FIG 2.

Creation of heterothallic K. marxianus strains. (A) Auxotrophic mating assay of Km1 strains. Shown are results from strains Km1 MATα α3^– kat1^– leu2^–, Km1 MATa α3^– kat1^– leu2^–, and homothallic Km1 leu2^–, streaked through strain Km1 MATa α3^– kat1^– trp1^– on 2% glucose plates and replica plated onto SCD − (Leu, Trp) plates after 2 days. Diploid growth is seen only upon sexual crossing between strains with opposite mating types or with homothallic haploid strains. (B) Auxotrophic mating assay of several α3^– kat1^– leu2^– triple-inactivation strains and Km1 MATa α3^– kat1^– trp1^– or Km1 MATα α3^– kat1^– trp1^–. Putative heterothallic strains were spotted over the negative control (−), the Km1 MATa α3^– kat1^– trp1^– reference (a), or the Km1 MATα α3^– kat1^– trp1^– reference (α) on glucose plates for mating. Replica plating onto SCD − (Leu, Trp) results in diploid growth. (C) The wild homothallic isolate Km18 was made trp^– by UV mutagenesis and crossed with heterothallic Km1 MATa α3^– kat1^– leu2^–. Diploids were sporulated, 16 spores were isolated (a through p) and germinated, and the resulting haploids were screened for heterothallic strains by crossing with Km1 MATa α3^– kat1^– trp1^– or Km1 MATα α3^– kat1^– trp1^–. Screened haploids were auxotrophic strains unable to mate (c, d, f, i, j, and o), possible trp^– revertants (h, k, n, and p), homothallic (g), or heterothallic (a, b, e, l, and m).