FIGURE 3.

Generation of plp1 knock-out and complementation strains for domain deletion analysis and immunofluorescence of complemented strains. A, the plp1 knock-out was generated in the RHΔku80Δhxg strain by replacing the PLP1 locus with the HXG drug selection marker. The plp1ko was complemented at the endogenous locus by double homologous recombination and negative selection. B, PCR on parasite genomic DNA demonstrated plp1 locus replacement with HXG in the knock-out and HXG replacement with PLP1 cDNAs in complemented strains. C, PLP1 domain structure and domain deletions tested in this study are shown. D, immunofluorescence with a polyclonal PLP1 antibody showed some PLP1 complementation constructs co-localized with MIC2, whereas other constructs were poorly detected. Scale bars, 2 μm. E, parasite strains investigated in this study are listed, including the genotype, common name used throughout this paper, and the presence or absence of spheres in postegressed cultures. Spheres were observed by bright field examination of egressed cultures, and images were acquired on bright field at a magnification of ×200 on a Zeiss Axio inverted microscope.