Yeast strain y187

Our yeast two-hybrid (Y2H) protocol was described in detail by Caignard et al. [48 (link)]. Briefly, the Y2H gold yeast strain (Clontech Laboratories, Mountain View, CA, USA) was transformed with the pDEST32 plasmid encoding Gal4-DB fused to SUB. After confirming that the sub ORF did not induce autonomous transactivation of the HIS3 reporter gene, screening was performed on synthetic medium lacking histidine ([His-] medium) and supplemented with only 5 mM of 3-amino-1,2,4-triazole (3-AT; Sigma-Aldrich, St. Louis, MI, USA). A mating strategy was used for screening an Ixodes ricinus tick cDNA library cloned into the Gal4-AD pDEST22 vector (Invitrogen) and previously established in Y187 yeast strain (Clontech Laboratories) [49 (link)]. Yeast cells were plated on a selective medium lacking histidine and supplemented with 5 mM 3-AT to select for interaction-dependent transactivation of the HIS3 reporter gene. AD-cDNAs from [His+] colonies were amplified by PCR and sequenced to identify the proteins interacting with SUB.

All mutants were generated from the wild-type M. acridum strain CQMa102 (WT) and grown on 1/4 SDAY media (10‰ glucose, 5‰ yeast extract, 2.5‰ peptone and 18‰ agar, wt/vol) or SYA media (5‰ yeast extract, 0.5‰ KCl, 1‰ KH₂PO₄, 30‰ sucrose, 0.5‰ MgSO₄, 3‰ NaNO₃, 0.01‰ MnSO₄, 0.01‰ FeSO₄ and 18‰ agar, wt/vol) at 28°C. The Y2HGold and Y187 yeast strains (Clontech, Palo Alto, CA, USA) were used in the autoactivation and yeast one-hybrid assays, respectively. Escherichia coli DH5α (Solarbio, Beijing, China) and Agrobacterium tumefaciens AGL1 (Solarbio, Beijing, China) were used for the recombinant plasmid manipulations and fungal transformations, respectively.

Bait (pGBKT7) and prey (pGADT7AD) vectors described above were transformed into Y2HGold and Y187 yeast strains (Clontech), respectively. Diploid yeast cells were generated by mating single colonies from bait and prey strains and then selected on SD/-Trp/-Leu plates. For Fig. 6, overnight cultures from single yeast colonies were diluted to an OD₆₀₀ of 0.2 and spotted on SD/-Trp/-Leu, SD/-Trp/-Leu/-His, and SD/-Trp/-Leu supplemented with 125 ng/ml Aureobasidin A (AbA). For Fig. 7a, overnight cultures from single yeast colonies were first diluted to an OD₆₀₀ of 0.2, from which ten-fold serial dilutions were spotted on SD/-Trp/-Leu supplemented with or without 125 ng/ml Aureobasidin A (AbA). The plates were incubated at 30 °C, and pictures were taken on the third day after plating.

Full length wild-type or mutant BRC-1 sequences were cloned into plasmid pBridge (Takara Bio), transformed into yeast strain Y2HGold (Takara Bio) and plasmids were selected on medium lacking tryptophan. Full length BRD-1 sequences were cloned into plasmid pACT2.2, transformed into yeast strain Y187 (Takara Bio) and transformants were selected on medium lacking leucine. Wild type or mutant BRC-1 expressing strains were mated with BRD-1 expressing strain and the diploids selected on -Trp-Leu double drop out plate at 30C. Diploid cells were grown in liquid -Trp-Leu medium overnight, and serial dilutions were plated on -His -Trp -Leu and -Trp -Leu solid media. For quantitative measurement of wild type or mutant BRC-1 and BRD-1 interactions, supernatants from liquid cultures were assayed using CPRG (chlorophenol red-b-D-galactopyranoside, RocheApplied Science Cat. NO.10884308001) as substrate and β-galactosidase units were calculated as described (Yeast Protocol Handbook, Takara Bio, https://www.takarabio.com/products/protein-research/two-hybrid-and-one-hybrid-systems/yeast-two-hybrid-system/matchmaker-gold-yeast-two-hybrid-system).

Open reading frames were amplified from cDNA prepared by reverse transcription of C. elegans total RNA using CloneAmp DNA polymerase (Takara Bio Inc) and the primers used are listed in Supplemental File 1. Amplicons were inserted into either pGBKT7 or pGADT7 cleaved with EcoRI and BamHI by In-Fusion cloning (Takara Bio Inc). The sequences were confirmed by Sanger sequencing (DNA sequencing and Services, Dundee University and Eurofins Genomics). pGADT7 and derivatives were transformed into yeast strain Y187 and pGBKT7 and derivatives into yeast strain Y2HGold (Takara Bio Inc.). Yeast transformation and subsequent mating of transformants were done using standard protocols. Protein-protein interactions were assayed by comparing growth on synthetic defined medium lacking leucine and tryptophan with growth on synthetic defined medium lacking in addition adenine and histidine.

The Matchmaker® Gold Yeast Two-Hybrid System (Clontech) was used to screen of a pre-transformed Mate and Plate™ Library—normalized Universal Human HeLa cDNA library (Clontech), following the instructions of the manufacturer. In summary, Saccharomyces cerevisiae Y2HGold (Clontech) was transformed with plasmid pFA147, a derivative of pGBKT7 (Table S1) and mated with yeast strain Y187 (Clontech) carrying the HeLa cDNA library cloned into pGADT7. The two strains were mated and plated in lower stringency or double dropout (DDO) media (SD/–Leu/–Trp) supplemented with X-α-Galactosidase (X) and Aureobasidin A (A) (all from Clontech) and incubated 3–5 days at 28–30°C. The blue colonies grown on DDO/X/A media were patched into a higher stringency or quadruple dropout (QDO) media (SD/–Ade/–His/–Leu/–Trp) supplemented with X-α-Galactosidase and Aureobasidin A and incubated 3–5 days at 28–30°C. The blue colonies that grew in QDO/X/A were further analyzed for autonomous system activation, and for identification of the target protein, by sequencing the plasmid DNA (primers listed in Table S2). We analyzed 1.43 × 10⁸ clones, patched 60 blue colonies and recovered 41 from high stringency media for further analysis.