Yeast two hybrid assay

The full-length cDNAs for ZmbHLH23, ZmbHLH61, ZmbHLH114, ZmbHLH163, and ZmbHLH180 were generated by PCR amplifying cDNA from the maize inbred line B73. All the primers used are listed in Additional file 1: Table S1. The amplified full-length fragments of ZmbHLH23, ZmbHLH114, and ZmbHLH180 were ligated into the pGADT7 and pGBKT7 vectors (Clontech). ZmbHLH61 was ligated into the pGBKT7 vector. ZmbHLH163 was ligated into the pGADT7 vector. The yeast two-hybrid assay was performed according to the manufacturer’s instructions (Clontech).

The open reading frames (ORFs) of three CrASRs were cloned into the vector pGBKT7 (Clontech, Mountain View, CA, USA) for transcription activation analysis with the yeast two-hybrid assay according to the manufacturer’s introduction (Clontech, CA, USA). In brief, the full CDSs of CrASRs were amplified from C. rosea leaves cDNA using the corresponding primers as listed in Supplementary Materials Table S1. Then all PCR products were inserted into GAL4-DBD vector pGBKT7 at EcoRI site and sequenced. These constructs along with the negative control pGBKT7 (containing the binding domain, BD) were transformed into yeast strain AH109 using the LiOAc/PEG method. The yeast clones were cultured in liquid SD-Trp medium to an OD600 value of 2, after which they were diluted using a gradient dilution (1:10, 1:100, and 1:1000). Two-microliter yeast cultures were spotted onto the corresponding synthetically defined (SD/-Trp and SD/-Trp/-Leu) medium plates for 2 days at 30 °C. Yeast transformation and determination of blue/white colonies were conducted according to the instructions of the manufacturer (Clontech), and X-α-Gal was used as a substrate for the reporter gene MEL1. The primers used in construction of pGBKT7 vectors for CrASRs transactivation activity assay in yeast are listed in Supplementary Materials Table S1.

For yeast two-hybrid (Y2H) analysis, the CDS for OsMADS57 and SLR1 were cloned into the pGBKT7 and pGADT7 vectors, respectively. Detecting the interaction between OsMADS57 and SLR1 in yeast was performed. The yeast two-hybrid assay was conducted following the manufacturer’s instructions (Clontech). Primers used for these constructs are listed in Additional file 5: Table S1.

The yeast two-hybrid assay was performed according to the manufacturer’s protocol (Clontech, Palo Alto, CA, USA). Briefly, HIV-1 and SIVpbj1.9 nef genes in a pLexA-binding domain (BD) fusion vector (His+) and a Jurkat cDNA library expressed in a pB42-activation domain (AD) fusion vector (Trp+) were introduced into yeast strain EGY48 by co-transformation, and positive colonies were screened twice to eliminate false positives. pB42AD-cDNA plasmids were then recovered from positive colonies, sequenced and introduced into EGY48/p8op-lacZ/nef by transformation to confirm the interaction with HIV-1 and SIVpbj1.9 Nefs. Except for the cells, the mammalian two-hybrid assay was performed essentially the same as the yeast two-hybrid assay. Briefly, nef expressers in a pM-BD fusion vector (Clontech) and UBE3A in a pVP16AD fusion vector were introduced by co-transfection into NIH 3T3 cells with a reporter gene, pG5CAT, and pCMV-β-gal to control for transfection efficiency. Three days after transfection, chloramphenicol acetyltransferase (CAT) enzymatic activity was measured as per the manufacturer’s protocol (Clontech).

The CDS of LOG was amplified and inserted into the prey vector pPR3-N. The CDS of OsDES1 was amplified and cloned into the bait vector pBT3-SUC. A yeast two-hybrid assay was performed according to the manufacturer’s instructions (Clontech). The DUALmembrane system was used for conducting the assays. All primers are listed in Supplementary Table S1.