Endogenous tagging of Tgatpβ and Tgatposcp genes with YFP-HA tags.
(A) Schematic representation of the Tgatpβ(TGME49_261950) gene locus showing coding region in light blue and 3′ UTR in gray. The hatched region shows the HR, which was cloned into a donor plasmid (dark blue) upstream of an in-frame coding region for YFP-HA tag. The donor plasmid also includes the DHFR cassette, which confers pyrimethamine resistance. Asterisk denotes the position of stop codon. The donor plasmid was linearized with the restriction enzyme BstZ17I to facilitate single-crossover homologous recombination, and the resulting modification of the gene locus was confirmed by PCR using the primers ConF and YFP_R. A similar strategy was used to tag the Tgatposcp gene locus. (B) and (C) Confirming the expression of the TgATPβ-YFP-HA and TgATPOSCP-YFP-HA proteins by SDS-PAGE western blotting (B) and microscopy (C) on respective clonal transgenic lines. Gel lanes in (B): 1, Cell lysate from TgATPβ-YFP-HA transgenic parasites; 2, Cell lysate from TgATPOSCP-YFP-HA transgenic parasites; 3, Cell lysate from parental parasites; M, Molecular weight size markers. Mitotracker was used to visualize the mitochondrion in (C). (D) Functional validation of mitochondrial ATP synthesis in wt parental strain, and TgATPβ-YFP-HA- and TgATPOSCP-YFP-HA-expressing transgenic parasites. All three strains exhibited similar response to atovaquone treatment in the presence and absence of glucose, thereby confirming that the YFP-HA tag had no effect on mitochondrial ATP synthesis. The raw luminescence reading from this experiment and its analysis are provided in S1 Data file. DHFR, dihydrofolate reductase; HR, homology region; TgATPβ, T. gondii ATP synthase β subunit; TgATPOSCP, T. gondii ATP synthase OSCP subunit; wt, wild type; YFP-HA, yellow fluorescent protein plus hemagglutinin.