Skip to main content
. 2022 Oct 20;18(10):e1010901. doi: 10.1371/journal.ppat.1010901

Fig 6.

Fig 6

PDE1 and PDE2 are important for lytic growth, with ΔPDE2 parasites displaying an A23187-mediated egress defect similar to ΔCDPK3 parasites (A) Schematic representation of PDE2 rapamycin mediated deletion of PDE cKO lines. Addition of rapamycin leads to excision of the entire gene, placing YFP under the control of the PDE promoter. Coloured triangles represent primer pairs used to detect unexcised (blue) and excised (brown) loci. (B) PCR analysis of DMSO- and RAP-treated PDE cKO parasite lines showing near complete excision for all lines. (C) Western blot analysis of PDE cKO lines showing near complete loss of the PDEs at the protein level following treatment with RAP. PDEs at their expected molecular weights as depicted by arrows (E) Representative images of plaque assays performed on DMSO- and RAP-treated WT and PDE cKO lines after a period of 5 days. Assays were performed in biological triplicates. (F) Measurement of plaque area shown in Fig 6E. Data are represented as mean ± s.d. (n = 3). Significance was assessed using multiple t-tests. ****, P ≤ 0.0001; **, P ≤ 0.01; n.s, not significant. (G) Quantification of plaque numbers shown in Fig 6E. Data are represented as mean ± s.d. (n = 3). Significance was assessed using multiple t-tests. ****, P ≤ 0.0001; n.s. not significant. (H) Egress assay of GFP-T2A-jRCaMP1b expressing WT, ΔCDPK3 and RAP-treated PDE-HA-cKO parasites following treatment with 8 μM A23187. Data are represented as mean ± s.d. (n = 3). Two-way ANOVA.****, P ≤ 0.0001; *, P ≤ 0.05. (I) Quantification of intracellular cAMP levels of PDE2-HA-cKO and PDE2-HA-cKO:ΔCDPK3 parasites treated with either DMSO or RAP. Data are represented as mean ± s.d. (n = 7). Two-way ANOVA with Turkey’s multiple comparisons. **, P ≤ 0.01; *, P ≤ 0.05. (J) Egress assay of DMSO- or RAP-treated GFP-T2A-jRCaMP1b expressing PDE2-HA-cKO and PDE2-HA-cKO:ΔCDPK3 parasites following treatment with (i) 8 μM A23187 or (ii) 50 μM BIPPO. Data are represented as mean ± s.d. (n = 3). Two-way ANOVA. ****, P ≤ 0.0001; ***, P ≤ 0.001; *, P ≤ 0.05.