Hifi dna assembly master mix

E. coli JM109 was used for all cloning steps. PCR steps were performed using Q5 polymerase. Initial plasmid construction for reporter gene expression was performed by conventional restriction digest – ligation protocols. Promoter and reporter gene sequences were amplified with primers listed in Additional file 1: Table S1. For the construction of ACS1, TUP1, FLC2 and open reading frame PAS_chr3_0669 overexpression strains, genes were PCR amplified with gene-specific primers using Q5 polymerase and ligated into EcoRI-NotI linearized pGAPzB vector using a HIFI DNA assembly mastermix (New England Biolabs). Constructs were verified by Sanger sequencing and after linearization with MfeI used for transformation of competent P. pastoris X-33, BG10 and BG10 ∆OCH1 cells. Q5 polymerase, T4 DNA ligase and HIFI DNA assembly mastermix were obtained from New England Biolabs. Primers used for amplification are listed in Additional file 1: Table S1. Growth tests for overexpressing and corresponding empty vector control strains were performed as described above.

Complementary site-directed mutagenesis primers were designed to alter the N of the antisense NGG PAM site at residue 104 of the firefly luciferase gene. PAM site mutagenesis targeted the wobble base of proline to ensure silent mutations. Mutagenesis PCR was performed using NEB Q5 High Fidelity Polymerase (cat. #M0492L). PCR products were digested with 1 μL of DpnI for 1 h at 37 °C. Samples were then Gibson assembled using NEB HiFi DNA Assembly Master Mix (cat. # E2621L) before transformation into NEB DH5α Chemically Competent E. coli (cat. # C2987H). All complete plasmids were confirmed by PCR and Sanger sequencing.

To express proteins with a desired tag protein at the N-terminus, the SpeI-sGFP (lacking the stop codons)-StuI-XbaI sequence was introduced into the XbaI-SacI site of pE2113_GW_SAS^{58 (link)} using NEBuilder HiFi DNA Assembly master mix, resulting in a pE2113_n_sGFP vector. For the co-IP experiment, the coding sequence of 3xFLAG was amplified by PCR using 3xFLAG/pCAMBIA as a DNA template^{64 (link)}, and the amplified FLAG sequences were inserted into the SpeI-StuI site of pE2113_n_sGFP using NEBuilder HiFi DNA Assembly master mix to generate the pE2113_n_FLAG vector. The coding sequence of GF14h was PCR-amplified and cloned into the XbaI-SacI site of pE2113_n_FLAG. HA-GF14h and cMYC-MFT2 were first amplified by PCR using GF14h/pGBKT7 and MFT2/pGADT7 plasmids as templates, HA-GF14h or cMYC-MFT2 were cloned into the SpeI-SacI site of OREB1/pE2113_n_FLAG.

A yeast system was utilized to examine the transcriptional activity of CsMYB15 according to a previously described method (Ma et al., 2009 (link)). The coding sequences of CsMYB15 were cloned into the bait vector pGBKT7 by homologous recombination method using HiFi DNA Assembly Master Mix (NEB, Ipswich, USA) according to the manufacturer’s protocol. The CsMYB15-pGBKT7 + pGADT7-AD, pGBKT7-lam + pGADT7-T (negative control), and pGBKT7-53 + pGADT7-T (positive control) were transformed into Y2H strain yeast independently. After being cultured on SD/-Trp medium at 30 °C for 3 days, single colonies were selected and further amplified. Subsequently, the positive transformants were grown on SD/-Leu/-Trp and SD/-Leu/-Trp/-His/-Ade media, and the growth of all the transformants were observed after 3 days. Additionally, the galactosidase assay was performed by adding X-α-Gal to the medium according to a previously described method (Ma et al., 2009 (link)).

Construction of S. epidermidis O47 ΔsadA was carried out using plasmid pBASE6^{64 (link)}. The 1 kb upstream downstream region of sadA were amplified using appropriate primers (Supplementary Table 2) and ligated with linearized pBASE6 (EcoRV restriction site) using Hi-Fi DNA Assembly Master Mix (New England Biolabs) and transformed into E. coli C2987 (New England Biolabs) and then into E. coli DC10B chemically competent cells. Colonies with the desired plasmid were picked and the respective plasmid was transformed into S. epidermidis O47 by protoplast transformation. Mutagenesis was conducted as reported by^{65 (link)}. Mutants were confirmed using PCR for the gene deletion and HPLC analyses for the overnight supernatants.

Expression plasmids were constructed using pET28a (Novagen). pET28a was linearized, using the restriction enzymes NotI and NcoI to add a histidine tag to the C-terminal end, and ligated with either of the 3 genes encoding SPSE_1761, SPSE_0802 or SPSE_0436 amplified from S. pseudintermedius ED99 genome (oligonucleotides listed in Supplementary Table S2). The ligations were performed using Hi-Fi DNA Assembly Master Mix (New England Biolabs), then transformed into E. coli DC10B by heat shock method. The colonies grown on selective agar media containing kanamycin were confirmed by plasmid isolation and sequencing. The plasmids were then transformed into the expression host E. coli BL21 (DE3). The clones containing the correct plasmid were cultured overnight at 25°C in LB supplemented with kanamycin. The cells were harvested and lysed using 0.1 mm glass beads in FastPrep instrument (MP Biomedicals). The cell lysate was centrifuged (15,000 g, 30 min, 4°C) and the proteins in the supernatant were subjected to protein purification using Ni-NTA superflow resin (IBA). The expression and purification steps were verified by 14% SDS-PAGE. The protein concentration was determined by the Bradford method using a protein assay dye (Bio-Rad, Hercules, CA, United States).