FIG 1.

MFM1.15 shows a partial Myr⁻ phenotype and has a mutation in ROP17. (A) Representative immunofluorescence assay (IFA) images of HFFs infected with wild-type RH (RH-WT), RHΔmyr1, or RH mutant MFM1.15. All three parasite strains express cytosolic td-tomato (red) and were transfected with a plasmid expressing HA-tagged GRA16 which was detected by probing with anti-HA (green). Hollow white arrows indicate vacuoles containing parasites expressing the GRA16HA transgene. Solid white arrows indicate the nuclei in cells containing such vacuoles. Only the RH-WT-infected cells show efficient translocation of GRA16HA to the host cell nucleus. Images from one of six biological replicates are shown. (B) Quantitation of nuclear GRA16HA was assessed by anti-HA tag staining followed by ImageJ to determine the intensity of nuclear staining in infected cells by transiently transfected parasites from at least 10 random fields. The MFM1.15 mutant shows a significantly reduced nuclear signal but still more than the essentially complete lack of signal in the nuclei of cells infected by the RHΔmyr1 mutant. Data presented are the pooled data of three independent biological replicates. Error bars indicate standard errors of the means. Values that are significantly different are indicated by a bar and asterisks as follows: **, P < 0.0001. (C) Mutations identified by whole-genome sequencing of mutant MFM1.15 and the subclone MFM2.1b. Coverage is the number of reads spanning the indicated nucleotide, and variant frequency is the fraction of reads showing the variant nucleotide relative to the reference (GT1). Both mutants show mutations relative to the annotated type I strain, GT1, in TGGT1_258580 (ROP17) with a missense mutation in MFM1.15 and a nonsense mutation in MFM2.1b.