Pgadt7 rec

For protein expression studies, we used pRSET-A and pFN2A Flexi vectors (Thermo Scientific) with competent BL21(DE3) and DH5α cells (NEB, USA). pGAD-T7 Rec (Clontech) was used for the cDNA library preparation, pGADT7-AD (Clontech) as prey vector for one to one assays, and were then transformed in Y2H Gold yeast cells. pGBKT7 (Clontech) was used as a bait vector (DNA-BD) and was transformed into Y187 yeast cells.

For protein expression studies, we used pRSET-A and pFN2A Flexi vectors (Thermo Scientific) with competent BL21(DE3) and DH5α cells (NEB, USA). pGAD-T7 Rec (Clontech) was used for the cDNA library preparation, pGADT7-AD (Clontech) as prey vector for one to one assays, and were then transformed in Y2H Gold yeast cells. pGBKT7 (Clontech) was used as a bait vector (DNA-BD) and was transformed into Y187 yeast cells.

Full-length and selected domains of Rfg1 cDNA were amplified from C08 and W05 and were subcloned into pGBKT7 (Clontech, United States). nopP coding sequences were amplified from both CCBAU25509 and CCBAU45436 and were cloned into pGADT7-rec (Clontech, United States). Primers for the amplification of target sequences can be found in Supplementary Table S2. The pGBKT7 and pGADT7-rec constructs were transformed into Y2H gold and Y187 (Takara Bio, Inc., Japan), respectively, using lithium acetate/polyethylene glycol method (Gietz and Schiestl, 2007 (link)). Yeast mating and interaction analyses were done according to Yeast Protocols Handbook (Clontech, United States).

Putative MYB cis‐element was identified in the promoter region of the ADC gene (Pbr022368.1) based on the pear genome sequence (http://peargenome.njau.edu.cn/). Yeast one‐hybrid assay was performed to investigate whether PbrMYB21 could interact with the MYB cis‐element. The full‐length ORF of PbrMYB21 was amplified by PCR using primers (GSP7, Table S1) and integrated into the BamHI and NcoI sites of pGADT7‐Rec (Clontech) to create a prey vector (pGADT7‐PbrMYB21). Based on the distribution of the MYB cis‐element, one fragment (P1, ‐673 to ‐668 bp) was PCR amplified using primers (GSP8, Table S1) containing SmaI and XhoI restriction sites (ADC‐P1) and cloned into the pAbAi vector to construct the bait. Both the effector vector and reporter vector were co‐transformed into yeast strain Y1H Gold following the manufacturer's instructions (Clontech). The transformed cells were spread on SD/‐Ura/‐Leu medium added with (0 or 300 ng/mL) AbA, and incubated for 3 days at 30 °C. Both positive (pGAD‐p53+ p53‐AbAi) and negative (pGADT7‐AD + P1) controls were included and processed in the same way.

Putative HSE cis-elements were identified in the promoter region of OsADC (LOC_Os08g33620) and AtADC1(At2g16500). Yeast one-hybrid assay was performed to investigate whether OsHSFA3 could bind with these HSE cis-elements in the promoters of OsADC and AtADC1. The full-length ORF of OsHSFA3 was amplified by PCR using gene-specific primers (Table S1) and cloned into pGADT7-Rec (Clontech, Mountain View, CA, USA) to create a prey vector (pGADT7-OsHSFA3). About 2000 bp promoter sequences of OsADC and AtADC1 genes were respectively cloned into the bait vector. The OsHSFA3 prey vector was cotransformed with either the OsADC or AtADC1 bait vector into yeast strain Y187. The cotransformed yeasts containing the bait and prey were cultivated on the SD/-Leu/-Trp/-His selective media supplemented with 0 or 3 mM 3-amino-1,2,4-triazole (3-AT) for 3 days according to the instructions for the Matchmaker^TM Gold Yeast One Hybrid System (Clontech, USA). Yeasts cotransformed with pGADT7-Rec2-53 (the pGADT7-Rec2-53 plasmid containing a murine p53 fused to GAL4 AD domain) and p53HIS2 (the p53HIS2 harboring three tandem copies of p53-binding DNA elements) were used as the positive control. The negative control was pGADT7-Rec2-OsHSFA3 and p53HIS2 cotransformation. The interaction between prey and bait was observed according to the growth of the yeast transformants in a series of 10-fold dilutions.

The full-length coding cDNA sequences of ScAP3-A, ScAP3-B, ScPI-A, and ScPI-B genes containing restriction sites at the 5’and 3’ ends were introduced into the GAL4-based yeast two-hybrid vectors pGBKT7 (Clontech) and pGADT7-Rec (Clontech) and co-transformed into the AH109 yeast strain by the LiAc/DNA/PEG method according to the Yeast Protocols Handbook from Clontech (http://www.clontech.com).