Codon usage in the plastid genome of <i>Plasmodium chabaudi chabaudi</i> isolates and <i>Plasmodium falciparum</i>.

a<p>The sum for each codon species present in all the protein coding sequences annotated in the apicoplast DNA.</p>b<p>AB649423.1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061778#pone.0061778-Arisue1" target="_blank">[7]</a>.</p>c<p>HF563595/HF563596 (this work).</p>d<p>X95275.2/X95276.2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061778#pone.0061778-Wilson1" target="_blank">[6]</a>.</p>e<p>Proposed tRNA species decoding the codon <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061778#pone.0061778-Preiser1" target="_blank">[10]</a>.</p>f<p>Termination codon.</p>g<p>It is not certain if tRNA-Ile(GAU) can decode ATC codons to Ile.</p

Alignment of HTS reads of <i>Plasmodium chabaudi chabaudi</i> isolate CB on available sequence data of <i>P. c. c.</i> isolate AS and a mouse single-copy gene.

a<p>Ratio of the average coverage to the average of 14 chromosomal contigs (chab01-14).</p>b<p>These preliminary genomic contigs (dated December 2012) were produced by the Wellcome Trust Sanger Institute and can be obtained from <a href="ftp://ftp.sanger.ac.uk/pub/pathogens/Plasmodium/chabaudi" target="_blank">ftp://ftp.sanger.ac.uk/pub/pathogens/Plasmodium/chabaudi</a>.</p>c<p><i>Plasmodium chabaudi</i> apicoplast DNA, partial sequence <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061778#pone.0061778-Arisue1" target="_blank">[7]</a>.</p>d<p>The genomic locus specifying chymotrypsin-like elastase family, member 2A. GRCm38.p1 C57BL/6J Chromosome: 4; NC_000070.6 (141814963.141826003, complement).</p

The rRNA/tRNA gene cluster of <i>Plasmodium chabaudi chabaudi</i> CB plastid DNA.

<p>(A) Part of the Pcc CB plastid DNA was amplified by PCR from total parasite DNA with the primers 1095 and 1096 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061778#pone-0061778-t004" target="_blank">Table 4</a> for details of each primer). The product was fractionated on an agarose gel along with DNA size markers (M). DNA that appeared as a single band (*) was collected and analyzed further. (B) Alignment of trace data obtained for the 6 kb PCR product by PCR-direct sequencing with primers that anneal at an end (1095, 1096) or an internal position (1005, 853). Three parts of the alignment are presented in boxes with arrows representing the direction of the sequencing reaction starting from each primer; the name of an encoded gene is given with a horizontal arrow representing the direction of transcription. Non-coding sequence and ambiguous sequencing data due to the presence of two different sequences are highlighted with blue and red, respectively. Because <i>rps4</i> and <i>sufB</i> share an identical sequence from the 1st to the 8th nucleotide of their coding sequence, the highlighted region in red starts at the 9th residue in the sequencing data from 1005 and 853. The C/T transition at position 28 of <i>trnT</i>, which is clearly identifiable in the sequencing data analyzed from inside (1005/853) but not in those from outside (1095/1096), is indicated with a vertical arrow. (C and D) Schematic representation of the PCR product. Selected genes in the region including the gene of the unusual tRNA-Arg(ACG) (<i>trnR(ACG)</i>*) and the 5' truncated <i>rrl</i> (<i>'rrl</i>) are indicated with color-coded thick arrows. The PCR product (horizontal thick black bar) amplified with primers 1095 and 1096 (red arrows) was a mixture of two DNA species. Because of the coexistence of two different types of molecule, the quality of sequencing data obtained (blue arrows) abruptly dropped at the end of the short IR sequence (highlighted with pink background) and gave a mixture of two sequences (dotted arrow). The sequence of two <i>trnT</i> genes (<i>trnT</i>-1 and -2) of Pcc CB is almost the same except for the variation at their 28th residue (T and C; circled). Each <i>trnT</i> is linked with its upstream gene but not with its downstream gene. Therefore the 28th residue of the gene was a mixture of C and T when the PCR product was sequenced from the outside toward the inside (C), but the residue was uniquely identified as either C or T when the same sample was sequenced from inside to outside (D). The length of each gene is not to scale with the others in this figure.</p

Proposed origin of the unique Pcc CB plastid DNA.

