Figure 5. Post-translational knockdown reveals a requirement for RhopH2 after invasion.
(A) Strategy for the introduction of a tandem 3xHA-DDD tag at the protein’s C-terminus. (B) Schematic showing the timeline for the stage-specific removal of TMP and harvest of trophozoite-stage parasites. Horizontal bars below the schematic indicate the presence of TMP. control, TMP is present continuously; max, maximal knockdown is achieved with TMP removal prior to the onset of gene transcription; ER, TMP is removed after invasion and synchronization of early rings; LS, TMP is removed from late schizonts after protein trafficking to rhoptries. (C) Sorbitol-induced osmotic lysis kinetics for each transfectant after continuous cultivation with TMP (con) or withdrawal for maximal knockdown (‘max’, as illustrated in panel [B]). (D) Osmotic lysis kinetics for R2-DDD with TMP removal as indicated in panel (B). Traces are color-coded to match panel (B). (E) Mean ± S.E.M. of the reciprocal of sorbitol-induced osmotic lysis halftime (t0.5) for control and each stage-specific R2-DDD knockdown. *p=0.02; **p<10−3. (F) Immunoblots of trophozoite-stage R2-DDD parasites after stage-specific knockdown as indicated. While late-schizont TMP removal (LS) reduces each protein’s abundance in newly invaded cells, early ring removal does not affect protein levels (ER). (G) Immunoblot showing CLAG3 protease susceptibility as an indicator of host membrane insertion for indicated stage-specific knockdowns. (H) Mean ± S.E.M. of fractional CLAG3 cleavage, quantified from three independent trials as in panel (G). *p<0.01. (I) Parasite expansion without or with TMP removal for two, four, or six days. Viable parasites could not be detected over a 60-day period when TMP was not restored.