Merit of integrating in situ transcriptomics and anatomical information for cell annotation and lineage construction in single-cell analyses of Populus.

Cell type annotation and lineage construction are two of the most critical tasks conducted in the analyses of single-cell RNA sequencing (scRNA-seq). Four recent scRNA-seq studies of differentiating xylem propose four models on differentiating xylem development in Populus. The differences are mostly caused by the use of different strategies for cell type annotation and subsequent lineage interpretation. Here, we emphasize the necessity of using in situ transcriptomes and anatomical information to construct the most plausible xylem development model.

Daily turnover of active giant virus infection during algal blooms revealed by single-cell transcriptomics.

Giant viruses infect many unicellular eukaryotes, including algae that form massive oceanic blooms. Despite the major impact of viruses on the marine ecosystem, the ability to quantify and assess active viral infection in nature remains a major challenge. We applied single-cell RNA sequencing, to profile virus and host transcriptomes of 12,000 single algal cells from a coccolithophore bloom. Viral infection was detected already at early exponential bloom phase, negatively correlating with the bloom intensity. A consistent percent of infected coccolithophores displayed the early phase of viral replication for several consecutive days, indicating a daily turnover and continuous virocell-associated metabolite production, potentially affecting the surrounding microbiome. Linking single-cell infection state to host physiology revealed that infected cells remained calcified even in the late infection stage. These findings stress the importance of studying host-virus dynamics in natural populations, at single-cell resolution, to better understand virus life cycle and its impact on microbial food webs.

Widespread Distribution and Evolution of Poxviral Entry-Fusion Complex Proteins in Giant Viruses.

Poxviruses are known to encode a set of proteins that form an entry-fusion complex (EFC) to mediate virus entry. However, the diversity, evolution, and origin of these EFC proteins remain poorly understood. Here, we identify the EFC protein homologs in poxviruses and other giant viruses of the phylum Nucleocytoviricota. The 11 EFC genes are present in almost all poxviruses, with the two smallest, G3 and O3, being absent in Entomopoxvirinae and basal lineages of Chordopoxvirinae. Five of the EFC genes are further grouped into two families, A16/G9/J5 and F9/L1, which are widely distributed across other major lineages of Nucleocytoviricota, including metagenome-assembled genomes, but are generally absent in viruses infecting algae or nonamoebozoan heterotrophic protists. The A16/G9/J5 and F9/L1 families cooccur, mostly as single copies, in 93% of the non-Poxviridae giant viruses that have at least one of them. Distribution and phylogenetic patterns suggest that both families originated in the ancestor of Nucleocytoviricota. In addition to the Poxviridae genes, homologs from each of the other Nucleocytoviricota families are largely clustered together, suggesting their ancient presence and vertical inheritance. Despite deep sequence divergences, we observed noticeable conservation of cysteine residues and predicted structures between EFC proteins of Poxviridae and other families. Overall, our study reveals widespread distribution of these EFC protein homologs beyond poxviruses, implies the existence of a conserved membrane fusion mechanism, and sheds light on host range and ancient evolution of Nucleocytoviricota. IMPORTANCE Fusion between virus and host membranes is critical for viruses to release genetic materials and to initiate infection. Whereas most viruses use a single protein for membrane fusion, poxviruses employ a multiprotein entry-fusion complex (EFC). We report that two major families of the EFC proteins are widely distributed within the virus phylum Nucleocytoviricota, which includes poxviruses and other double-stranded (dsDNA) giant viruses that infect animals, amoebozoans, algae, and various microbial eukaryotes. Each of these two protein families is structurally conserved, traces its origin to the root of Nucleocytoviricota, was passed down to the major subclades of Nucleocytoviricota, and is retained in most giant viruses known to infect animals and amoebozoans. The EFC proteins therefore represent a potential mechanism for virus entry in diverse giant viruses. We hypothesize that they may have facilitated the infection of an animal/amoebozoan-like host by the last Nucleocytoviricota common ancestor.

Single-cell transcriptomics unveils xylem cell development and evolution.

