Fig. 4
Toxoplasma RASP2 depletion impairs parasite invasion. a Strategy for conditional depletion of TgRASP2 using the Tet-OFF system. b Immunoblot of KD-TgRASP2-HA3 (±ATc). SAG1, loading control. c IFA of KD-TgRASP2-HA3 shows depletion of RASP2 after 2 days ATc treatment. d Plaque assays of KD-TgRASP2 and complemented cKD-TgRASP2 parasites (±ATc) shows that the strong phenotype induced by TgRASP2 depletion (no plaques) can be rescued by a complementing copy of the gene. CRISPR fitness score was derived from Ref. 20. e Intracellular replication of TATi_TgRASP2-HA3 (Ctrl) and KD-TgRASP2-HA3 ± 48 h ATc. The percentage of vacuoles containing 2, 4, 8, 16 or 32 parasites was determined on 200 vacuoles (n = 3). f Percentage of egressed vacuoles in TATi_TgRASP2-HA3 (Ctrl) and KD-TgRASP2-HA3+ATc. Parasite egress was induced 30 h post-invasion by addition of A23187 (3 μM) for 8 min before samples were fixed and processed for IFA (anti-GRA3 antibodies). Egress events (GRA3 in the PV) were quantified on 30 vacuoles. g Gliding assays were performed with KD-TgRASP2 ± ATc 48 h. Trails were revealed by IFA using anti-SAG1 antibodies. h 5 min-invasion assay of TATi_TgRASP2-HA3 (Ctrl) and KD-TgRASP2-HA3 parasites (±ATc) shows a strong invasion defect when TgRASP2 is affected. (d, e, f, h) Values represent means ± SD, n = 3, from a representative experiment out of 2 independent assays. Source data are provided as a Source Data file
