Punctuated chromatin states regulate Plasmodium falciparum antigenic variation at the intron and 2 kb upstream regions

2 kb upstream region FAIRE-Seq signal comparison between var genes and different gene families (P-value is calculated based on Wilcoxon-Rank-Sum test, FDR indicates false discovery rate). (XLSX 53 kb

Quantitative insertion-site sequencing (QIseq) for high throughput phenotyping of transposon mutants

Genetic screening using random transposon insertions has been a powerful tool for uncovering biology in prokaryotes, where whole-genome saturating screens have been performed in multiple organisms. In eukaryotes, such screens have proven more problematic, in part because of the lack of a sensitive and robust system for identifying transposon insertion sites. We here describe quantitative insertion-site sequencing, or QIseq, which uses custom library preparation and Illumina sequencing technology and is able to identify insertion sites from both the 5' and 3' ends of the transposon, providing an inbuilt level of validation. The approach was developed using piggyBac mutants in the human malaria parasite Plasmodium falciparum but should be applicable to many other eukaryotic genomes. QIseq proved accurate, confirming known sites in >100 mutants, and sensitive, identifying and monitoring sites over a >10,000-fold dynamic range of sequence counts. Applying QIseq to uncloned parasites shortly after transfections revealed multiple insertions in mixed populations and suggests that >4000 independent mutants could be generated from relatively modest scales of transfection, providing a clear pathway to genome-scale screens in P. falciparum QIseq was also used to monitor the growth of pools of previously cloned mutants and reproducibly differentiated between deleterious and neutral mutations in competitive growth. Among the mutants with fitness defects was a mutant with a piggyBac insertion immediately upstream of the kelch protein K13 gene associated with artemisinin resistance, implying mutants in this gene may have competitive fitness costs. QIseq has the potential to enable the scale-up of piggyBac-mediated genetics across multiple eukaryotic systems

Malaria infected red blood cells release small regulatory RNAs through extracellular vesicles

The parasite Plasmodium falciparum causes the most severe form of malaria. Cell communication between parasites is an important mechanism to control population density and differentiation. The infected red blood cells (iRBCs) release small extracellular vesicles (EVs) that transfer cargoes between cells. The EVs synchronize the differentiation of the asexual parasites into gametocytes to initiate the transmission to the mosquito. Beside their role in parasite communication, EVs regulate vascular function. So far, the exact cargoes responsible for cellular communication remain unknown. We isolated EVs from cultured iRBCs to determine their small RNA content. We identified several types of human and plasmodial regulatory RNAs. While the miRNAs and tRNA-derived fragments were the most abundant human RNAs, we also found Y-RNAs, vault RNAs, snoRNAs and piRNAs. Interestingly, we found about 120 plasmodial RNAs, including mRNAs coding for exported proteins and proteins involved in drug resistance, as well as non-coding RNAs, such as rRNAs, small nuclear (snRNAs) and tRNAs. These data show, that iRBC-EVs carry small regulatory RNAs. A role in cellular communication is possible since the RNAs were transferred to endothelial cells. Furthermore, the presence of Plasmodium RNAs, in EVs suggests that they may be used as biomarker to track and detect disease

Uncovering the essential genes of the human malaria parasite Plasmodium falciparum by saturation mutagenesis

Malaria is caused by eukaryotic Plasmodium spp. parasites that classically infect red blood cells. These are difficult organisms to investigate genetically because of their AT-rich genomes. Zhang et al. have exploited this peculiarity by using piggyBac transposon insertion sites to achieve saturation-level mutagenesis for identifying and ranking essential genes and drug targets (see the Perspective by White and Rathod). Genes that are current candidates for drug targets were identified as essential, in contrast to many vaccine target genes. Notably, the proteasome degradation pathway was confirmed as a target for developing therapeutic interventions because of the several essential genes involved and the link to the mechanism of action of the current frontline drug, artemisinin

Chemogenomic profiling of a Plasmodium falciparum transposon mutant library reveals shared effects of dihydroartemisinin and bortezomib on lipid metabolism and exported proteins

