Fig. 6: Effect of TgCet depletion on mRNA capping and parasite growth.

A Strategy for tagging of TgCet protein in RH-TIR1-3FLAG parental line. B Diagnostic PCR for confirmation of 3′ integration of mAID-3HA in the TgCet gene locus (n = 3). C Western blot analysis of TgCet-mAID-HA expression levels in tachyzoites with or without IAA at indicated time-points (n = 3). D Immunofluorescence analysis of TgCet-mAID-HA grown with IAA or vehicle for 1 h (n = 3). Scale bar 5 µm. E, F Immuno-dot blot and quantification analyzes of m⁷G levels of RNA extracted from TgCet-mAID-HA parasite (−IAA/+IAA). G TgCet-mAID-3HA parasites were transfected with TgTub-FLuc plasmid DNA. Luciferase activity was measured at indicated time-points. H RT-qPCR analysis of FLuc mRNA in TgCet-mAID-HA −IAA and +IAA parasites. I Percent parasite viability assessed with trypan blue in TgCet-mAID-HA −IAA and +IAA parasites. J Representative IFA images of TgCet-mAID-HA tachyzoites treated with either vehicle, IAA, or CHX and incubated with puromycin prior to parasite fixation (n = 3). Scale bar, 5 µm. K Parasite replication. TgCet-mAID-3HA parasites were grown in HFF monolayers for 18 h with IAA or vehicle. L TgCet-mAID-3HA parasites were grown with IAA or vehicle for 8 h. Number of parasites with defective morphology per vacuole was counted and plotted as a percentage of normal and abnormal parasites. M Crystal violet-stained images of plaques formed by RH-TIR1 and TgCet-mAID-3HA parasites on HFF monolayer treated with IAA or vehicle. Quantification of plaque numbers (N) and plaque areas (O). P Quantification of plaques after IAA withdrawal at indicated time-points in TgCet-mAID-3HA parasites. Q Parasite invasion. The graph shows the percentage invasion of RH-TIR1 and TgCet-mAID-3HA parasites on HFF monolayer. R Parasite egress. The graph shows the percentage of egress of RH-TIR1 and TgCet-mAID-3HA parasites cultured with IAA or vehicle, followed by calcium ionophore treatment. E–I: n = 3 replicates; mean ± S.D; two-way ANOVA with Tukey’s post hoc test; ns not significant; p values: *<0.05; *** <0.001; ****<0.0001. Figure 6K-R; n = 3 replicates; mean ± S.D; one-way ANOVA; Dunnett’s comparison test; ns not significant; p values: *<0.05; *** <0.001; ****<0.0001. Source data are provided as a source data file.