Fig. 2

Generation and identification of TgPPKL-AID and its complementation line. A CRISPR-mediated C-terminal AID tagging at the endogenous locus of the 3′-terminus of ppkl in the RHΔku80Δhxgprt/TIR1 line. The homologous regions (HR1 and HR2) derived from upstream of the stop codon and downstream of single-guide RNA (sgRNA) contain about 40 base pairs (bp) of DNA sequences, while the amplicon was amplified from a generic plasmid that contained a DNA fragment encoding an AID-Ty-3′UTR-resistant expression cassette. The amplicon and pCAS9-3′sgRNA were combined and transfected into the parasites. B Schematic diagram of the plant auxin-inducible degron system for the PPKL-AID fusion protein. C, D IFA and western blot detection of the PPKL-AID-6Ty fusion. The parasites were harvested for detection of the fusion protein by IFA (C) and western blot (D). GAP45 was used as the control for the IFA, while actin antibodies served as the loading control on the western blots. Scale bars = 2 μm. E, F A PPKL-COMP line was generated by transfection of a plasmid expressing PPKL-3HA. The parasites were grown for 24 h, followed by incubation in the presence or absence of auxin for 12 h. The expression was detected by western blots and IFA, where control antibodies were used for the detection. The intensity of the bands was quantified, followed by comparing the intensity of PPKL-AID-Ty and PPKL-HA with the intensity of actin, resulting in the relative expression of the endogenous and ectopic PPKL in the parasites (E). The parasites as described in (E) were analyzed by IFA using antibodies as listed in the figures. Scale bars = 2 μm