Matchmaker yeast two hybrid system

The Y2H assays were conducted using the MatchMaker yeast two-hybrid system (www.clontech.com). The ORF regions of TaCBL and TaCIPK were respectively sub-cloned into pGBKT7 and pGADT7 vectors and co-transformed into the Y187 yeast strain. The transformants were grown on double-dropout medium (DDO: SD/-Trp/-Leu) and selected on triple-dropout medium (TDO: SD/-Trp/-Leu/-His) containing 10 mM 3-amino-1, 2, 4-triazole (3-AT). For BiFC assays, the ORF regions of TaCBL and TaCIPK were sub-cloned into 35S-SPYCE and 35S-SPYNE vectors, respectively. After confirmation by sequencing, these constructs were separately transformed into Agrobacterium tumefaciens GV3101, then into tobacco leaves by Agrobacterium infiltration. Freshly transformed Agrobacterium cell cultures were re-suspended in suspension medium (10 mM MES-KOH (pH 5.6), 10 mM MgCl₂, and 0.1 mM acetosyringone), adjusted to an OD₆₀₀ of 0.5-0.8, and left at room temperature for 3 h before infiltration into tobacco leaves. Infiltrated leaf discs were collected 3–5 d later for observation under a confocal microscope. The TaCBLs and TaCIPKs primers used for vector construction are listed in Additional file 2: Table S1.

The Matchmaker yeast‐two‐hybrid system (Clontech, Kusatsu, Japan) was used to study the interaction of OsCUC1, OsCUC3, OMTN4 and OMTN6 with CLD1. The deduced amino acid sequences of OsCUC1, OsCUC3, OMTN4 and OMTN6 were separately cloned into pGADT7 (AD) vectors, while CLD1 was inserted into pGBKT7 (BD). Yeast strain Y2HGold was transformed with bait plasmid and strain Y187 was transformed with prey plasmid. Co‐transformants were plated on synthetic defined (SD)/–Leu/–Trp/–His/–Ade medium plates and SD/–Leu/–Trp/–His/–Ade/X‐α‐Gal medium plates for examination of growth. Primer sequences for the constructions are listed in Table S2.

For testing direct protein-protein interactions, the Matchmaker Yeast Two-Hybrid System (Clontech) was used according to the manufacturer’s instructions. To that end, ORFs of interest (TUT3, 4EIP, and Dis3L2) were PCR-amplified from genomic DNA and cloned into both pGBKT7 and pGADT7 plasmids. Prey and bait plasmids were co-transformed pairwise into AH109 yeast strains, and selected initially on double drop-out (DDO) plates (i.e., SD medium lacking Trp and Leu) or quadruple drop-out (QDO) plates (i.e., lacking Trp, Leu, His and Ade). Growth on QDO plates indicated positive interactions. The interaction between p53 and SV40 large T-antigen and the combination of LaminC and SV40 large T-antigen served as positive and negative controls, respectively. Western blotting was used to confirm expression of c-myc-tagged BD-domain proteins and HA-tagged AD-domain proteins.

To validate homodimer formation of the EF domain, a yeast two-hybrid assay was performed using the Matchmaker Yeast Two-Hybrid System (Clontech) following the manufacturer’s instructions. The cDNA encoding the EF domain was cloned into the AD and BD vectors. The recombinant plasmids were cotransformed into AH109 cells and their growth examined on DDO and QDO medium. The pGADT7-T/pGBKT7-53 (AD-T/BD-53) plasmid was used as a positive control, and pGADT7/pGBKT7 (AD/BD) was used as a negative control.

To confirm the interaction between TET1 and TDG, yeast two-hybrid assay was performed using he Matchmaker yeast-two hybrid system (Clontech). TET1 was divided into four overlapping fragments (TET1-1 aa 1–491; TET1-2 aa 397–931; TET1-3 aa 870–1403; TET1-4 aa 1367–2057) that were cloned into the BD (pAS2.1 BD FLAG) of the Gal4 protein and TDG was cloned into the AD (pACT2 AD) of the Gal4 protein. The Saccharomyces cerevisiae strain AH109 was co-transformed with 50–500 ng of bait and prey plasmids according to the Clontech manual. Interactions were assessed by spotting serial dilutions of cells on selective medium (SC-LEU-TRP-ADE-HIS) supplemented with 2.5 mM 3AT (3-Amino-1,2,4-triazole), a competitive inhibitor of the HIS3 gene product. Cells were incubated at 30 °C for 6–7 days.