Figure 2.
Generation and characterization of iΔFd strain.A, scheme of genetic constructs used to generate iΔFd strain by replacement of the endogenous gene locus through double-crossover homologous recombination. The construct for complementation via insertion at the UPRT locus is shown at the bottom. B, analytical PCR on iΔFd genomic DNA that confirms proper integration of the inducible construct and replacement of the endogenous TgFd gene. WT DNA served as control. Primer combinations and expected amplicon sizes are given in A. C, top left, TgFdmyc; top right, streptavidin Alexa Fluor 488; bottom left, merged DNA stain (DAPI) with green and magenta (false color) fluorescent images, with colocalized apicoplast signal appearing white (all three signals). The scale bar represents 10 μm. Arrowheads mark the biotin signal from biotinylated proteins likely located in the host mitochondria, which adhere to the PVM in strain RH. Fd, ferredoxin; iΔ, inducible knock-down; UPRT, uracil phosphoribosyltransferase.