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Kilias, E. S.

Publications and source records attributed to Kilias, E. S..

6 recordsLinked to original sources

Pre-existing genome architecture partitions viral takeover in the smallest free-living eukaryote

Nuclear DNA viruses colonize host genomes already structured into complex spatial domains, yet whether established spatial boundaries dictate viral progression remains unresolved--in part because sequence composition and transcriptional output are intertwined in animal models. Here, we tracked productive lytic infection of the marine picoeukaryote Ostreococcus tauri by the giant double-stranded DNA prasinovirus OtV5, recording transcription, accessibility, and 3D contact dynamics across parallel time courses. Although total cellular transcript pools collapsed 18-fold by 10 h post-infection, the host transcriptional shutdown, decay of insulation, early chromatin opening, and viral DNA positioning were all bounded by architectural coordinates defined prior to viral arrival. Across two low-GC outlier domains, chromatin accessibility spiked rapidly, spatial insulation eroded, and loci exhibited relative upregulation by 6 hpi, with the smaller outlier region retaining elevated transcript levels per cell compared to the rest of the genome. Unexpectedly, viral DNA was preferentially depleted from these accessible outlier cores, contacting instead the standard-GC telomeric flanks situated immediately beyond the pre-existing spatial boundaries. Domain identity, rather than local accessibility or active host expression, thus governs viral positioning, demonstrating that pre-existing nuclear architecture partitions the physical and transcriptional takeover of the host cell.

evolutionary biology↗

Long-read single-cell genomics: resolving chimeras in multiple displacement amplification

Multiple displacement amplification (MDA) enables whole-genome amplification from single cells, but introduces chimeric artifacts that severely compromise downstream analyses, particularly with long-read sequencing. Here, we systematically evaluate long-read PacBio HiFi sequencing of MDA amplified DNA from single cells using the model green alga Chlamydomonas reinhardtii. We show that MDA-derived libraries exhibit highly uneven coverage and extreme chimera rates impacting up to 70% of reads, leading to thousands of artefactual structural variants and misassemblies when assembled using algorithms designed for bulk sequencing. To overcome these challenges, we developed lrSAGA (long-read Single Amplified Genome Assembly), a novel tool to assemble long-read MDA sequencing datasets. Assemblies generated using lrSAGA are more complete, more contiguous, and have 75-95% fewer misassemblies compared to conventional assembly algorithms. Although overall contiguity is limited by MDA coverage dropouts, we demonstrate that up to 68% of the C. reinhardtii genome can be accurately assembled from just a single haploid cell. We further validated lrSAGA using published Oxford Nanopore and PacBio HiFi data from single or half Caenorhabditis elegans worms, generating accurate and highly complete assemblies. Applying our approach to single protist cells isolated from environmental water samples, we performed PacBio HiFi single-cell genome sequencing of four uncultivated microbial eukaryotes: an amoeboflagellate from the Naegleria genus, a flagellate from the Bodo genus, and two deep-branching flagellates from the enigmatic CRuMs supergroup, Collodictyon triciliatum and Diphylleia rotans. From single cells, we generated high-quality draft genome assemblies estimated to be 70-84% complete, demonstrating the potential of long-read single-cell genomics to unlock genome diversity from uncultivated microbial eukaryotes.

genomics↗

Single-cell sequencing reveals unexpected genetic diversity among Bodo spp. flagellates and their bacterial endosymbionts

Bodo is a cosmopolitan genus of free living bacterivorous single-celled flagellates in the class Kinetoplastea. Members of genus Bodo are considered the closest free-living relatives to the parasitic lineages Trypanosoma and Leishmania, the causative agents of the human diseases sleeping sickness, Chagas disease, and leishmaniasis. Currently, a single genome exists for the one formally described species in the genus, Bodo saltans. Previous studies on B. saltans have shown that it is dependent on an endosymbiotic bacterium from the order Holosporales, "Candidatus Bodocaedibacter vickermanii". Using single cell-sequencing, we isolated, sequenced, and assembled genomes for seven uncultured Bodo spp. cells from a single freshwater sample from Royal Leamington Spa, UK. By using comparative genomics, we show that these seven cells represent three potentially novel Bodo species and exhibit unexpected levels of diversity at the genome level. Our results indicate that Small Subunit (SSU) rDNA sequencing, often used to classify Bodo flagellates, is insufficient for determining species delimitation in this genus. In addition, we recovered a Holosporales bacterium genome from all seven Bodo spp. cells. Surprisingly, these seven bacterial endosymbionts also represent three potentially novel species and one novel genus of Holosporales bacteria. This diversity would be indistinguishable in routinely-used SSU ribosomal DNA (rDNA) metabarcoding or bulk sequencing pipelines, thus demonstrating the utility of using single-cell sequencing to reveal the level of genomic diversity within lineages of microbial eukaryotes and their cobionts.

