<i>ppkl</i> expression and activity.

<p>A. Schematic representation of PPKL structure showing N-terminal Kelch repeats and C-terminal catalytic domain. Residues of the first motif of the catalytic domain are indicated B. Phosphatase assay demonstrates PPKL is an active phosphatase whereas the catalytic site mutant (-GAXNE-) shows no activity. C. RNA-Seq data represented as both tags/kb of gene and fragments per kilobase of exon model per million mapped reads (FPKM) indicates <i>ppkl</i> mRNA levels are highest in schizonts and gametocytes D. Immunoblots of parasite extracts from rings, trophozoite, schizont stages from a gametocyte non-producer and, gametocyte and ookinete stages using a α-PPKL polyclonal antibody. Protein expression is detected only in schizonts and sexual stages.</p

Expression and localization of structural and invasive proteins of the ookinete.

<p>A. Direct immunofluorescence detection of alveolar and motor proteins (red) B. Total protein expression of the same proteins; GFP expression was used to normalize loading. C. Micronemal proteins CTRP and chitinase were spread throughout the cell body (red) in the mutant and not localized to the apex. D. Total protein expression of CTRP and chitinase in mutant ookinetes is identical to <i>wt</i>. E. Secretion of CTRP and chitinase remains similar to <i>wt</i>. Enolase, an abundant non-secreted protein is not detected in the ookinete medium. F. Abnormally intense α-tubulin staining was observed in <i>ppkl^–</i> apical end (green). Ookinetes were processed 24 hour post-activation. The nucleus was counter-stained using DAPI (blue). Bar = 1 µm.</p

PPKL localization during <i>Plasmodium</i> life-cycle.

<p>A. PPKL expression in merozoites and activated gametocytes. No expression is observed in the male gametocyte. B. PPKL localization during zygote-ookinete development and in sporozoites. Expression is predominantly cytoplasmic with a focussed expression observed only in emerging tip of the retort and ookinete stages. Bar = 1 µm.</p

Ultrastructure analysis of wild type and <i>ppkl^–</i> ookinetes.

<p>A. Scanning electron micrograph (SEM) of wild type ookinete. B. Transmission electron micrograph (TEM) detailing longitudinal section through wild type ookinete. C. SEM of <i>ppkl^–</i> exhibits an elongated apical and no basal constriction. D. TEM longitudinal section through <i>ppkl^–</i> further shows micronemes dispersed through the cell body. (A–D, bar = 1 µm) E. Detail of wild type apical complex depicts a well-defined collar, attached microtubules and concentrated micronemes. F. Detail of <i>ppkl^–</i> apical end reveals fewer micronemes and an underdeveloped apical complex. G. Longitudinal section of the elongated apical end shows collapsed microtubule bundles H. Loss of contact of microtubules with the IMC (arrowed). (E–F, bar = 0.25 µm). Abbreviations: Micronemes, Mn; Nucleus, Nu; Collar, Co; Microtubules, MT; Inner membrane complex, IMC.</p

Pbtert deletion and selection of tert- mutants.

