FIG 5.

ROP17 catalytic activity appears necessary for its role in effector translocation. (A) HFFs were infected with the indicated strains transiently expressing GRA24MYC, and then 17 to 18 h later, the presence of GRA24MYC in the nuclei of cells infected with GRA24MYC-expressing parasites was assessed by IFA. The results shown are the averages and standard deviations of three technical replicates for each condition and are representative of three biological replicates performed for RH, RHΔrop17, and RHΔrop17::ROP17 and two biological replicates for the catalytically dead mutants. Differences were assessed by ANOVA with a post hoc Tukey’s test. **, P < 0.0001; ns, not significantly different. (B) HFFs infected for 18 h with the indicated strains transiently expressing GRA16HA were assessed for the presence of GRA16 in the nucleus. Data shown are from one representative biological replicate of three performed, all of which gave similar results. Differences were assessed by ANOVA with a post hoc Tukey’s test. **, P < 0.0001. (C) Assessment of the effect of mutating ROP17 on host c-Myc upregulation upon infection. HFFs were infected with the indicated strain, and 20 h later, c-Myc expression in the nuclei of infected cells was assessed using IFA and anti-c-Myc antibodies (red). Expression of the variants of ROP17-3xHA was assessed by staining with anti-HA (green). None of the three catalytic mutants rescued the Myr⁻ phenotype of the Δrop17 mutant. (D) Quantitation of Toxoplasma’s ability to upregulate host c-Myc was performed by assessing a minimum of 110 random fields from each of three technical replicates for whether an infected cell did or did not express c-Myc. The results shown are from one biological replicate of two performed. Differences between groups were assessed by ANOVA with a post hoc Tukey’s test. **, P < 0.0001.