<p>An intramolecular recombination event probably between <i>trnI</i> and <i>trnV</i> caused partial deletion of one of the two IR units in the ancestral DNA molecule (thick black arrow). The chimeric <i>trnI</i>/<i>trnV</i> in the intermediate molecule (1) was lost probably because it was a pseudogene. Subsequent truncation of the 5' region of <i>rrl</i> in the affected IR unit (thick blue arrow) and degradation of the <i>trnR(ACG)</i> in the other IR unit (thick red arrow) results in the Pcc plastid DNA. The order of these two events as well as which intermediate molecule (2) was generated in this process, are unknown. Finally, differentiation between <i>trnM-1</i> and <i>-2</i> as well as between <i>trnT-1</i> and <i>-2</i> occurred and these resulted in the Pcc CB plastid DNA. Switching between forms A and B could have happened at any point of this process, and may happen frequently as suggested by the fact that the apparent molar ratio between the two forms is 1∶1. Genes are color-coded as in panels A and B, and those changing/changed are indicated with a green circle.</p

Oligonucleotides used for PCR amplification and sequencing.

a<p>Gene containing the annealing site.</p>b<p>Of the target gene.</p

Unique forms of the plastid DNA of <i>Plasmodium chabaudi chabaudi</i> CB and their proposed origin.

<p>Schematic diagram of the two forms of the plastid DNA of Pcc CB. Each gene is represented with a box color-coded in white (one specifying a protein), blue (rRNA gene), yellow (tRNA gene) or red (a pseudogene). The colour of the name of each gene represents the transcription direction (black, clockwise; red, counter-clockwise). The bulge connecting two parts of the <i>trnL</i>(UAA) coding sequence indicates the intron. The nucleotide sequences of the complete Pcc CB plastid DNA in forms A (A) and B (B) were deposited to the DDBJ/EMBL/GenBank databases with the accession numbers HF563595 and HF563596, respectively.</p

Variations identified in the plastid DNA sequence between <i>Plasmodium chabaudi chabaudi</i> isolates CB and AS.

a<p>AB649423.1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061778#pone.0061778-Arisue1" target="_blank">[7]</a>.</p>b<p>–: gap. For variation that was not unique, the two most major variations are given at each side of/.</p>c<p>SNV: single nucleotide variation. Variation that was not unique is shown in parenthesis.</p>d<p>Name of the gene containing each variation. For variation in the non-coding region, nearest genes are shown in parenthesis.</p>e<p>Position in the coding sequence. Position is that in the AS sequence, and those which were not unique are given in parenthesis. –: a variation in a non-coding region.</p>f<p>Change in the amino acid sequence of the CB version caused by each variation. Position is that in the AS sequence. Synonymous variations are shown in parenthesis. –: a variation outside a protein coding sequence.</p

Autophagy-Related Atg8 Localizes to the Apicoplast of the Human Malaria Parasite <em>Plasmodium falciparum</em>

<div><p>Autophagy is a membrane-mediated degradation process, which is governed by sequential functions of Atg proteins. Although Atg proteins are highly conserved in eukaryotes, protozoa possess only a partial set of Atg proteins. Nonetheless, almost all protozoa have the complete factors belonging to the Atg8 conjugation system, namely, Atg3, Atg4, Atg7, and Atg8. Here, we report the biochemical properties and subcellular localization of the Atg8 protein of the human malaria parasite <em>Plasmodium falciparum</em> (PfAtg8). PfAtg8 is expressed during intra-erythrocytic development and associates with membranes likely as a lipid-conjugated form. Fluorescence microscopy and immunoelectron microscopy show that PfAtg8 localizes to the apicoplast, a four membrane-bound non-photosynthetic plastid. Autophagosome-like structures are not observed in the erythrocytic stages. These data suggest that, although <em>Plasmodium</em> parasites have lost most Atg proteins during evolution, they use the Atg8 conjugation system for the unique organelle, the apicoplast.</p> </div

PfAtg8 is associated with the apicoplast membrane.

<p>(A) <i>P. falciparum</i> FCR3 parasites at the schizont stage were analyzed by immunoelectron microscopy (immunogold and silver enhancement method) with an antibody against PfAtg8 (#1). (a) A schizont in an erythrocyte. (b) A magnified image of the area indicated in (a). (c) Another typical image of a PfAtg8-positive structure. (B) <i>P. falciparum</i> transfectant expressing ACP-GFP was analyzed as in panel (A) with an antibody against GFP. A, apicoplast; Mt, mitochondrion. Scale bars, (A, a) 1 μm, (A, b and c, and B) 200 nm.</p

PfAtg8 localizes to the apicoplast.

<p><i>P. falciparum</i> FCR3 (A–E) and <i>P. falciparum</i> 3D7 transfected with ACP-GFP (F–H) were stained with the indicated organelle markers and visualized by confocal microscopy (because ACP-GFP was not uniformly expressed, some merozoites displayed only faint GFP signals). Anti-PfAtg8 antibody #1 was used in (A–F), and anti-PfAtg8 antibody #2 was used in (G). Apical membrane antigen 1 (AMA1) as a microneme marker (A), rhoptry-associated protein 1 (RAP1) as a rhoptry body marker (B), rhoptry neck protein 2 (RON2) as a rhoptry neck marker (C), the ring-infected erythrocyte surface antigen (RESA) as a dense granule marker (D), MitoTrackerRed CMXRos as a mitochondria marker (E), ACP-GFP (F–H) and the organellar histone-like protein PfHU (H) as an apicoplast marker were used. Scale bar, 1 μm.</p