Figure 6

Barcode sequencing of thiabendazole-treated deletion library led to the identification of a previously uncharacterized gene required for centromere silencing. (a) The deletion mutant of SPBP8B7.28c displayed TBZ sensitivity and a centromere silencing defect. Five-fold serial dilution of wild type (WT; DY2776), raf1Δ (DY2781), swi6Δ (DY2784), and SPBP8B7.28cΔ (DY2792) cells were spotted on agar plates of YES medium, YES supplemented with 10 mg/l TBZ, YE medium (ade6 mutant colonies turn pink on YE plates due to a low level of adenine), and EMM supplemented with uracil, leucine, and arginine (no adenine). These strains all harbor the otr1R(SphI)::ade6+ marker which, when expressed, allows the strains to grow in the absence of adenine and form white colonies on YE plates [50]. (b) The protein encoded by SPBP8B7.28c shares a conserved domain with proteins from other fungi species. The multiple sequence alignment was created with T-COFFEE [72] and visualized with BOXSHADE 3.21. Six cysteine residues are invariant in the alignment and two FSKxQ motifs are also highly conserved. Accession numbers are [NP_596535.1] (Schizosaccharomyces pombe), [XP_002173616.1] (Schizosaccharomyces japonicus), [EEQ92506.1] (Ajellomyces dermatitidis), [XP_002583495.1] (Uncinocarpus reesii), [XP_002379665.1] (Aspergillus flavus), [XP_384593.1] (Gibberella zeae), [EEU42643.1] (Nectria haematococca), [XP_955929.2] (Neurospora crassa), [XP_001588826.1] (Sclerotinia sclerotiorum).