UIS4 protein expression is upregulated in <i>puf2</i> salivary gland sporozoites.

<p><b>(A)</b> UIS4 was detected by immunofluorescence assay with a goat anti-UIS4 antibody (SICGEN) in salivary gland sporozoites from days 18, 22 and 27 post-mosquito infection in the indicated parasite lines. Note the change in morphology of the <i>puf2</i> sporozoite. Scale bars = 5 μm. <b>(B)</b> Fluorescence images representing the quantification method used with ImageJ software. UIS4 immunofluorescence of a <i>puf2</i> sporozoite at day 22 post-mosquito infection is shown. The shape of the sporozoite was outlined; the same shape was placed on a neutral area of the image to obtain a background value. Fluorescence intensities of the sporozoite and background were determined with ImageJ software as explained in the Material and Methods. <b>(C)</b> Scatter plot representation of the UIS4 fluorescence intensity measurements (arbitrary units a.u.) are shown (n = 20 for each timepoint and parasite line). P-values were obtained by Mann-Whitney test.</p

RNA immunoprecipitation of PUF2::GFP from <i>puf2</i>::<i>gfp</i> salivary gland sporozoites from day 21 post mosquito infection reveals binding of <i>uis4</i> by PUF2::GFP.

<p><b>(A)</b> Schematic of the GFP-Trap_A Kit (Chromotek) IP protocol used. (<b>B)</b> RT-PCR performed on RNA isolated from the input and IP eluates as outlined in A to verify the presence or absence of <i>uis4</i> and control genes in IP eluates. The material used for the input RT-PCR represents 6.7% of bound as well as unbound samples. RT+ and RT- indicate cDNA synthesis set-up in the presence (+) or absence (-) of Reverse Transcriptase.</p

PUF2::GFP behave like wildtype parasites and maintain a latent salivary gland parasite population.

<p><b>(A)</b> Schematic of genetic modification of the <i>puf2</i> locus. Shown are position of primers (red: reverse) and genotyping PCRs (italics) as shown in B. <b>(B)</b> PCR genotyping of <i>puf2</i>::<i>gfp</i> parasite clone compared to wild type; from left to right are shown: 5’ and 3’ integration sites of the plasmid construct; wildtype <i>puf2</i> locus; human <i>dhfr</i> selection marker; a control reaction. See A for position of primer pairs. <b>(C)</b> Field inversion gel electrophoresis followed by Southern blot analysis detects correct integration of the plasmid construct into chromosome 7. <b>(D)</b> Parasitemia development of <i>puf2</i>::<i>gfp</i> (n = 6) in Balb/C mice following mosquito bite (10 mosquitoes per mouse were allowed to feed for 30 minutes). days p.i. = days post mosquito infection. Mean ± s.d. <b>(E)</b> RT-PCR performed on RNA isolated from the <i>puf2</i>::<i>gfp</i> parasite line sporozoites and wild type (PbA259) sporozoites from days 20/21 post-mosquito infection confirm transcription of <i>puf2</i>::<i>gfp</i> mRNA in the <i>puf2</i>::<i>gfp</i> parasites and not the wildtype, untagged gene. <i>hsp70</i> and <i>18S</i> rRNA were amplified as control genes. <b>(F)</b> Immunofluorescence assay (IFA) of salivary gland sporozoites from days 21/22 post-mosquito infection. Rabbit anti-GFP antibody ab6556 (Abcam), mouse anti-CSP (3D11), or goat anti-UIS4 antibody (SICGEN) were used. Scale bars = 5μm. <b>(G)</b> IFA of <i>puf2</i>::<i>gfp</i> and <i>puf2</i> sporozoites at days 18, 22 and 27 post-mosquito infection; mouse anti-CSP (3D11) was used. Note the morphological change (rounding up) of day 27 <i>puf2</i> parasites. Scale bars = 5 μm.</p

Expression of fluorescently-tagged proteins of multigene families in liver stages of single-gene tagging (SGT) mutants at 48hpi in cultured hepatocytes (Huh7).

<p><b>A</b>. Fluorescence-microscopy analysis of members of the <i>fam-a</i>, <i>fam-b</i> and <i>pir</i> multigene family in live liver-stages. The parasites expressing mCherry-tagged Fam-b2 and PIR1 also express cytoplasmic GFP (cyt GFP; green). <b>B</b>. IFA-analysis of fixed liver-stages using anti-mCherry (red) anti-PbEXP1 (green) antibodies. PbEXP1 is a parasitophorous vacuole membrane resident protein. <b>C</b> Fluorescence-microscopy analysis of expression of SMAC and IBIS, exported proteins encoded by single-copy genes in live liver-stages. Nuclei are stained with Hoechst-33342 (blue); scale bar: 10μm.</p

Simultaneous expression (and export) of two proteins of the same family in a single blood stage parasite (trophozoites or schizonts) of double-gene tagging (DGT) mutants.

<p>These mutants contain the following pairs of genes tagged with either mCherry or GFP: <i>fam-a1/fam-a2</i> (2 independent mutants; panel A, B), <i>fam-b1/fam-b2</i> (panel C<i>) and pir1/pir3</i> (panel D); RMgm ID as indicated in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005917#ppat.1005917.t002" target="_blank">Table 2</a></b>. Localisation of mCherry-tagged members in the trophozoite stage (upper two rows) and in the schizont (low row) stage. Parasite nuclei are stained with Hoechst, BF bright field. scale bar: 5μm.</p

Expression of proteins encoding blood-stage exported proteins in blood- and liver-stages

<p>Expression of proteins encoding blood-stage exported proteins in blood- and liver-stages</p

Maximum likelihood phylogeny of <i>fam-a</i> gene sequences from <i>Plasmodium</i> spp.

