Matchmaker gold two hybrid system

Yeast two-hybrid assay (Y2H) was performed using the Matchmaker® Gold Two-Hybrid System (Clontech Laboratories, Inc., USA). Briefly, GmMYB176 was cloned into the vector pGBKT7 as the bait. The cDNA library as prey was generated by SMART™ cDNA Synthesis technology (Clontech Laboratories, Inc., USA) from soybean embryos (50–60 days after pollination) and fused to GAL4 activation domain. Screening was performed by co-transformation of bait and prey using Mate & Plate^TM library system (Clontech Laboratories, Inc., USA). After transformation, yeast cells were spread on SD/Ade/-His/-Leu/-Trp agar plates, and incubated at 30 °C for 5 days.

The full-length coding regions of the GIF genes were amplified by PCR using gene-specific primers containing homologous recombination sites and were cloned into the bait vector pGBKT7. The full-length coding regions of the GRF genes were cloned into the prey vector pGADT7 by gene-specific primers containing homologous recombination sites. Each pair of bait–prey vectors was co-transformed into the yeast strain AH109 following the instructions of Matchmaker Gold Two-hybrid System (Clontech, Mountain View, CA, USA). The transformed yeasts were plated on an SD medium lacking leucine and tryptophan (SD/-Trp-Leu). After the yeast cells grew at 30 °C for 3–4 days, colonies were picked and transferred to an SD medium lacking leucine, histidine, adenine, and tryptophan (SD/-Trp-Leu-His-Ade). The yeast concentrations were estimated by measuring their optical densities at 600 nm. These were maintained at the same concentration (OD₆₀₀:1) for protein interactions assay. The strength of the interaction depended on the yeast growth conditions [19 (link)]. The combination of SlGIFs introduced into the pGBKT7 vector and the empty pGADT7 vector were used as negative controls, as well as the combination of empty pGBKT7 and SlGRFs introduced in the pGADT7 vector. pGBKT7-53 and pGADT7-RceT were used as positive controls The primers for the yeast two-hybrid assays are presented in in Table S1.

After treating the samples with 30% PEG6000 (W/V) for five hours, we extracted the total RNA in the peanut roots, stems, and leaves to construct the yeast cDNA library. The cDNA fragments were ligated to pGADT7 plasmid and stored in yeast cells. Full-length AhHDA1 cDNA was amplified using PCR and the following primer pairs: AhHDA1-EcoRI-F: 5′-TACGAATTCATGGGGATAGAAGAAGAGAG-3′, and AhHDA1-SacI-R: 5′-GAGCTCTCAGCAGCATCCATGTGG-3′. The PCR fragment was ligated into the vector pGBKT7 (Clontech, Mountain View, CA, USA) after cleavage by the restriction endonucleases Eco RI and Sac I (New England Biolabs, Ipswich, MA, USA). After all cloned fragments were transformed and screened using SD/Trp ⁻/Leu ⁻/His ⁻ selective medium, they were checked by PCR and sequenced. We performed the two-hybrid assays using the Matchmaker™ Gold two-hybrid system (Clontech) following the manufacturer’s instructions (Liu et al., 2016a (link)).

Protein interactions in vivo in yeast were assayed using the ‘Matchmaker Gold Two-hybrid System’ (Clontech) using the protocols provided by the supplier. Diploids formed by mating the ‘bait’- and ‘prey’-containing strains were spotted by serial dilution on appropriate selection media to test for the expression of reporter genes.

Y2H assays were performed using the Matchmaker Gold Two-Hybrid system (Clontech). The full-length CDS fragments of CcFL01489, CcFL03547, CcFL06843, CcFL09745, and CcFL13362 were amplified from C. chingii cDNA. These fragments were then fused as BamHI/EcoRI fragments into pGKBT7 (BD) and pGADT7 (AD) using the Basic Seamless Cloning and Assembly Kit (TransGen, Beijing, China) to generate CcFL01489-BD (bait vector), CcFL03547-AD (prey vector), CcFL06843-AD (prey vector), CcFL09745-AD (prey vector), and CcFL13362-AD (prey vector), respectively. The bait vector and prey vector were co-transformed into the Y2H Gold strain performed as previously described [73 (link)], with the empty vector as a negative control. Positive transformants were selected on SD/-Trp/-Leu dropout medium and further screened on SD/-Leu/-Trp/-His/-Ade + X-α-gal (40 μg mL⁻¹) dropout medium. The cultures were then incubated at 30 °C for 3 days. The primers used in the Y2H assay are listed in Additional file 2: Table S8.