Rescue of growth of the pmr1-Δ ret1-27 double mutant by CaCl2 and MnCl2.

Since the ret1-27 pmr1-Δ double mutant was inviable on regular media, it was obtained from the MC39-MOX strain, which carried a PMR1-contaning plasmid. To allow the MC39-MOX and MC39-RET-MOX strains to lose the PMR1-contaning plasmid, they were streaked on YPD plate supplemented with 10 mM CaCl2. Equal amounts of cells from single colonies obtained on this medium were suspended in sterile water and spotted onto test plates. Growth of only one subclone for each case is shown in the figure. Growth of three additional subclones is shown in S4 Figpmr1-Δ ret1-27, a MC39-MOX subclone lacking the plasmid; PMR1 ret1-27, a MC39-MOX subclone retaining the plasmid; pmr1-Δ RET1, a MC39-RET-MOX subclone lacking the plasmid; PMR1 RET1, a MC39-RET-MOX subclone retaining the plasmid.