<p>(A) Schematic representation of the construct used to delete the <i>tert</i> gene. The construct, containing the <i>Tgdhfr-ts</i> selectable marker (SM) cassette, targets the <i>tert</i> gene at the flanking regions (red) by double cross-over integration. The red arrows indicate primers used for diagnostic PCR to confirm correct disruption of <i>tert</i>. Boxes correspond to lanes on the PCR gels in (B), (D) and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108930#pone.0108930.s001" target="_blank">Fig. S1A</a>. (B) Diagnostic PCR of uncloned parasites transfected with a DNA construct to delete the <i>tert</i> gene. Parasites were collected and analysed directly after transfection and selection with pyrimethamine (parent populations). Diagnostic PCR shows the presence of parasites with correct disruption of the <i>tert</i> gene. In all experiments (1065, 1078, 1138, 1207, 1217) the 5′ and 3′ integration fragments (lanes 5′, 3′), as well as the <i>Tgdhfr-ts</i> fragment (lane SM) were amplified. However, all populations contained parasites with a wild type <i>tert</i> gene as shown by amplification of the wild type <i>tert</i> fragment (lane wt). The primer pairs used are shown in (A) and expected fragment sizes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108930#pone.0108930.s004" target="_blank">Table S2</a>. <i>pbs21</i>-specific primers were used as a positive control for all the PCR reactions (“+”). The water control is marked as “-“. (C) Southern analysis of separated chromosomes using the 3′UTR <i>Pbdhfr-ts</i> probe shows only in experiment 1065 and 1217 hybridisation with chromosome 14 on which the <i>tert</i> gene is located. This probe recognizes the endogenous <i>Pbdhfr-ts</i> gene on chromosome 7 in all populations and additional chromosomes in experiments 1078, 1138, 1207 (possible episomal construct signal). (D) Diagnostic PCR of uncloned and propagated parasites transfected with a DNA construct to delete the <i>tert</i> gene. The parent parasite populations of experiment 1065, 1207 and 1217 [see (B)] were propagated in mice (m0  =  mouse 0, m1  =  mouse 1) for another 1–2 weeks. Parasite populations collected were analysed by diagnostic PCR for the presence of parasites with correct disruption of the <i>tert</i> gene [primers same as in (B)]. In all populations no parasites with a disrupted <i>tert</i> gene could be detected by diagnostic PCR after 1 week (1207 all populations) or after two weeks of propagation (1065 uncl.2, 1217 uncl.2 m0 and 1217 uncl.2 m1).</p

Pbtert gene structure (A) and PbTERT (B) and PbTR (C) expression.

<p>(A) The <i>tert</i> gene of <i>P. berghei</i> and homology (percentage identity) of TERT proteins in different <i>Plasmodium</i> species. Sequencing of the gap between two adjacent <i>tert</i> gene models available in PlasmoDB revealed a sequence duplication of 57 nt (19aa). The complete Pb<i>tert</i> gene encodes a protein of 2312aa, which is comparable to the size of other <i>Plasmodium tert</i> genes. (B) Western analysis of PbTERT protein in mixed blood stages. Two bands with a size between 150 and 250 kDa were detected (expected size of the TERT protein is ∼240 kDa). (C) Northern analysis of Telomerase-associated RNA (TR) in different blood stages of <i>P. berghei</i>. RNA was hybridized with a probe recognizing TR (upper panel) (the expected size of TR is 2 kb) and as a loading control with a probe recognizing <i>large subunit ribosomal RNA</i> (expected size 0.8 kb). The “% loading” refers to the quantity of the loading control signal detected for each stage relative to the “late trophozoite” lane which is set as 100%.</p

<i>P. berghei</i> telomere characterisation.

<p>(A) Determination of telomere length by Telomere Restriction Fragment (TRF) analysis. Left Panel: Southern analysis of separated chromosomes of <i>P. berghei</i> (Pb), <i>P. chabaudi</i> (Pc), <i>P. vinckei</i> (Pv) and <i>P. yoelii</i> (Py) showing hybridization of all chromosomes to a telomere-specific probe. The same probe was used for TRF analysis (middle, right panels). Middle panel: Southern analysis of digested <i>P. yoelii</i> (size control) and <i>P. berghei</i> gDNA probed with the telomeric probe showing the characteristic “smeared” hybridisation pattern in TRF analysis. Right panel: The average telomere length was measured as the highest peak of the signal intensity along the smear. Using the molecular marker (“M”, grey line) as a size reference (relevant marker bands sizes are noted on the graph), the mean telomere length was estimated to be ∼2500 bp and ∼950 bp for <i>P. yoelii</i> (blue line) and <i>P. berghei</i> (red line), respectively. Complete digestion of gDNA was confirmed by hybridisation with a 5′ <i>d-type small unit ribosomal RNA</i> probe. (B) Fluorescence <i>in situ</i> hybridisation with a telomere-specific probe. Fixed late blood stages of <i>P. berghei</i>. The telomeric probe (1.5 kb) was labelled with fluorescein (green). Hoechst (blue) was used for nuclear staining. The size bar is 5 µm.</p

Gametocyte conversions and exflagellation in <i>P</i>. <i>berghei</i> mutant parasites.