<p>The tree was estimated using RAxML and a GTR+Γ model. Branches subtended by nodes with >75 bootstrap support are shown in bold. Robust basal nodes are indicated by black squares with bootstrap proportions (above node) and Bayesian posterior probabilities (beneath node). At right, coloured blocks indicate the species to which a terminal node belongs. Clades of orthologs that display positional conservation are indicated with green blocks; where a sequence has been lost secondarily in a species is shown by an ‘X’. The tree is rooted using an out-group comprising single copy <i>fam-a</i> orthologs from primate <i>Plasmodium</i> species. The phylogeny is subdivided into four sections: genes located at the conserved, ‘ancestral’ locus on chromosome 13 (below line <i>i</i>); genes found at loci conserved across RMP species (between lines <i>i</i> and <i>ii</i>); and a robust clade of species-specific paralogs derived from a conserved locus on chromosome 6 or 13 (between lines <i>ii</i> and <i>iii</i>); a robust clade of species-specific paralogs derived from a conserved locus on chromosome 8 (above line <i>iii</i>).Transcription levels (shown as different coloured and sized circles) in blood stages are shown for individual genes based on RNAseq data (FPKM values) (from [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005917#ppat.1005917.ref033" target="_blank">33</a>] and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005917#ppat.1005917.s007" target="_blank"><b>S1</b> Table</a>). Expression levels as shown by four different sized circles: Class 1 (smallest circle): 2-8x the threshold level; class 2: 8-16x the threshold; class 3 (largest circle): >16x the threshold.</p

Features of tagged members of the <i>pir</i>, <i>fam-a</i> and <i>fam-b</i> multigene families

<p>Features of tagged members of the <i>pir</i>, <i>fam-a</i> and <i>fam-b</i> multigene families</p

Maximum likelihood phylogeny and chromosomal location of <i>fam-a</i> genes that have an internal, syntenic chromosomal location

<p><b>A.</b> The tree was estimated using RAxML and a GTR+Γ model. Nodes support is indicated by bootstrap proportions and Bayesian posterior probabilities. Genes that occupy the conserved, chromosomal-internal locus are labelled in bold and accompanied by a symbol, colour-coded by species (repeated in <b>B</b>). In <i>P</i>. <i>yoelli</i> and <i>P</i>. <i>chabaudi</i>, these genes are paraphyletic with other genes that have been transposed to subtelomeric locations of other chromosomes (these paralogs are labelled in italics). The tree is rooted with the single copy <i>fam-a</i> orthologs from primate <i>Plasmodium</i> spp.<b>B.</b> Chromosomal organisation of the internal copies of <i>fam-a</i> genes on chromosome 14 of <i>P</i>. <i>falciparum</i> 3D7 (Pf14; green), chromosomes 13 of <i>P</i>. <i>berghei</i> ANKA (Pb13; black), <i>P</i>. <i>yoelii yoelii</i> 17X (Py13; red) and <i>P</i>. <i>chabaudi</i> AS (Pc13; blue). <i>Fam-a</i> genes are interspersed with cyclin (lilac), cyclin fragments (violet), ribosomal protein S6 (yellow) and conserved <i>Plasmodium</i> protein (orange). The internal <i>fam-a</i> region is bordered by RNA polymerase III subunit RPC4 (PF3D7_1463400, PBANKA_1327000, PY17X_1330800, PCHAS_133030) and YL1 protein (PF3D7_1464000, PBANKA_1327400, PY17X_1332100, PCHAS_133200) (shown in grey). Synteny between RPC4 and YL1 is shown with grey lines. The arrows indicate the location on forward and reverse strands.</p

Transcription of <i>pir</i>, <i>fam-a</i> and <i>fam-b</i> genes based on RNAseq data of different blood stages of two <i>P</i>. <i>berghei</i> ANKA reference lines.

<p><b>A</b>. Features of transcription of <i>pir</i>, <i>fam-a</i> and <i>fam-b</i> genes in the two <i>P</i>. <i>berghei</i> reference lines (line 1 and line 2) based on RNAseq data (from[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005917#ppat.1005917.ref033" target="_blank">33</a>] and shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005917#ppat.1005917.s007" target="_blank">S1 Table</a>). Transcribed genes are genes with an FPKM value above the cut-off level of 21. Total transcript abundance is the sum of all FPKM values observed in the different blood stages (see <b>B</b>). The fold up-down regulation is based on the difference in FPKM values of individual genes between blood stages of the two different parasite lines (see <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005917#ppat.1005917.s001" target="_blank">S1 Fig</a></b>). <b>B.</b> Percentage of genes transcribed in the different blood stages (see <b>A</b>). Ring, red; trophozoite, green; schizont, purple; gametocyte, black. <b>C.</b> Total transcript abundance in the different blood stages: mean and standard deviation of total transcript abundance of all FPKM values observed in the different blood stages (see <b>A</b>). <b>D.</b> Percentage of non-transcribed genes (light grey) and genes with less (grey) or more (black) than 1.5x difference in transcript abundance between blood stages of two different parasite lines (see <b>A</b>). The coloured circles show the genes with >1.5 fold down-or upregulation in the four different blood stages (see <b>B</b>).</p