The antimalarial activity of the frontline drug artemisinin involves generation of reactive oxygen species (ROS) leading to oxidative damage of parasite proteins. To achieve homeostasis and maintain protein quality control in the overwhelmed parasite, the ubiquitin-proteasome system kicks in. Even though molecular markers for artemisinin resistance like pfkelch13 have been identified, the intricate network of mechanisms driving resistance remains to be elucidated. Here, we report a forward genetic screening strategy that enables a broader identification of genetic factors responsible for altering sensitivity to dihydroartemisinin (DHA) and a proteasome inhibitor, bortezomib (BTZ). Using a library of isogenic piggyBac mutants in P. falciparum, we defined phenotype-genotype associations influencing drug responses and highlighted shared mechanisms between the two processes, which mainly included proteasome-mediated degradation and the lipid metabolism genes. Additional transcriptomic analysis of a DHA/BTZ-sensitive piggyBac mutant showed it is possible to find differences between the two response mechanisms on the specific components for regulation of the exportome. Our results provide further insight into the molecular mechanisms of antimalarial drug resistance

Genome sequence of the necrotrophic plant pathogen Pythium ultimum reveals original pathogenicity mechanisms and effector repertoire

Background: Pythium ultimum (P. ultimum) is a ubiquitous oomycete plant pathogen responsible for a variety of diseases on a broad range of crop and ornamental species. Results: The P. ultimum genome (42.8 Mb) encodes 15,290 genes and has extensive sequence similarity and synteny with related Phytophthora species, including the potato blight pathogen Phytophthora infestans. Whole transcriptome sequencing revealed expression of 86% of genes, with detectable differential expression of suites of genes under abiotic stress and in the presence of a host. The predicted proteome includes a large repertoire of proteins involved in plant pathogen interactions although surprisingly, the P. ultimum genome does not encode any classical RXLR effectors and relatively few Crinkler genes in comparison to related phytopathogenic oomycetes. A lower number of enzymes involved in carbohydrate metabolism were present compared to Phytophthora species, with the notable absence of cutinases, suggesting a significant difference in virulence mechanisms between P. ultimum and more host specific oomycete species. Although we observed a high degree of orthology with Phytophthora genomes, there were novel features of the P. ultimum proteome including an expansion of genes involved in proteolysis and genes unique to Pythium. We identified a small gene family of cadherins, proteins involved in cell adhesion, the first report in a genome outside the metazoans. Conclusions: Access to the P. ultimum genome has revealed not only core pathogenic mechanisms within the oomycetes but also lineage specific genes associated with the alternative virulence and lifestyles found within the pythiaceous lineages compared to the Peronosporaceae

A SLC6 transporter cloned from the lion's mane jellyfish (Cnidaria, Scyphozoa) is expressed in neurons.

In the course of recent comparative genomic studies conducted on nervous systems across the phylogeny, current thinking is leaning in favor of more heterogeneity among nervous systems than what was initially expected. The isolation and characterization of molecular components that constitute the cnidarian neuron is not only of interest to the physiologist but also, on a larger scale, to those who study the evolution of nervous systems. Understanding the function of those ancient neurons involves the identification of neurotransmitters and their precursors, the description of nutrients used by neurons for metabolic purposes and the identification of integral membrane proteins that bind to those compounds. Using a molecular cloning strategy targeting membrane proteins that are known to be present in all forms of life, we isolated a member of the solute carrier family 6 from the scyphozoan jellyfish Cyanea capillata. The phylogenetic analysis suggested that the new transporter sequence belongs to an ancestral group of the nutrient amino acid transporter subfamily and is part of a cluster of cnidarian sequences which may translocate the same substrate. We found that the jellyfish transporter is expressed in neurons of the motor nerve net of the animal. To this end, we established an in situ hybridization protocol for the tissues of C. capillata and developed a specific antibody to the jellyfish transporter. Finally, we showed that the gene that codes for the jellyfish transporter also expresses a long non-coding RNA. We hope that this research will contribute to studies that seek to understand what constitutes a neuron in species that belong to an ancient phylum

De novo sequence assembly of Albugo candida reveals a small genome relative to other biotrophic oomycetes