BACKGROUND: Xylem, the most abundant tissue on Earth, is responsible for lateral growth in plants. Typical xylem has a radial system composed of ray parenchyma cells and an axial system of fusiform cells. In most angiosperms, fusiform cells comprise vessel elements for water transportation and libriform fibers for mechanical support, while both functions are performed by tracheids in other vascular plants such as gymnosperms. Little is known about the developmental programs and evolutionary relationships of these xylem cell types. RESULTS: Through both single-cell and laser capture microdissection transcriptomic profiling, we determine the developmental lineages of ray and fusiform cells in stem-differentiating xylem across four divergent woody angiosperms. Based on cross-species analyses of single-cell clusters and overlapping trajectories, we reveal highly conserved ray, yet variable fusiform, lineages across angiosperms. Core eudicots Populus trichocarpa and Eucalyptus grandis share nearly identical fusiform lineages, whereas the more basal angiosperm Liriodendron chinense has a fusiform lineage distinct from that in core eudicots. The tracheids in the basal eudicot Trochodendron aralioides, an evolutionarily reversed trait, exhibit strong transcriptomic similarity to vessel elements rather than libriform fibers. CONCLUSIONS: This evo-devo framework provides a comprehensive understanding of the formation of xylem cell lineages across multiple plant species spanning over a hundred million years of evolutionary history.

Complete Genome Sequence of Phaeobacter sp. Strain G2, Isolated from a Lab Culture of the Calcifying Alga Emiliania huxleyi.

We report the complete genome sequence of Phaeobacter sp. strain G2, which was isolated from a lab culture of the coccolithophore alga Emiliania huxleyi strain RCC 1216. The 4,963,472-bp sequence has a G+C content of 58.85% and was assembled using Illumina short reads and Oxford Nanopore Technologies long reads.

Rampant nuclear-mitochondrial-plastid phylogenomic discordance in globally distributed calcifying microalgae.

Incongruent phylogenies have been widely observed between nuclear and plastid or mitochondrial genomes in terrestrial plants and animals. However, few studies have examined these patterns in microalgae or the discordance between the two organelles. Here we investigated the nuclear-mitochondrial-plastid phylogenomic incongruence in Emiliania-Gephyrocapsa, a group of cosmopolitan calcifying phytoplankton with enormous populations and recent speciations. We assembled mitochondrial and plastid genomes of 27 strains from across global oceans and temperature regimes, and analyzed the phylogenomic histories of the three compartments using concatenation and coalescence methods. Six major clades with varying morphology and distribution are well recognized in the nuclear phylogeny, but such relationships are absent in the mitochondrial and plastid phylogenies, which also differ substantially from each other. The rampant phylogenomic discordance is due to a combination of organellar capture (introgression), organellar genome recombination, and incomplete lineage sorting of ancient polymorphic organellar genomes. Hybridization can lead to replacements of whole organellar genomes without introgression of nuclear genes and the two organelles are not inherited as a single cytoplasmic unit. This study illustrates the convoluted evolution and inheritance of organellar genomes in isogamous haplodiplontic microalgae and provides a window into the phylogenomic complexity of marine unicellular eukaryotes.

Unraveling gene content variation across eukaryotic giant viruses based on network analyses and host associations.

The nucleocytoplasmic large DNA viruses (NCLDVs, phylum Nucleocytoviricota) infect vertebrates, invertebrates, algae, amoebae, and other unicellular organisms across supergroups of eukaryotes and in various ecosystems. The expanding collection of their genome sequences has revolutionized our view of virus genome size and coding capacity. Phylogenetic trees based on a few core genes are commonly used as a model to understand their evolution. However, the tree topology can differ between analyses, and the vast majority of encoded genes might not share a common evolutionary history. To explore the whole-genome variation and evolution of NCLDVs, we dissected their gene contents using clustering, network, and comparative analyses. Our updated core-gene tree served as a framework to classify NCLDVs into families and intrafamilial lineages, but networks of individual genomes and family pangenomes showed patterns of gene sharing that contradict with the tree topology, in particular at higher taxonomic levels. Clustering of NCLDV genomes revealed variable granularity and degrees of gene sharing within each family, which cannot be inferred from the tree. At the level of NCLDV families, a correlation exists between gene content variation, but not core-gene sequence divergence, and host supergroup diversity. In addition, there is significantly higher gene sharing between divergent viruses that infect similar host types. The identified shared genes would be a useful resource for further functional analyses of NCLDV-host interactions. Overall this study provides a comprehensive view of gene repertoire variation in NCLDVs at different taxonomic levels, as well as a novel approach to studying the extremely diverse giant virus genomes.

