCS-mRFP vector. The resulting plasmids were transformed PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25447644 into A. tumefaciens GV3101 [74], which was subsequently infiltrated into tobacco (N. benthamiana) leaves [83]. For colocalization analysis, N. benthamiana plants coexpressing mRFP-tagged SlVPE3 (SlVPE3-mRFP) and PRpHluorin-tagged KTI4 (KTI4-PRpHluorin) under the control of the CaMV 35S promoter were generated. The plasmid containing PRpHluorin was kindly provided by Dr. Liwen Jiang (School of Life Sciences, The C.