Fig. 3
Construction and characterization of conditional knockdown lines SFA4-mAID and SFA5-mAID (A) mutation of DNA sequences encoding position 74 at the TIR1. The DNA sequence of TIR1 encoding the 74th amino acid was sequenced and shown was the mutation site. B the new version of TIR1-Flag was expressed in the parasite to generate a parental line TIR1F74G. C Schematic of the CRISPR/Cas9 system used for inserting the mAID-6Ty-DHFR at the C-terminus of the SFA4 or SFA5. The pCas9-sgRNA (that encodes a sgRNA targeting to the downstream of the stop codon) and amplicon (that contains homologous regions and the mAID-6Ty-DHFR) were transfected into the TIR1F74G line, resulting in the mAID-6Ty-DHFR fragments fusing at the C-terminus of SFA4 or SFA5. Note that DHFR indicates the resistant expression cassette. D–E Immunofluorescence microscopy and Western blot of SFA4-mAID-Ty and SFA5-mAID-Ty lines grown with/without 5-Ph-IAA treatment for 16 h. The parasites were stained using mouse anti-Ty (SFA4 for green, SFA5 for red) and rabbit anti-GAP45 (the SFA4-mAID line for red, the SFA5-mAID line for green). Scale bar = 2 μm. SFA4-mAID-6Ty and SFA5-mAID-6Ty tachyzoites were grown on HFF with/without 5-Ph-IAA treatment for 16 h. Labels were detected by mouse anti-Ty and rabbit anti-Actin (or Tubulin) as control. F–G Plaque formation with TIR1, SFA4-mAID and SFA5-mAID lines grown with/without 5-Ph-IAA for 7 days. The numbers of plaques were scored using ImageJ. Three independent experiments were performed. ****, p < 0.0001. H Parasites were grown in HFF cells in treatment (+ 5-Ph-IAA) or control (ethanol) for 24 h. IFA was performed for scoring vacuoles containing different numbers of parasites. Means ± SEM of three biological replicates with Two-way ANOVA analysis. ***, p < 0.001