Yeastmaker yeast transformation system 2

The bait sequence double E (p184), synthesized in tandem, was cloned into pAbAi to generate the bait plasmid (Figure 4A), which was transformed into yeast strain Y1HGold according to the instructions of the Yeastmaker^TM Yeast Transformation System 2 (Clontech, USA). As a negative control, the E (p184)-M sequence was mutated from AAAG to AATC. Bait strains were selected from colonies that grew well on a synthetic defined (SD) medium lacking uracil. The coding sequence of ZmDof30 was cloned into the EcoRI and BamHI sites of pGADT7 to generate the prey vector. After screening for the minimal inhibitory concentration of aureobasidin A (AbA), the bait strains were transformed with the prey vector and cultured on SD medium lacking leucine and containing the minimal inhibitory concentration of AbA at 30°C for 3 days.

The yeast two-hybrid library of R P. fugax (cloned into the prey vector pGADT7) was constructed by selecting three P. fugax R plants randomly at the early flowering stage and using the Matchmaker^® Gold Yeast Two-Hybrid System (Clontech) according to the manufacturer’s instructions. Full-length PfMADS16 was cloned into vector pGBKT7 (bait vector) and then transformed into the yeast strain Y2HGold using the Yeastmaker^TM Yeast Transformation System 2 (Clontech).
The constructed R P. fugax yeast two-hybrid library was used to screen the interaction proteins of PfMADS16 according to the manufacturer’s instructions (Matchmaker^® Gold Yeast Two-Hybrid System). The primers used for pGBKT7 vector construction are listed in Supplementary Table S1. To confirm protein interactions, the screened prey and bait vectors were validated by one-to-one interaction hybridization.

The sequence verified clones were transformed into yeast cells (Y2H gold for pGBKT7 vector with ZPR genes and Y187 for pGADT7 vector with GmHD-ZIPIII-1 and 2) using protocols described in Yeastmaker^Tm Yeast Transformation system 2 user manual (Clontech, CA, USA). Yeast mating and screening of the interaction partners using different selection media was performed following the user manual of Matchmaker^® Gold Yeast Two-Hybrid System (catalogue #630489, Clontech, CA, USA). All growth assays were reconfirmed using three independent yeast transformants. Enzymatic assays to determine the strength of interaction were performed in triplicate according to the “Yeast protocol handbook” (Clontech, CA, protocol PT4084-1, USA) and using 2-Nitrophenyl β-D-Galactopyranoside/ONPG (Catalogue# 73660, Clontech, CA, USA) as the substrate. Millers units were calculated and statistical significance was determined using one-way ANOVA and Tukey HSD in JMP software (SAS Institute Inc., Cary, NC, USA, 1989-2019).

Plasmid NpGBKT7-SELENOF’ was used to screen the human fetal brain cDNA library via the yeast two-hybrid system. Yeast transformation and library screening were performed following the procedures described in the user manuals for the assays (YeastmakerTM Yeast Transformation System 2 and Matchmaker™ Gold Yeast Two-hybrid System, Clontech, U.S.A). Positive yeast colonies from library screening were selected from the quadruple dropout solid medium SD/−Ade/-His/−Leu/−Trp containing X-α-Gal and Aba. Plasmids extracted from the yeast cells were transformed into E. coli Top10 competent cells. Prey plasmids were amplified in LB medium containing ampicillin and extracted from E. coli cells. The obtained prey plasmids and the NpGBKT7-SELENOF’ bait plasmid were co-transformed into Y2HGold yeast cells and grown on SD /−Leu /−Trp /x-α-gal /Aba solid medium to determine whether the colonies turned blue. The positive prey plasmids were then sequenced and analyzed with the basic local alignment search tool (BLAST).

The ORF sequences of PgCBF3 and PgCBF7 were cloned into pGBKT7. According to the procedure of Yeastmaker^TM Yeast transformation system 2 (Clontech, Takara, Kusatsu, Japan), the recombinants of pGBKT7-PgCBF3 and pGBKT7-PgCBF7 were transformed into the yeast strains Y2HGold stain, which were screened on SD/-Trp-Leu-His-Ade/X-α-Gal medium. The yeast with pGADT7-T + pGBKT7-Lam was used as the negative control and with pGADT7-T + pGBKT7-p53 as the positive control.

The sequence of pglA, excluding signal peptide sequence (1–81 bp), was obtained through direct gene synthesis. The resulting fragment was subsequently inserted into the pGBKT7 vector. The recombinant vector, pGBKT7-pglA, was then introduced into Saccharomyces cerevisiae strain Y2HGold using Yeastmaker^TM Yeast Transformation System 2 (Clontech, CA, USA). To evaluate the potential toxicity and autoactivation of the pglA bait, the transformed cells were plated onto three distinct media of SD/-Trp, SD/-Trp/X, and SD/-Trp/X/A, respectively. Following plating, the cells were cultured at 30 °C for 3–5 days. The strain of Y2HGold containing the pGBKT7-BD vector was spread on an SD-Trp medium, as a positive control.
To determine the expression of pglA bait, the transformed cells were cultured in an SD/-Trp broth medium at 30 °C under 200 rpm for 4–8 h, until the OD₆₀₀ value reached the range of 0.4–0.6. As positive control, the Y2HGold strain carrying the pGBKT7-53 vector and the Y2HGold strain carrying the pGBKT7-BD vector were also cultured in an SD/-Trp broth medium. For negative control, the wild type Y2HGold strain was cultured in a YPDA broth medium. The various yeast cells were harvested via centrifugalization, and the total protein was extracted using the Yeast Protein Extraction Reagent (Takara, Dalian, China).

To screen for effector-interacting proteins, a cDNA library was prepared from the susceptible V. vinifera cultivar ‘Pinot Noir’ inoculated with P. viticola. Screening was carried out using the Yeastmaker^TM Yeast Transformation System 2 (Clontech) according to the manufacturer’s protocol. For analyses of effector-importin-α interactions, the P. viticola effector sequence was inserted into the plasmid pGBK-T7 (BD), and the importin-α sequence was ligated into the plasmid pGAD-T7 (AD).