FIG 5.
PfPMRT1 is essential for early gametocyte development. (A) Live cell microscopy of 3D7-iGP-PfPMRT1-GFP parasites across the complete gametocyte development. White arrowheads indicate remaining GDV1-GFP signal observed in close proximity to the Hoechst signal, as previously reported (59, 94, 102, 103). (B) Live cell microscopy of PfPMRT1-GFP parasites expressing the PPM marker Lyn-mCherry. Nuclei were stained with Hoechst 33342. Scale bar, 2 μm. (C) Experimental setup of gametocyte induction upon GDV1-GFP-DD expression (+shield-1) and conditional PfPMRT1 knockout (+rapalog) and elimination of asexual blood stage parasites (+GlcNac). (D) Gametocyte development over 12 days of condΔPMRT1/GDV1-GFP-DD or 3D7-iGP parasites without (control) or with rapalog addition at day 3 (3 dpi) or day 5 (5 dpi) after induction of sexual commitment by conditional expression of GDV1-GFP upon addition of shield-1. Scale bar, 5 μm. (E) Diagnostic PCR to verify the excision at the genomic level at 5 dpi and 12 dpi. Black arrowhead, original locus; red arrowhead, excised locus. (F) Representative Giemsa smears and quantification of parasite stage distribution at day 10 postinduction for parasites treated without (control) or with rapalog at day 3 postinduction. For each condition, the distributions of parasitemia and parasite stages in erythrocytes of three independent experiments were determined and are displayed as percentage (ΔPMRT1, ncontrol = 3,370, 2,304, and 2,759, and nrapalog = 3,010, 1,830, and 2,387; 3D7-iGP, ncontrol = 4,985, 4,685, and 5,206, and nrapalog = 4,930, 4,332, and 5,384). Nuclei were stained with Hoechst 33342. Scale bar, 10 μm.