Figure 4.

Complementation of the PP2C-deficient yeast mutant strain TM126 (ptc1Δ). For each strain, 3 μl of cell suspension of each yeast strain, containing approximately 3 × 10⁴ cells, was spotted onto agar plates (see Materials and Methods) and incubated at either 28°C or 37°C for 2 days. Strain TM126 was transformed with the yeast expression vector pDBL2 as a control; plasmid pDBC1 contains the wild-type yeast PTC1 gene (19), plasmids pDBL3 and pDBL3M contain either wild-type or mutant L. japonicus LjNPP2C1 genes, and plasmid pDBL5 contains the LjNPP2C1 gene in the antisense orientation with respect to the yeast ADH promoter. The poor growth phenotype of strain TM126 at the nonpermissive temperature (37°C) is evidenced by a less dense (partially translucent) spot; complementation of the temperature-sensitive growth phenotype is evidenced by denser (nontranslucent) spots.