Fig. 5

Parasite motility and egress were strongly impaired upon depletion of PPKL. Parasites were grown for 24 h in total, and induced in auxin for 6 h prior to processing for assays of parasite motility and egress. After parasite purification, extracellular parasites on poly-lysine-coated coverslips were allowed to move freely for 20 min at 37 °C, and processed for IFA using antibodies against SAG1 for visualization. For the parasite egress assay, intracellular parasites were stimulated in DMEM containing 3 μM A23187 for 5 min at 37 °C, followed by IFA using antibodies against GRA7 and GAP45. A Representative images of parasites with tails are shown. (B, C) Quantification of parasite motility tail lengths and egress percentages for the TIR1 and PPKL-AID lines. Data are shown with mean ± SD and analyzed by two-way ANOVA with Tukey’s multiple comparisons. Scale: 5 μm. ****P < 0.0001. D Depletion of PPKL resulted in blocked secretion of the microneme protein MIC2. The parasites, including PPKL-AID and the complementation line (PPKL-COMP: PPKL-AID/PPKL-HA), were grown in ±IAA for 12 h, followed by harvesting of the parasites for incubation with 1% ethanol and 1% BSA for 10 min. The parasites were subjected to centrifugation to collect the supernatant and sediment, which were examined by western blot with antibodies against MIC2, ROP5, and GRA7. E The parasites incubated in ±IAA for either 6 or 12 h were harvested for examination of conoid protrusion under stimulation by 3 μM A23187. Examples of the parasites with normal protrusion and abnormal appearance of the apical region are shown. The red arrowhead points to the apical region of the parasite. The parasites were scored and plotted as percentage of conoid protrusion. Data were analyzed by two-way ANOVA of Tukey’s multiple comparisons. ****P < 0.0001, scale bar: 2 μm