Fig. 6

PPKL is essential for parasites in the type II strain ME49. A, B PPKL was efficiently depleted by the addition of auxin. The PPKL-AID line was grown in auxin for 24 h, followed by IFA using antibodies against the Ty epitope and GAP45. Western blotting was performed for parasites induced in auxin for the indicated times, and actin served as the loading control. Scale bars = 2 μm. C, D Parasite growth was examined by plaque formation assay. The parasites were grown on HFF host cell monolayers for 10 days, followed by fixation and staining. Three independent experiments were performed in triplicate. The plaque areas were scored using ImageJ software, and data were expressed as mean ± SD and analyzed by two-way ANOVA with Tukey’s multiple comparisons. ****P < 0.0001. Scale = 0.5 cm. E, F Depletion of PPKL resulted in defects in parasite replication and morphology. Parasites were grown in ±IAA for 24 h, followed by IFA using antibodies against GAP45 for imaging and scoring of parasites in vacuoles (E). At least 200 vacuoles were scored for each replicate, and three independent experiments were performed in triplicate. Vacuoles with 1–3 parasites are shown from the IFA of parasite replication (F). Scale = 2 μm. ****P < 0.0001