FIG 6.

Ectopic expression of RRL mutants of GRA44, GRA45, and MYR4 rescues the translocation defect in Δgra44, Δgra45, and Δmyr4 parasites. (A) Representative immunofluorescence microscopy images of transiently expressed GRA44, GRA45, and MYR4 RRL → ARL-mutated proteins. The indicated strains were transiently transfected with plasmids expressing the corresponding C-terminally V5-tagged protein under the control of its native promoter, and the tachyzoites were allowed to infect HFFs for 18 to 22 h before the infected monolayers were fixed with methanol. The localization of the V5-tagged proteins and rescue of the GRA16-HA host nuclear translocation were assessed by IFA using mouse anti-V5 and rat anti-HA antibodies, respectively. White arrows indicate a GRA16-HA-positive host nucleus in a cell infected with tachyzoites expressing the indicated V5-tagged protein. Bars = 10 μm. (B) Quantification of the data represented in panel A, showing the percentage of infected cells showing GRA16-HA in the host nucleus via IFA. The indicated strains were transiently transfected with either an empty plasmid (−) or plasmids expressing the corresponding C-terminally V5-tagged protein (+) under the control of its native promoter. The data for the untransfected parental strain, the empty plasmid-transfected strains, and the wild-type protein-transfected strains are the same as those in Fig. 4B and are included here for ease of comparison. Scoring and statistics are as described in the legend to Fig. 3B, except that the single asterisk indicates a P value of 0.017.