Ah109

Full-length coding sequences of the OsNF-YAs (OsNF-YA1, OsNF-YA2, OsNF-YA10, OsNF-YA11) genes were amplified by PCR using the primers listed in S1 Table and inserted into the bait vector pGBKT7. The full-length coding sequences of the OsMYCs (OsMYC2/3/4) and their different truncated variants were described in our previous reports [32 (link)]. Different combinations of plasmids were co-transformed into the yeast strain AH109 according to the product specification (Takara, Japan). The transformants were cultivated on SD/-Leu/-Trp plates for about 3 days, and then positive clones were transferred to SD/-Leu/-Trp/-His/-Ade plates for interaction tests. Yeast cells were photographed after 3 days at 30°C to record growth. All experiments were repeated three times with similar results.

Cep131-FL (1–1114 aa), Cep131-N1 (1–480 aa), Cep131-M1 (481–781 aa), Cep131-C1 (782–1114 aa), Cep162-N (1–447 aa), Cep162-C (448–897 aa), Cep290-N (1–887 aa), and Cep290-ΔN (888–1978 aa) were introduced into either pGBKT7 or pGADT7 vector. Clones in pGADT7 and pGBKT7 were transformed into yeast strain AH109 (Takara Bio). The yeasts were grown on SD-Leu-Trp plates at 30°C. After 3 to 4 days incubation, 2 independent positive colonies were picked and diluted with TE buffer (10 mM Tris-HCl, 1 mM EDTA (pH 7.5)), and then transferred to SD-Ade-Leu-Trp-His or SD-Leu-Trp-His plates with 3-amino-1,2,4-triazole and incubated for 5 days at 30°C.

The CDS sequences of TaABI5 and TaICE1 were cloned and full-length cDNA were respectively inserted into bait vector pGBDT7 and prey vectors pGADT7 for yeast two-hybrid experiments. As both of them are transcription factors with transcriptional activation activities, transcriptional activation experiments are indispensable. The recombinant pGBDT7:TaABI5 was transformed into strain AH109 (Takara, Shiga, Japan).
In the yeast two-hybrid experiment, recombinant plasmids pGADT7:TaICE1 and pGBDT7:TaABI5 were co-transformed into yeast strain AH109 and cultured on SD/-Trp-Leu medium for 60–80 h, then transferred to SD/-Trp-His-Leu-Ade medium with AbA (400 ng/mL) (Takara, Shiga, Japan) for 80–90 h.
The full-length TaABI5 sequence was inserted into the green fluorescent protein (GFP) vector. The pTaABI5:GFP vector was constructed using the above methods. Nicotiana benthamiana epidermal cells were infected by Agrobacterium-mediated. The transient expression of TaABI5 was detected using laser confocal microscopy.

Cells of A. tumefaciens GV3101 were transformed with the pGWB405 vector carrying GhNAC204 under the control of the CaMV 35 S promoter to produce GhNAC204 with a C-terminal green fluorescent protein (GFP) tag. The transformed GV3101 cells were cultured and used to infiltrate leaves of Nicotiana benthamiana. The infiltrated plants were incubated for 48 h; then, green fluorescence was visualized under a confocal laser scanning microscope as previously described [26 (link)]. For the assay of the transactivation activity of GhNAC204 protein, the pGBKT7-GhNAC204 recombinant plasmid and the pGBKT7 empty vector were both transformed into the yeast strain AH109 (Takara, Dalian, China). Transformants were grown on SD/-Trp medium for the selection of positive clones and then transferred to SD/-Trp/-His/-Ade medium (Takara, Dalian, China) for the transactivation assay at 30 °C for 3 days.

The transcription activation experiment was performed according to the methods used in previous research [36 (link)] (Yamaji et al., 2009). In order to further explore whether AGB1 affects the transcriptional activation of MYB62, we carried out transcriptional activation experiments in yeast cells (Figure 5B). MYB62 was inserted into pBridge vector to construct pBridge-MYB62, and MYB62 was fused with the binding domain of the GAL4 transcription factor (GAL4-BD), which can bind target sequences upstream of the reporter genes (histidine-deficient reporter gene) in yeast chromosomes. When MYB62 was inserted into pBridge, the reporter gene could be activated depending on the transcriptional activation activity of MYB62 (Figure 5B). The yeast transformed with the pBridge-MYB62 vector could grow on the screening medium. When AGB1 was inserted into another expression cassette of the pBridge-MYB62 vector to make pBridge-MYB62-AGB1, the effect of AGB1 on the transcriptional activation of MYB62 could be detected (Figure 5B). These constructed bodies were then introduced into the yeast reporter strain AH109 (Yeast Protocols Handbook; TaKaRa, Japan). The transformed yeast cells were selected on the selective medium (SD/-Trp, SD/-Trp-Ade, SD/-Trp-His-Ade) and the growth status was recorded.

A yeast one-hybrid system was employed to investigate transcriptional activation driven by SK using the reporter gene (HIS3) with the C-termini of SK1, SK2, and SKL1. These fragments were fused to the GAL4-DNA binding domain in pGBKT7 (Clontech-Takara Bio, Tokyo, Japan). The resulting plasmids were transformed into the yeast strain AH109 (Takara Bio, Tokyo, Japan). The yeast liquid cultures were diluted to an absorbance at 600 nm of 0.6, and 2 μL of each dilution were inoculated onto tryptophan- and histidine-negative synthetic dropout medium.