Generation and confirmation of complemented PbPL-knockout parasites.

<p>A) Schematic representation of the plasmid pL0017.1.2-5’FR-PbPL-V5 used to transfect marker-free PbPL-knockout (KO-MF) parasites, thereby generating complemented PbPL-KO (CMP) parasites. The PbPL coding sequence and the endogenous promoter region (1067 bp upstream of the start codon) were amplified from wild-type (WT) parasite gDNA by PCR and cloned in frame with a c-terminal V5-tag into a pL0017-derived plasmid. This vector integrates into the <i>c-</i> or the <i>d-ssu-rRNA</i> locus via single crossover recombination and conveys resistance to pyrimethamine. B) Diagnostic PCR of complemented parasite lines. Successful integration of the transfected plasmid into either of two possible loci in the <i>P</i>. <i>berghei</i> genome was tested by PCR. Locations of primers used for PCR analysis are shown. For each locus, one primer pair (1 and 4, 2 and 4, respectively) yields a PCR product of 3 kb if no integration has taken place. In case of successful integration, the primers are too far apart (>14 kb) to result in a complete PCR product under the chosen conditions. To further confirm integration, additional primer pairs (1 and 3, 2 and 3) were used, which only generate a PCR product of 3 kb if the plasmid has integrated. C) Complemented parasites express PbPL-V5 under the endogenous promoter. HepG2 cells were infected with complemented PbPL-KO sporozoites (CMP2) constitutively expressing cytosolic mCherry (red), fixed at 54 hpi and analyzed by IFA using an antiserum against PbPL (green, upper panel) or a monoclonal antibody against the V5-tag (green, lower panel) in combination with an antiserum against the PVM marker protein ExpI (purple). The merged channels additionally contain DAPI-stained nuclei (blue). Scale bars = 10 μm. IFAs are representative for the CMP1 and CMP3 parasite lines, which showed similar PbPL- and V5-stainings. All primer sequences are listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004760#ppat.1004760.s008" target="_blank">S1 Table</a>.