Interaction between Pho85 bait and Pcl5 prey detected using high-throughput mating C2H screening and further checked with the two-step low-throughput confirmation protocola

Bait protein (pC2HB-XX)Prey protein (pC2HP-XX)C2H result for spot assay of SC2H3 diploids
NameORF no.BCCM accession no.NameORF no.BCCM accession no.SC-M-HSC-M-C-H
Pho85orf19.6846LMBP 10482Pcl5orf19.4012LMBP 10501G (W)G
Pcl5orf19.4012LMBP 10577Pho85orf19.6846LMBP 10593G (W)G
  • a During the high-throughput screen, the interaction was detected on an SC-Met-Cys-His agar plate. On the SC-Met-His agar plate, the growth was very weak and difficult to distinguish against the background growth. The interaction was then further validated with a low-throughput screen using tetraploids followed by a cotransformation and a spot assay of the diploid SC2H3 strain transformed with both the bait and prey plasmids. The interactions between bait Pho85 and prey Pcl5 were also shown using a co-IP experiment. We were also able to show the interaction with a cotransformation experiment when switching bait and prey. W, weak; G, growth; M, Met; C, Cys; H, His. See text for more details. BCCM accession numbers are given; plasmids are accessible at the Belgian Co-ordinated Collections of Microorganisms (BCCM;