Easy yeast plasmid isolation kit

The preconstructed cDNA library was transformed into the Y1H bait strain (pBait-AbAi) using the Yeastmaker Transformation System 2 (Clontech, Cat. No. 630439) [22 (link)]. The transformation reaction was spread on SD/-Leu plates with minimal inhibitory concentration of AbA. The candidate clones were restreaked onto SD/-Leu/AbA at least three times to generate a single colony. To eliminate duplicate clones, we conducted yeast colony PCR with Matchmaker Insert Check PCR Mix 2 (Clontech, Cat. No. 630497). The prey vectors that showed a single band in yeast colony PCR were isolated by an Easy Yeast Plasmid Isolation Kit (Clontech, Cat. No. 630467) and sequenced using T7 primers. For verification of positive interactions, each prey should be transformed into bait and mutant bait strains on selective media with side-by-side positive and negative controls.

Phenotypic confirmation was performed by re-streaking blue colonies onto the high stringency quadruple dropout selective media containing X-α-gal (for colorimetric identification) to confirm growth. A single blue colony was picked and the prey plasmid was isolated using the Easy Yeast Plasmid Isolation Kit (Clontech) and transformed into Escherichia coli for propagation. E. coli transformants were plated on Luria-Bertani (LB) media supplemented with 250 μg / ml carbenicillin which selected for clones containing the prey plasmid containing the ampicillin resistance gene. Prey plasmid was isolated from selected E. coli transformants using Qiagen Mini-Prep Kit (Qiagen, Valencia, CA). The positive protein-protein binding interaction was confirmed by cotransforming the pGADT7-FH2/Daam1 prey plasmid and the pGBKT7-BBA66 bait into yeast strain Y2HGold. As the confirmatory controls, yeast strain Y2HGold was cotransformed with i) pGADT7-FH2/Daam1 prey plasmid and bait plasmid pGBKT7 with no cloned insert, or ii) pGADT7-FH2/Daam1 prey plasmid with pGBKT7 containing the bba64 coding sequence.

To construct the bait vector for TF-Centered Y1H, the coding sequence of BpERF2 (birch ethylene-responsive factor 2) was amplified and cloned into vector pGADT7-Rec2-ERF2 using the yeast recombination method. The PCR reaction to amplify BpERF2 comprised 0.5 µl of cDNA template, 0.5 µM of primers BpERF2-F and BpERF2-R (Table 1), 2.5 mM each dNTP, 2 µl of 10 × buffer, 0.5 U Taq, with sterile distilled water to a volume of 20 µl. The PCR thermal profile was 94 °C for 3 min; followed by 25 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 2 min. The PCR product was purified using a Column PCR product purification kit (Qiagen, Hilden, Germany) according to the supplier’s protocol. pGADT7-Rec2 was linearized using Sma I. The linearized pGADT7-Rec2 and the PCR product of BpERF2 were cotransformed into Y187 using the Yeastmaker™ Yeast Transformation System 2 with small scale transformation to generate the recombined pGADT7-Rec2-ERF2. The recombined pGADT7-Rec2-ERF2 was isolated from yeast cells using an Easy Yeast Plasmid Isolation Kit (PT4073–1, Clontech), and was transformed into DH5α Escherichia coli. The recombined pGADT7-Rec2-ERF2 vector was isolated from E. Coli using E.Z.N.A.TM Plasmid Mini Kit (OMEGA).

In order to screen host proteins that interact with the SipC bait in the yeast two-hybrid system, a cDNA library of dGCs, pGBKT7-sipC, and the library plasmid were co-transformed into Y2HGold using a Yeastmaker™ Library Construction & Screening Kit according to the manufacturer protocol. Co-transformants were then grown on higher-stringency SD/-Ade/-His/-Leu/-Trp/X-α-Gal/ABA agar plates at 30 °C for 3–5 days. Single blue colonies were selected and serially passaged three times on SD/-Ade-His-Leu-Trp/X-α-Gal/ABA plates. Positive colonies were thereafter re-seeded three times in SD/-Ade-His-Leu-Trp liquid media. Prey plasmids were extracted from putatively positive clones using the Easy Yeast Plasmid Isolation Kit (Clontech).

To confirm interactions, co-transformations of pGBKT7-sipC bait into Y2HGold with each prey plasmid in putatively positive hits were performed. Briefly, the prey plasmids were extracted from putatively positive clones using an Easy Yeast Plasmid Isolation Kit (Clontech, Mountain View, CA, USA). Subsequently, each prey plasmid was transformed into E. coli DH5α competent cells (Transgen, Beijing, China) and purified from transformants growing on selected LB/ampicillin agar plates using a Plasmid Mini Kit I (Omega Bio-Tek, Norcross, GA, USA). Each putatively positive prey plasmid was co-transformed with pGBKT7-sipC bait and pGBKT7 plasmids into Y2HGold. Cotransformants were grown on SD/-Ade-His-Leu-Trp/X-α-Gal/ABA plates to test for interactions. The cotransformant containing pGADT7-T and pGBKT7-Lam grown on an SD/-Ade-His-Leu-Trp/X-α-Gal/ABA plate was used as a negative control, and the cotransformant containing pGADT7-T and pGBKT7-53 grown on SD/-Ade-His-Leu-Trp/X-α-Gal/ABA was used as a positive control. True positive interactions were indicated by blue colonies under these conditions. Positive prey plasmids were sequenced, and results were blasted against NCBI databases.

The blue colonies grown on QDO/X/A agar plates were preliminarily detected with PCR using the Matchmaker™ Insert Check PCR Mix (Clontech Laboratories). The potential prey plasmids were then isolated from the identified blue colonies using the Easy Yeast Plasmid Isolation Kit (Clontech Laboratories). A 3-μl aliquot of prey plasmids extracted from yeast cells was transformed into competent cells (E. coli DH5α) for plasmid rescue and sequencing. The gene fragments were analyzed with BLAST from the US National Center for Biotechnology Information (NCBI) to identify the host genes. The biological processes and structures of the identified genes were also analyzed using the UniProt database (http://www.uniprot.org/) and SMART server (http://smart.embl-heidelberg.de/).