FIG 3.

GRA44, GRA45, and MYR4 are required for Toxoplasma effector translocation and fully efficient growth in vitro. (A) Schematic of CRISPR-mediated gene disruption of candidate genes, followed by insertion of the pTKO2 plasmid carrying the gene for mCherry and a chloramphenicol acetyltransferase (CAT) gene for selection in chloramphenicol. UTR, untranslated region. (B) Quantification of the percentage of infected cells showing GRA16-HA in the host nucleus via IFA. Tachyzoites were allowed to infect HFFs for 16 h before the infected monolayers were fixed with methanol and stained with rat anti-HA antibodies. The averages are based on examination of at least 25 infected host cells per experiment from 2 to 5 biological replicates, and error bars indicate the standard deviation (SD). Statistics were performed using one-way analysis of variance and Dunnett’s multiple-comparison test. P values are for the indicated strain relative to the parental control. **, P < 0.0001; ns, not significant (P > 0.05). (C) Quantification of the percentage of infected cells showing the upregulation of the human c-Myc protein in the host nucleus via IFA. Tachyzoites were allowed to infect HFFs in serum-free medium for 20 h before the infected monolayers were fixed with methanol and stained with rabbit anti-c-Myc antibodies. Scoring and statistics are as described in the legend to panel B. (D) Quantification of the percentage of transfected, infected cells showing GRA16-HA in the host nucleus via IFA. Wild-type RHΔhpt and RHΔmaf1 tachyzoites were transiently transfected with a plasmid expressing GRA16-HA, and transfected parasites were allowed to infect HFFs for 16 h before the infected monolayers were fixed with methanol and stained with rat anti-HA antibodies. The averages are based on the examination of 25 vacuoles from 2 biological replicates, and error bars indicate the SD. Statistics are as described in the legend to panel B. (E) Western blot of human cyclin E1 protein in infected cells. HFFs were infected with the indicated tachyzoites or mock treated with uninfected HFF lysate for 18 h before lysates were generated for immunoblotting. Lysates were analyzed by Western blotting using mouse anti-cyclin E1 antibodies. Rabbit anti-SAG2A and mouse anti-GAPDH were used to assess the levels of parasite and host proteins in the lysate, respectively. Lanes −, empty lanes. (F) Quantification of plaque size. HFFs were infected with tachyzoites of the indicated strain for 7 days, fixed with methanol, and then stained with crystal violet. Plaque size was measured using ImageJ software. Plaque areas were normalized to the median for the parental strain for each biological replicate. The averages are based on the results of at least 3 independent biological replicates, each with 2 to 3 technical replicates, and error bars represent the standard error of the mean. Statistics are as described in the legend to panel B.