Two hybrid system 3

The transactivation analysis was performed using Matchmaker Two-Hybrid System 3 according to the manufacturer’s instructions (Clontech, Mountain View, CA, United States). Full length and truncated protein-coding regions of CbABF1 were amplified with specific primers harboring NdeI and SalI restriction sites (Supplementary Table S1, YH1 and YH393 for CbABF1, YH1, and YH89 for CbABF1Δ1, YH90, and YH393 for CbABF1Δ2). After double digestion, the resultant fragments were purified and sub-cloned into vector pGBKT7 which has a GAL4 DNA-binding domain to get fusion constructs (pGBKT7-CbABF1, pGBKT7-CbABF1Δ1, and pGBKT7-CbABF1Δ2). These constructs and empty pGBKT7 were separately co-transformed with pGADT7 into yeast strain AH109, and the transformants were cultivated at 30°C for 4 days on the SD/-Trp/-Leu or SD/-Trp/-Leu/-His medium supplemented with 35 mM 3-AT. Growth status evaluation and X-gal assay were performed to determine the transcriptional activation, as described in the manual.

Yeast two-hybrid assays was performed with the matchmaker Two-Hybrid System 3 (Clontech) using a modified set of plasmids compatible with Gateway technology and conditions specified by the manufacturer. MKKs and chimeras were cloned as DNA Binding Domain fusions and MPKs were cloned as Activation Domain fusions. Three independent yeast colonies were tested for each pairwise comparison at 1, 1:10 and 1:100 dilutions after incubation of 2, 3 and 4 days in plates containing 1 mM 3-amino-1/2/4-triazole. Experiments were repeated three times with yeast cultures at OD600 of 1, 2 and 4. Interactions were evaluated as positive if significant growth was detected in 1:100 dilution at day 3.

The Clontech Matchmaker Two-Hybrid System 3 was used. For library screening, binding domain (BD)-PTLΔC1 bait (plasmid pGBKT7) with the C-terminal AD removed (Kaplan-Levy et al., 2014 (link)) in cells of yeast strain Y187 were mated with the AD-library prey (plasmid pGADT7) in strain AH109, and approximately 4×10⁷–1×10⁸ diploids selected for each mating. 3-Amino-1,2,4-triazole (3-AT; Sigma) was included to suppress leaky growth of His3p and any activation by bait sequences, with the required concentration determined by trial runs. A concentration of 2.5mM 3-AT was found to be sufficient to inhibit auto-activation by full- length BD–PTL. For pairwise interaction tests, a full-length clone of the protein-coding sequence of AKIN10 (At3g01090.2) was obtained from the Arabidopsis Biological Resource Center (U24028; see Supplementary Table S1 at JXB online for primer sequences). Full-length AKIN11 coding sequence (At3g29160) was obtained from the inflorescence cDNA library by reverse transcription (RT)-PCR and 5′-rapid amplification of cDNA ends. Truncations and deletions of PTL used to locate sites of interaction have been described elsewhere (Kaplan-Levy et al., 2014 (link)). Activation was quantified using triplicate technical assays of α-galactosidase activity of the MEL1 reporter in liquid cultures.