microbiology↗

Immune-like glycan-sensing and horizontally-acquired glycan-processing orchestrate host control in a microbial endosymbiosis

Endosymbiosis was a key factor in the evolution of eukaryotic cellular complexity. Yet the mechanisms that allow host regulation of intracellular symbionts, a pre-requisite for stable endosymbiosis and subsequent organelle evolution, are largely unknown. Here, we describe an immune-like glycan-sensing/processing network, partly assembled through horizontal gene-transfers (HGTs), that enables Paramecium bursaria to control its algal endosymbionts. Using phylogenetics, RNA-interference (RNAi), and metabolite exposure experiments, we show that P. bursaria regulates endosymbiont destruction using glycan-sensing/processing - a system that includes a eukaryotic-wide chitin-binding chitinase-like protein (CLP) localized to the host phago-lysosome. RNAi of CLP alters expression of eight glycan-processing genes, including two prokaryote-derived HGTs, during endosymbiont destruction. Furthermore, glycan-sensing/processing dynamically regulates endosymbiont number in P. bursaria, plasticity crucial to maximize host fitness across ecological conditions. CLP is homologous to a human phagocyte-associated innate immune factor, revealing how immune functions can be alternatively adapted and expanded, partly through HGT, enabling endosymbiotic control. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=186 HEIGHT=200 SRC="FIGDIR/small/613017v2_ufig1.gif" ALT="Figure 1"> View larger version (77K): org.highwire.dtl.DTLVardef@13aeaaorg.highwire.dtl.DTLVardef@302e6dorg.highwire.dtl.DTLVardef@138224forg.highwire.dtl.DTLVardef@54aca8_HPS_FORMAT_FIGEXP M_FIG C_FIG

evolutionary biology↗

De novo genome sequence assembly of the RNAi-tractable endosymbiosis model system Paramecium bursaria 186b reveals factors shaping intron repertoire

How two species engage in stable endosymbiosis is a biological quandary. The study of facultative endosymbiotic interactions has emerged as a useful approach to understand how endosymbiotic functions can arise. The ciliate protist Paramecium bursaria hosts green algae of the order Chlorellales in a facultative photo-endosymbiosis. We have recently reported RNAi as a tool for understanding gene function in Paramecium bursaria 186b, CCAP strain 1660/18 [1]. To complement this work, here we report a highly complete host genome and transcriptome sequence dataset, using both Illumina and PacBio sequencing methods to aid genome analysis and to enable the design of RNAi experiments. Our analyses demonstrate Paramecium bursaria, like other ciliates such as diverse species of Paramecia, possess numerous tiny introns. These data, combined with the alternative genetic code common to ciliates, makes gene identification and annotation challenging. To explore intron evolutionary dynamics further we show that alternative splicing leading to intron retention occurs at a higher frequency among the smaller number of longer introns, identifying a source of selection against longer introns. These data will aid the investigation of genome evolution in the Paramecia and provide additional source data for the exploration of endosymbiotic functions.

genomics↗

Identification of a Non-Canonical Ciliate Nuclear Genetic Code Where UAA and UAG Code for Different Amino Acids

The genetic code is one of the most highly conserved features across life. Only a few lineages have deviated from the "universal" genetic code. Amongst the few variants of the genetic code reported to date, the codons UAA and UAG virtually always have the same translation, suggesting that their evolution is coupled. Here, we report the genome and transcriptome sequencing of a novel ciliate, belonging to the Oligohymenophorea class, where the translation of the UAA and UAG stop codons have changed to specify different amino acids. Genomic and transcriptomic analyses revealed that UAA has been reassigned to encode lysine, while UAG has been reassigned to encode glutamic acid. We identified multiple suppressor tRNA genes with anticodons complementary to the reassigned codons. We show that the retained UGA stop codon is enriched in the 3UTR immediately downstream of the coding region of genes, suggesting that there is functional drive to maintain tandem stop codons. Using a phylogenomics approach, we reconstructed the ciliate phylogeny and mapped genetic code changes, highlighting the remarkable number of independent genetic code changes within the Ciliophora group of protists. According to our knowledge, this is the first report of a genetic code variant where UAA and UAG encode different amino acids.

genomics↗