<p>(A) Gametocyte conversion was observed during blood stage development in mutant <i>P</i>. <i>berghei</i> parasites over 5 days post infection by either using a wt parent line which expresses GFP in male gametocytes and RFP in female gametocytes (RMgm-164) with <i>P</i>. <i>berghei</i> mutants generated in the same genetic background and analysed using FACS determining the number of gametocytes in infected blood or by observing mature gametocytes in Giemsa stained smears. No significant difference was seen between wt and mutants parasites. Error bars indicate SD of n = 2 biological replicates. (B) Exflagellation (male gamete formation) in mutant <i>P</i>. <i>berghei</i> parasites normalised to wt in an <i>in vitro</i> activation assay. Error bars indicate SD of n = 3 biological replicates. P-values **p < 0.005, *p < 0.05 unpaired two tailed t-test compared to wt.</p

Mosquito stage development of <i>P</i>. <i>berghei</i> mutant parasites.

<p>(A) <i>In vitro</i> ookinete conversion of mutant <i>P</i>. <i>berghei</i> parasites as compared to wt. The error is given as the SD of n = 3 independent biological replicates. P-value **p < 0.005 unpaired two tailed t-test compared to wt. (B) Number of mature oocysts at 7–12 days post-<i>P</i>. <i>berghei</i> mutant parasite-infected blood feed in mosquito mid guts. n = 40 mosquitoes cumulative of two independent biological replicates. P-values ****p < 0.00005, ***p < 0.0005, **p < 0.005, *p < 0.05 unpaired two tailed t-test compared to wt. (C) <i>in vitro</i> ookinete conversion assay to measure fertility of <i>aco</i>^- <i>P</i>. <i>berghei</i> gametocytes. Fertility of <i>aco</i>^- <i>P</i>. <i>berghei</i> gametocytes was analysed by their capacity to form ookinetes by crossing gametes with RMgm-348 (Pb270, <i>p47</i>^<i>-</i>) which produces viable male gametes but non-viable female gametes and RMgm-15 (Pb137, <i>p48/45</i>^<i>-</i>) which produces viable female gametes but non-viable male gametes. <i>p47</i>^<i>-</i> and <i>p48/45</i>^<i>-</i> self crosses serve as negative controls and <i>p47</i>^<i>-</i> x <i>p48/45</i>^<i>-</i> cross is the positive control. <i>aco</i>^- cross with either <i>p47</i>^<i>-</i> or <i>p48/45</i>^<i>-</i> did not produce any ookinetes. The error is given as the S.D. of n = 2 independent biological replicates. P values ***p<0.0005, **p<0.005 unpaired two tailed t-test compared to cross <i>p47—</i>x <i>p48/45 –</i>. (D) Ookinete motility assay. Mature ookinetes were embedded in matrigel and tracks were constructed on Image J. Displacement in 10.5 min was calculated for ookinetes moving in a straight line and represented as speed of motility in μm/min. (n = mean 40 ookinetes).</p

U-¹³C-glucose and U-¹³C¹⁵N-glutamine labelling of glycolytic and TCA cycle intermediates in <i>P</i>. <i>berghei</i> gametocytes during activation.

<p>(A) Gametocytes were activated and then metabolically labelled with U-¹³C-glucose and U-¹³C¹⁵N-glutamine for indicated times during activation (0, 10, 20, 30 min post activation). Percentage labelling (mol% containing one or more ¹³C carbons after correction for natural abundance) in indicated metabolites was determined by GC-MS. (B) Fractional labelling of TCA-cycle intermediates in unactivated gametocytes (0 min) and activated gametes (30 min post activation) cultured in the presence of U-¹³C-glucose and U-¹³C¹⁵N-glutamine. The x-axis indicates the number of ¹³C atoms in each metabolite (the ion used to analyse aspartate contains 3 of the 4 carbons as the 4-carbon fragment was below the limit of quantification). Due to the presence of a labelled nitrogen atom when labelling with U-¹³C¹⁵N-glutamine, the isotopologue analyses of nitrogen-containing metabolites include an isotope 1 dalton higher than the U-¹³C-glucose equivalent; i.e. aspartate (Asp, +4), glutamine/glutamate (Glx, +6) and ɣ-aminobutyric acid (GABA, +5). Error bars indicate SD of n = 3 biological replicates. Abbreviations are same as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006094#ppat.1006094.g001" target="_blank">Fig 1</a>.</p