Yeast protocols handbook

The N.benthamiana leaves expressing the interested proteins were respectively harvested 48 hours post transformation. Protein extraction and immunoblotting analysis with corresponding antibodies as indicated were performed according to the method described previously [105 (link)].
For determination of the protein express in yeast, the total protein extract was prepared and analyzed with anti-Myc and anti-HA antibodies, separately, following the Yeast Protocols Handbook from Clonetech (Otsu, Shiga, Japan).

The yeast reporter strain PCY2 pex14Δ was transformed with two-hybrid plasmids pPC86 and pPC97 (Chevray and Nathans, 1992 (link)) or derivatives thereof and grown on synthetic medium lacking tryptophan and leucine for 3 days at 30°C. ß-galactosidase activity of transformed cells was determined by a filter assay as described before (Rehling et al., 1996 (link)) using X-Gal as substrate. For quantification, β-galactosidase activity was determined by performing a liquid assay using 2-nitrophenyl β-D-galactopyranoside as substrate according to the manufacturer’s instructions (Clonetech, Yeast Protocols Handbook, 2009).

For interaction analysis of TaCIPK10 and TaCBLs or TaNH2, the MatchMaker yeast two‐hybrid system was performed (Takara, Dalian, China). The coding sequences of TaCBLs, TaNH2, TaNH2‐ΔAKR and the AKR motif of TaNH1/2/3 were subcloned into pGADT7 (activation domain, AD), the coding sequences of TaCIPK10 and TaCIPK10Δ were subcloned into pGBKT7 (DNA‐binding domain, BD) vector. The pairs of recombinant plasmids of AD and BD were co‐transformed into yeast strain AH109 following the Yeast Protocols Handbook (Takara, Dalian, China). Protein expression in yeast was confirmed by Western blots following a previous study (Wang et al., 2016).

The yeast two‐hybrid assay was carried out as described previously (Wu and Li, 2017). The TaDA1‐A and TaGW2‐B CDSs were amplified and sub‐cloned into the pGBKT7 and pGADT7 vectors, respectively, to generate the TaDA1‐A‐BD and TaGW2‐B‐AD plasmids. After the co‐transformation of both plasmids into yeast AH109 cells, the interaction between the expressed proteins was determined by the growth of the co‐transformants on a selection medium (SD/‐Trp/‐Leu/‐His/‐Ade), following the Yeast Protocols Handbook (Takara Bio).
The LCI assay for the interaction between TaDA1‐A and TaGW2‐B was performed in N. benthamiana leaves as described previously (Liu et al., 2017). The full‐length TaDA1‐A and TaGW2‐B CDSs were fused with the N‐terminal and C‐terminal regions of the LUC reporter gene, respectively (TaDA1‐A‐nLUC and cLUC‐TaGW2‐B), and transformed into Agrobacterium tumefaciens strain GV3101. Agrobacteria harbouring the nLUC and cLUC derivative constructs were co‐infiltrated into N. benthamiana, and the LUC activity was imaged and analysed 48–72 h after infiltration using the NightSHADE LB 985 Plant Imaging System (Berthold Technologies, Bad Wildbad, Germany).

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.