Fig. 2

Phenotypic characterisation of the act1 conditional knockout (cKO) at various time points after rapamycin-induced gene excision. a Quantification of apicoplast loss in the act1 cKO over a time range. Loss of the apicoplast was quantified at 0, 24, 48 and 96 h after induction. Vacuoles were stained with α-ACT1 and α-HSP60 and scored for the correct ratio of apicoplasts per parasite. The apicoplast is lost in > 50% of the act1 cKO parasites as early as 24 h after rapamycin induction. Error bars represent ± standard error of the mean (SEM). b Representative images of apicoplast loss. Yellow dotted lines indicate the edge of the vacuoles in greyscaled images. Apicoplast loss occurs even when there is still ACT1 present (24 h). The apicoplast was stained with an antibody against HSP60 [62]. c Egress in the act1 cKO at different time points (0, 36, 72 and 96 h). Vacuoles were stained with α-SAG1 under non-permeabilising conditions and scored. Three conditions were considered: lysed and moved out of the vacuole, lysis of the membranes without the release of parasites, and no lysis of the parasitophorous vacuole membrane or host membrane. Error bars represent ± SEM. d Representative images of egress; at 72–96 h post induction, the act1 cKO parasites are unable to lyse the membranes, as determined by positive SAG1 staining without prior permeabilisation and are not released from the vacuoles. Scale bars: 10 μm. e and f Trail deposition and invasion assays for the act1 cKO compared to wt (RH) at time points 0, 24, 48 and 96 h after induction. After 48 h post induction, no significant change of trail deposition or invasion were observed, Error bars represent ± standard error of the mean. All experiments were performed in biological triplicate and compared with a two-tailed Student’s t-test, **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05