Fig 2: ROP1 knockout alters rhoptry morphology but not ROP secretion.A. TEM images of the rhoptries of intracellular tachyzoites. White arrowheads indicate rhoptries. Scale bar = 500 µm. B. Normalised median anti-phospho-STAT6 fluorescence intensity of infected HFFs. HFFs were infected for 2 h, fixed with methanol and stained with anti-phospho-STAT6 and anti-T. gondii, then analysed by flow cytometry. p-values were calculated by two-sided Wilcoxon rank sum test with Benjamini-Hochberg adjustment. C. Normalised median nuclear anti-cMyc fluorescence intensity of infected HFFs. HFFs were infected for 24 h in 0.1% FBS medium, fixed and stained with anti-cMyc and anti-T. gondii, and the median nuclear anti-cMyc fluorescence intensity was determined from immunofluorescence microscopy images. p-values were calculated by two-sided Wilcoxon rank sum test with Benjamini-Hochberg adjustment. D. Recruitment of host IRGB6 to T. gondii vacuoles in BMDMs. BMDMs were stimulated with IFN? for 24 h, infected with T. gondii cell lines for 1 h, fixed and stained with anti-IRGB6. The percentage of vacuoles decorated with IRGB6 was determined by automated fluorescence imaging and analysis. p-values were calculated by paired two-sided t-test with Benjamini-Hochberg adjustment.

Fig 3: ROP1 co-immunoprecipitates with host C1QBP.A. Immunofluorescence verification of C-terminal HA-tagging of ROP1. Scale bar = 10 µm. B. Enrichment of proteins that co-immunoprecipitate with ROP1. Primary MEFs were infected with PRU?KU80 or PRU ROP1-HA parasites for 24 h, following which ROP1 was immunoprecipitated with anti-HA agarose matrix and co-immunoprecipitated proteins were identified and quantified by mass spectrometry. L2FCs were calculated from the geometric mean of the iBAQ intensities across replicates, and p-values calculated by two-sided Welch’s t-test. Proteins with p-value < 0.05 and L2FC > 3 are annotated. C, D. Co-immunoprecipitation of C1QBP with ROP1 in C primary MEFs and D HFFs infected with RH ROP1-HA, PRU ROP1-HA, and PRU 202200-HA. S = post-immunoprecipitation supernatant, IP = immunoprecipitate. Note that the immunoprecipitate fraction represents 3x the relative amount of the total lysate compared to the post-IP supernatant fraction. E, F. Immunofluorescence localisation of ROP1 and C1QBP in RH?KU80 and PRU?KU80 infected E primary MEFs and F HFFs at 24 hours post-infection. Scale bar = 10 µm.

Fig 4: ROP1 contributes to T. gondii resistance to IFN? in murine and human macrophages.A, B. T. gondii growth restriction in IFN?-stimulated BMDMs infected at an MOI of 0.3 for 24 h, quantified by high-content imaging and automated analysis. Parasite growth in IFN?-stimulated macrophages is shown as a percentage of that in unstimulated macrophages in terms of A total parasite number and B vacuole number. p-values were calculated by paired two-sided t-test with Benjamini-Hochberg adjustment. C, D. T. gondii growth restriction in IFN?-stimulated THP-1-derived macrophages infected at an MOI of 0.3 for 24 h, quantified by high-content imaging and automated analysis. Parasite growth in IFN?-stimulated macrophages is shown as a percentage of that in unstimulated macrophages in terms of A total parasite number and B vacuole number.p-values were calculated by paired two-sided t-test with Benjamini-Hochberg adjustment. E. F. Propidium iodide uptake of IFN?-stimulated BMDMs infected at an MOI of E 0.3 or F 3 for 12 h. p-values were calculated by paired two-sided t-test with Benjamini-Hochberg adjustment.