Background: Albugo candida is a biotrophic oomycete that parasitizes various species of Brassicaceae, causing a disease (white blister rust) with remarkable convergence in behaviour to unrelated rusts of basidiomycete fungi. Results: A recent genome analysis of the oomycete Hyaloperonospora arabidopsidis suggests that a reduction in the number of genes encoding secreted pathogenicity proteins, enzymes for assimilation of inorganic nitrogen and sulphur represent a genomic signature for the evolution of obligate biotrophy. Here, we report a draft reference genome of a major crop pathogen Albugo candida (another obligate biotrophic oomycete) with an estimated genome of 45.3 Mb. This is very similar to the genome size of a necrotrophic oomycete Pythium ultimum (43 Mb) but less than half that of H. arabidopsidis (99 Mb). Sequencing of A. candida transcripts from infected host tissue and zoosporangia combined with genome-wide annotation revealed 15,824 predicted genes. Most of the predicted genes lack significant similarity with sequences from other oomycetes. Most intriguingly, A. candida appears to have a much smaller repertoire of pathogenicity-related proteins than H. arabidopsidis including genes that encode RXLR effector proteins, CRINKLER-like genes, and elicitins. Necrosis and Ethylene inducing Peptides were not detected in the genome of A. candida. Putative orthologs of tat-C, a component of the twin arginine translocase system, were identified from multiple oomycete genera along with proteins containing putative tatsecretion signal peptides. Conclusion: Albugo candida has a comparatively small genome amongst oomycetes, retains motility of sporangial inoculum, and harbours a much smaller repertoire of candidate effectors than was recently reported for H. arabidopsidis. This minimal gene repertoire could indicate a lack of expansion, rather than a reduction, in the number of genes that signify the evolution of biotrophy in oomycetes

Ancient origin of elicitin gene clusters in Phytophthora genomes

The genus Phytophthora belongs to the oomycetes in the eukaryotic stramenopile lineage and is comprised of over 65 species that are all destructive plant pathogens on a wide range of dicotyledons. Phytophthora produces elicitins (ELIs), a group of extracellular elicitor proteins that cause a hypersensitive response in tobacco. Database mining revealed several new classes of elicitin-like (ELL) sequences with diverse elicitin domains in Phytophthora infestans, Phytophthora sojae, Phytophthora brassicae, and Phytophthora ramorum. ELIs and ELLs were shown to be unique to Phytophthora and Pythium species. They are ubiquitous among Phytophthora species and belong to one of the most highly conserved and complex protein families in the Phytophthora genus. Phylogeny construction with elicitin domains derived from 156 ELIs and ELLs showed that most of the diversified family members existed prior to divergence of Phytophthora species from a common ancestor. Analysis to discriminate diversifying and purifying selection showed that all 17 ELI and ELL clades are under purifying selection. Within highly similar ELI groups there was no evidence for positively selected amino acids suggesting that purifying selection contributes to the continued existence of this diverse protein family. Characteristic cysteine spacing patterns were found for each phylogenetic clade. Except for the canonical clade ELI-1, ELIs and ELLs possess C-terminal domains of variable length, many of which have a high threonine, serine, or proline content suggesting an association with the cell wall. In addition, some ELIs and ELLs have a predicted glycosylphosphatidylinositol site suggesting anchoring of the C-terminal domain to the cell membrane. The eli and ell genes belonging to different clades are clustered in the genomes. Overall, eli and ell genes are expressed at different levels and in different life cycle stages but those sharing the same phylogenetic clade appear to have similar expression patterns.This work was financially supported by grants to F.G. from Nederlandse Organisatie voor Wetenschappelijk Onderzoek Aspasia (015.000.057), to B.M.T. from National Resources Inventory–United States Department of Agriculture Cooperative State Research, Education and Extension Service (00-52100-9684 and 2002-35600-12747) and National Science Foundation (MCB-0242131) and by funding to B.M.T. from the Virginia Bioinformatics Institute, the US Department of Energy Joint Genome Institute and the Willie Commelin Scholten Foundation. S.C.W. was financially supported by the Scottish Executive Environment and Rural Affairs Department, and R.H.Y.J. received a travel grant from the Stichting Technische etenschappen for attending the Phytophthora annotation jamboree