Yeast two-hybrid (Y2H) assays were used to determine the direct interaction between paired proteins using a Matchmaker™ GAL4 Two-Hybrid System 3 Kit (Clontech Laboratories, Mountain View, CA, USA) [26 (link)]. All primers are listed in Table S1 . In brief, BbPEX6 and BbPEX26 were amplified and cloned into the vector pGBKT7, whereas BbPEX1 and BbPEX6 were cloned into the vector pGADT7. Paired plasmids were transformed into yeast strain YH109, and the resultant transformant was screened on SD/Leu–Trp medium. The positive interaction was determined when the transformants grew well on SD/Trp–Leu–His–Ade plates. Strains for positive and negative control were provided by the kit.
Matchmaker gal4 two hybrid system 3 kit
Manufactured by Takara Bio
Sourced in United States
The Matchmaker GAL4 Two‐Hybrid System 3 kit is a tool for studying protein-protein interactions. It provides a set of plasmids and yeast strains to identify novel protein interactions using a yeast-based system.
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7 protocols using matchmaker gal4 two hybrid system 3 kit
1
Yeast Two-Hybrid Protein Interaction
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Yeast Two-Hybrid System for Protein Interaction Analysis
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The Matchmaker GAL4 Two‐Hybrid System 3 kit (Clontech) was used. Activation domain (AD)‐fused AP1 and SEP1, SEP2 (aa 87–220), SEP3, and SEP4 (aa 57–257), RAD23C and BD‐fused PHYL1OY, PHYL1PnWB, and PHYL1PYR were constructed previously (Maejima et al., 2014 ; Kitazawa et al., 2017 ; Iwabuchi et al., 2019 ). To construct AD‐fused RAD23D, the RAD23D gene of A. thaliana (NM_001085211) was cloned into the pGADT7 vector (Clontech) using NdeI and EcoRI sites. To construct DNA‐binding domain (BD)‐fused PHYL1231/09, PHYL1FBP, PHYL1JWB, PHYL1MD, PHYL1PvWB, PHYL1PWB, PHYL1PnWB, and PHYL1SY and their mutants, these fragments were cloned into the pGBKT7 vector (Clontech) using the same restriction sites. Co‐transformation of yeast cells and evaluation of protein interaction were performed as described by Iwabuchi et al. (2019 ).
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DINO Library Screening for Protein Interactions
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In order to probe whether the DINO library was suitable to screen for protein interactions, we made a screen using the AH109 yeast strain harboring the bait construction pGBKT7-SmicRACK1 as bait, and the DINO library as the prey in the Y187 yeast strain. The screening was carried out with the MATCHMAKER GAL4 Two-Hybrid System 3 kit following the instructions of the manufacturer (Clontech).
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Yeast Two-Hybrid Assay for Protein-Protein Interactions
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The SlTPR4 cDNAs were amplified and cloned into pGADT7, and atpA cDNAs were amplified and cloned into pGBKT7. A yeast two-hybrid assay was performed following Matchmaker GAL4 two-hybrid system 3 kit instructions (Clontech, Mountain View, CA, USA). Yeast transformants were grown on SD-Trp-Leu-His medium. Primers used for the constructs were listed in Table S1 .
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Yeast Two-Hybrid Screening of Plant Transcription Factors
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The Matchmaker GAL4 Two-Hybrid System 3 kit (Clontech; http://www.clontech.com ) was used for Y2H assays, as described previously (Yamaji et al., 2006 (link)). The BD-fused PHYL1OY and AD-fused SEP3 were constructed as previously reported (Maejima et al., 2014a (link)). For construction of the other AD-fused MTFs, the MTFs were amplified using the primers shown in Supplementary Table S1 , and cloned into the pGADT7 vector (Clontech) digested by NdeI and EcoRI-HF (NEB), as described above. The BD-fused PHYL1PnWB was constructed in the same manner, using the pGBKT7 vector (Clontech), digested by NdeI and EcoRI-HF.
