FIG 2.

Ptp2 is required for the growth and differentiation of C. neoformans as a negative-feedback regulator of Hog1. (A) Growth of ptp1Δ and ptp2Δ mutants at diverse temperatures. Each strain (WT [strain H99] and hog1, ptp1 [YSB1704], ptp2 [YSB275], ptp2::PTP2 [YSB2195], ptp1 ptp2 [YSB2058], and hog1 ptp2 [YSB1617] strains) was grown overnight at 30°C in liquid YPD medium, 10-fold serially diluted, and spotted onto YPD agar medium. Cells were incubated at 30°C, 37°C, and 39 to 40°C for 4 days and photographed. (B) The WT strain (H99) and ptp1, ptp2, and ptp1 ptp2 mutants were grown to mid-logarithmic phase, and total protein extracts were prepared for immunoblot analysis. The anti-pY-Hog1 and anti-pTY-Hog1 antibodies were used for monitoring phosphotyrosine and phosphotyrosine/threonine levels, respectively. The same blots were stripped and then reprobed with polyclonal anti-Hog1 antibody for loading controls. (C) Ptp2 was essential for the sexual differentiation in C. neoformans. Serotype A MATα and MATa strains were cocultured on V8 medium (pH 5) for 15 days at room temperature in the dark. The images were photographed after 11 and 15 days. (D) Ptp2 modulated mating by negatively controlling Hog1. Each indicated α strain was cocultured with KN99a on V8 medium in the dark at room temperature and photographed after 8 and 12 days. (E) The deletion of PTP2 resulted in decreased levels of a pheromone gene, MFα1. Northern blot analysis for measuring the expression of MFα1 was performed with total RNA isolated from the coculture of the indicated strains, which were grown for 24 h under mating conditions. (F) Ptp2 was required for cell-cell fusion during mating response. Cell fusion efficiency was calculated relative to the control strains (WT α [YSB119] × WT a [YSB121]).