Conditional depletion of TgPP6C and TgPP6R produces a phenotype similar to that observed in RHΔpp6c and RHΔpp6r.

(A) Plaque assay of the indicated strains treated with or without IAA for 7 days. The arrows indicate plaque formed by PP6C-mAID and PP6R-mAID with IAA. (B) The relative size of the plaques for each strain. Data represent mean ± SD of three independent experiments and statistical differences were analyzed by the unpaired t-test. ****P < 0.0001; n.s., not significant. (C) and (D) Immunofluorescence staining shows PP6C-mAID (red) and PP6R-mAID (red) strains, cultured with or without IAA for 24 h, stained against TgIMC1 (green). Knock-down of TgPP6C and TgPP6R resulted in asynchronous division of tachyzoites. Scale bars: 2 μm. (E) Replication of PP6C-mAID and PP6R-mAID parasites after growth in the presence or absence of IAA for 32 h. Data are mean ± SD from three independent experiments, > 200 vacuoles were counted per replicate and were analyzed by a two-way ANOVA; *P = 0.0178; ***P = 0.0001; ****P < 0.0001; n.s., not significant. (F) and (G) Invasion and egress assays showed that PP6C-mAID and PP6R-mAID pretreated with IAA slightly affected parasite invasion, but not egress. Data represent mean ± SD and The three independent data were analyzed for statistical significance by the unpaired t-test. *P = 0.0237 (PP6C-mAID +IAA vs. PP6C-mAID–IAA); *P = 0.0232 (PP6R-mAID +IAA vs. PP6R-mAID–IAA); n.s., not significant.

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