Lithium acetate-treated yeast cells (strain AH109) were co-transformed with pairs of appropriate pGADT7 and pGBKT7 vectors. Successful co-transformants were selected on synthetically defined medium (SD) lacking tryptophan and leucine (SD/–LW). To evaluate the protein interaction, the co-transformants were cultured on three selective media: leucine/tryptophan/histidine-lacking SD (SD/–LWH), SD/–LWH containing 5 mM 3-amino-1,2,4-triazole (Sigma-Aldrich) (SD/–LWH+3AT), and leucine/tryptophan/adenine/histidine-lacking SD (SD/–LWAH).
Lithium acetate-treated yeast cells (strain AH109) were co-transformed with pairs of appropriate pGADT7 and pGBKT7 vectors. Successful co-transformants were selected on synthetically defined medium (SD) lacking tryptophan and leucine (SD/–LW). To evaluate the protein interaction, the co-transformants were cultured on three selective media: leucine/tryptophan/histidine-lacking SD (SD/–LWH), SD/–LWH containing 5 mM 3-amino-1,2,4-triazole (Sigma-Aldrich) (SD/–LWH+3AT), and leucine/tryptophan/adenine/histidine-lacking SD (SD/–LWAH).
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Yeast Two-Hybrid Screening of Rice MADS Proteins
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The Matchmaker GAL4 Two‐Hybrid System 3 kit (Clontech) was used to evaluate protein–protein interactions in yeast cells. OsMADS5, OsMADS8 and OsMADS18 genes in pENTA were PCR‐amplified using their respective forward primers and pENTA‐to‐pGADT7‐EcoRI‐R reverse primers (Table S4 ). OsRAD23c was amplified with OsRAD23c‐to‐pGADT7‐F and R primers. Each amplified fragment and pGADT7 vector (Clontech) digested with NdeI and EcoRI‐HF (New England Biolabs) were ligated using the SLiCE method to construct AD‐fused OsMADSs and OsRAD23c. Other genes were previously cloned into pGADT7 and pGBKT7 vectors, as shown in Table S3 (Iwabuchi et al., 2019 (link), 2020 (link); Kitazawa et al., 2017 (link), 2022 (link), 2023 (link); Maejima, Iwai, et al., 2014 (link)). Appropriate pairs of pGADT7 and pGBKT7 vectors were co‐transformed into yeast strain AH109, and protein interaction was evaluated on selective media, as described previously (Kitazawa et al., 2017 (link)).
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Yeast Two-Hybrid Assay for Protein Interactions
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The Matchmaker GAL4 Two-Hybrid System 3 kit (Clontech) was used for Y2H assays as described previously (Yamaji et al., 2006) . Briefly, lithium acetate-treated yeast cells (strain AH109) were cotransformed with appropriate pairs of pGADT7 and pGBKT7 vectors. The cotransformants were plated on synthetically defined (SD) leucine/tryptophan/histidine-lacking medium containing 5-mM 3-amino-1,2,4-triazole (Sigma-Aldrich, St. Louis, MO, USA) (SD/-LWH + 3AT). The plates were incubated for 4 days at 30°C. Some of constructions used in the experiments were created in previous reports (Maejima et al., 2014; Kitazawa et al., 2017; Iwabuchi et al., 2019 Iwabuchi et al., , 2020)) . For further constructions, RAD23A and RAD23B were amplified by PCR from Arabidopsis (col-0) cDNA using the primers listed in Supplemental Table S1 and cloned into pGADT7 digested with NdeI and EcoRI-HF (NEB), using the GeneArt Seamless Cloning and Assembly Kit. In the same way, domains of the Arabidopsis MTF and RAD23 used in the experiments were also amplified from Col-0 using the appropriate pair of primers (Supplemental Table S1) and cloned into NdeI/EcoRI-HF-digested pGADT7. OsRAD23 or its UBA2 domain was amplified from rice (cv. Koshihikari) cDNA and cloned in the same way. SEP3 94-145 or SEP3 146-
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