Phis2 vector

The CDS of MdAGO4‐1/2 was recombined into the pGADT7 vector (Clontech, Palo Alto, CA), and the MdMYB1 promoter sequence was inserted into the pHIS2 vector (Clontech). The primers used to amplify the CDS and promoter fragments are listed in Table S1. To determine the suitable concentration of 3‐amino‐1,2,4‐triazole(3‐AT) to suppress background histidine leakiness of the pHIS2 vector, the yeast strain Y187 transformed with each recombinant pHIS2 vector was grown on ‐Trp/‐His (‐T/‐H) media containing 3‐AT at different concentrations. The interactions between the MdAGO4‐1/2 and four promoter fragments were detected on ‐Trp/‐Leu/‐His (‐T/‐L/‐H) medium containing 3‐AT at the suitable concentration for each vector construct. The empty pGADT7 vector was used as the control.

For the Yeast-One-Hybrid assay with a 2CPA promoter bait the previously described pONE1-derived construct was used, which expresses HIS3 under the control of GAL1, 10 minimal promoter, if transcription factors bind to the promoter [10 (link)]. For tAPx promoter reporter plasmid construct, the promoter was divided into two parts, which overlap 110 bp. tAPx-I (−868 to −227) and tAPx-II (−337 to +41) and the sAPx promoter (−908 to −33) were cloned into the pHIS2 vector (Clontech) upstream of the auxotrophic marker HIS3 and the GAL4 minimal promoter. The yeast strain Y187 was transformed with the bait constructs. Prey constructs were generated by cloning the coding sequences of the RAP2.4 transcription factors into the pACT2 vector downstream of the cDNA for the GAL4 activation. Prior to the interaction analysis on SD media lacking leucine (LEU), tryptophan (TRP) and histidine (HIS), the bait strains were co-transformed with empty pAct2 vectors. On 0–100 mM 3-AT, the constructs were tested for autoactivation and suppression of autoactivation by yeast proteins. Afterwards, the yeast-one-hybrid analysis of the RAP2.4 transcription factors was performed on 3-AT concentrations guaranteeing specificity. Colonies were re-assayed on the same auxotrophic medium for interaction confirmation.

The CDSs of PheIDD21α and PheIDD21β were cloned into the prey vector (pGADT7), and the 230–340-bp promoter fragments of IDD-binding sites (four putative elements in PH02Gene17121, three putative elements in PH02Gene35441,and two putative elements in PH02Gene11386) were amplified and ligated into the pHIS2 vector (Clontech). The corresponding pGADT7 and pHIS2 constructs were co-transformed into yeast strain Y187 following the manufacturer’s instructions for the yeast one-hybrid library screening system (Clontech). Interaction of pHIS53 with pGADT753 was used as a positive control, and the empty AD and pHIS-pro-frag were used as negative controls. The transformed strains were cultured at 30 °C for 2–3 days on SD/-Trp/-Leu media and were used for spot assays on SD/-Trp/-Leu/-His media with 10 mM 3-AT.The primers are listed in Table S3.

The coding sequence of MdMYC2 (2097 bp) was ligated into the pGADT7 vector. A MdD1 promoter fragment (30 bp) was ligated into the pHIS2 vector (Clontech). All constructs were transformed into yeast strain AHY187 by the lithium acetate method. The Y1H assay was conducted as described according to the manufacturer's instructions. All primers used are listed in Additional file 13: Table S2.

The full-length coding sequence of OsWRKY54 was amplified by PCR and introduced by ligation into the pGBKT7 vector, generating the pGBKT7-OsWRKY54 construct to investigate the transactivation activity of OsWRKY54. The empty vector pGADT7 plus the pGBKT7 or pGBKT7-OsWRKY54 construct was co-introduced into AH109 yeast cells. The yeast colonies were grown on the plates containing SD/Trp- or SD/Trp-/His-/Ade- medium and incubated at 30 °C.
A yeast one-hybridization assay was employed to determine the binding ability of OsWRKY54 to the OsHKT1;5 promoter. A 2036-bp promoter sequence of OsHKT1;5 was cloned in front of the HIS3 reporter gene in pHIS2 vector (Clontech, Shiga, Japan), generating the pHIS-ProOsHKT1;5 construct. The full-length OsWRKY54 was cloned in-frame after the GAL4 activation domain in pGADT7 (Clontech) to produce the pGAD-OsWRKY54 construct. A pair of these plasmids (pGAD-OsWRKY54 and pHIS2-ProOsHKT1;5, or control pGADT7 and pHIS2) was transformed into yeast strain AH109. The yeast colonies were spotted on SD/-Leu/-Trp and SD/-Leu/-Trp/-His medium containing 0–75 mM 3-amino-1,2,4-triazole and cultured at 30 °C for 3 days. The primers used for constructing the vectors are listed in Supplemental Table S2.

The MdGSTF6 promoter sequence (1515 bp) was inserted into the pHIS2 vector (Clontech, Palo Alto, CA, USA), and the coding sequence (CDS) of MdMYB1 was cloned into the pGADT7 vector (Clontech). The primers used to amplify the CDS of MdMYB1 and promoter of MdGSTF6 are listed in Table S2. To determine the suitable concentration of 3-AT to suppress background histidine leakiness of the pHIS2 vector, the recombinant pHIS2 vectors transformed into the yeast strain Y187 were grown on −Trp/−His (−T/−H) medium containing different concentrations of 3-AT. The empty pGADT7 plasmid was used as the control.

Two tandem copies of the NACRS with the core sequence “CGT[A/G]” and calmodulin-binding NAC (CBNAC) binding site with the core sequence “GCTT” were separately inserted into the pHIS2 vector (Clontech, USA) as the reporter constructs. The coding region of ThNAC13 gene was cloned into the pGADT7-Rec2 vector as the effector construct. The effector construct was co-transformed together with each reporter construct into the yeast strain Y187. The DNA-protein interactions were evaluated according to the growth ability of the cotransformants on SD medium lacking leucine (Leu) and tryptophan (Trp; SD/-Leu/-Trp, DDO) and SD medium without leucine (Leu), tryptophan (Trp) and histidine (His; SD/-Leu/-Trp/-His, TDO) containing 3-AT (3-amino-1,2,4-triazole).