Pjg4 5 vector

The MdARF8, MdARF26, and MdARF27 CDSs were inserted into the pJG4-5 vector (Clontech, USA), and the MdSAUR76 promoter (full-length, −2001 to −33 bp; P1 fragment, −2001 to −1850; P2 fragment, −467 to −341; P3 fragment, −341 to −258) and MdLBD16 promoter were cloned into the pLacZi vector. The constructed fusion and empty vectors were introduced into yeast strain EGY48 (Weidi Biotechnology 86 Co, Ltd., Shanghai, China). The Y1H assay was conducted as previously described [67 (link)]. The yeast cells were grown on SD/-Leu/-Trp-deficient medium at 28 °C for 3 days.
For the Y2H assay, MdIAA27T and MdIAA27C CDSs were cloned into pGBKT7 and MdARFs CDSs were cloned into pGADT7 vectors. All constructs and empty vectors were introduced into the yeast strain Y2H (Weidi Biotechnology 86 Co, Ltd., China), as described in the Yeast Protocols Handbook (Clontech, Shanghai, China). Yeast cells were cultured on a minimal Leu and Trp medium for 3 days at 28 °C and then plated onto the SD (-Leu, -Trp, -His, and -ade) medium to analyze possible interactions. Primers are listed in Supplementary Table S1.

To generate AD‐GbBM, the full‐length GbBM was amplified using KOD polymerase (Toyobo, Japan), and subcloned into the EcoRI and XhoI sites of the binary vector pJG4‐5 vector (Clontech). To generate GbPALp::LacZ, Gb4CLp::LacZ, GbCHSp::LacZ, GbCHIp::LacZ, GbF3Hp::LacZ, GbFLSp::LacZ, GbDFRp::LacZ, GbANSp::LacZ, and GbUFGTp::LacZ, reporter constructs, the promoter fragments were amplified by PCR and then separately cloned into the pLacZi2μ vector. The primer sequences are listed in Table S2. Transformants were grown on proper drop‐out plates containing X‐gal (5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐ galactopyranoside) for blue color development. Representative data was shown from one of the three biological replicates, which yielded similar results.

The 2-kb promoter regions (upstream of start codon) of CzDGAT1A and CzDGTT5 were individually cloned into pLacZi2μ (Clontech), while the coding sequences of TFs including eight MYBs and six bZIPs were each cloned into pJG4-5 vector (Clontech). The yeast one-hybrid assay was performed by co-introducing pLacZi2μ-promoter and pJG-TF into the yeast strain EGY48, as described in the Yeast Protocols Handbook (Clontech). Transformants were grown on the synthetic dextrose plates lacking Ura and Trp, but containing X-gal (5-bromo-4-chloro-3-indolyl-b-dgalactopyranoside). The plates were incubated at 30 °C for 2 days for color development.