Giant Virus-Eukaryote Interactions as Ecological and Evolutionary Driving Forces.

Giant DNA viruses of eukaryotes are notable for their extraordinary genome size and coding capacity. Once thought to be oddities in the virus world, these elusive microbes have turned out to be widely occurring in marine, freshwater, and terrestrial ecosystems and are commonly associated with diverse hosts, in particular microbial eukaryotes. This commentary discusses how new sequencing techniques and information can inform us about the interactions between giant viruses and eukaryotic hosts during the viral replication cycle and their implications for ecological and evolutionary processes across different spatiotemporal scales.

Host Range and Coding Potential of Eukaryotic Giant Viruses.

Giant viruses are a group of eukaryotic double-stranded DNA viruses with large virion and genome size that challenged the traditional view of virus. Newly isolated strains and sequenced genomes in the last two decades have substantially advanced our knowledge of their host diversity, gene functions, and evolutionary history. Giant viruses are now known to infect hosts from all major supergroups in the eukaryotic tree of life, which predominantly comprises microbial organisms. The seven well-recognized viral clades (taxonomic families) have drastically different host range. Mimiviridae and Phycodnaviridae, both with notable intrafamilial genome variation and high abundance in environmental samples, have members that infect the most diverse eukaryotic lineages. Laboratory experiments and comparative genomics have shed light on the unprecedented functional potential of giant viruses, encoding proteins for genetic information flow, energy metabolism, synthesis of biomolecules, membrane transport, and sensing that allow for sophisticated control of intracellular conditions and cell-environment interactions. Evolutionary genomics can illuminate how current and past hosts shape viral gene repertoires, although it becomes more obscure with divergent sequences and deep phylogenies. Continued works to characterize giant viruses from marine and other environments will further contribute to our understanding of their host range, coding potential, and virus-host coevolution.

A single-cell view on alga-virus interactions reveals sequential transcriptional programs and infection states.

The discovery of giant viruses infecting eukaryotes from diverse ecosystems has revolutionized our understanding of the evolution of viruses and their impact on protist biology, yet knowledge on their replication strategies and transcriptome regulation remains limited. Here, we profile single-cell transcriptomes of the globally distributed microalga Emiliania huxleyi and its specific giant virus during infection. We detected profound heterogeneity in viral transcript levels among individual cells. Clustering single cells based on viral expression profiles enabled reconstruction of the viral transcriptional trajectory. Reordering cells along this path unfolded highly resolved viral genetic programs composed of genes with distinct promoter elements that orchestrate sequential expression. Exploring host transcriptome dynamics across the viral infection states revealed rapid and selective shutdown of protein-encoding nuclear transcripts, while the plastid and mitochondrial transcriptomes persisted into later stages. Single-cell RNA-seq opens a new avenue to unravel the life cycle of giant viruses and their unique hijacking strategies.

Using single-cell transcriptomics to understand functional states and interactions in microbial eukaryotes.

Understanding the diversity and evolution of eukaryotic microorganisms remains one of the major challenges of modern biology. In recent years, we have advanced in the discovery and phylogenetic placement of new eukaryotic species and lineages, which in turn completely transformed our view on the eukaryotic tree of life. But we remain ignorant of the life cycles, physiology and cellular states of most of these microbial eukaryotes, as well as of their interactions with other organisms. Here, we discuss how high-throughput genome-wide gene expression analysis of eukaryotic single cells can shed light on protist biology. First, we review different single-cell transcriptomics methodologies with particular focus on microbial eukaryote applications. Then, we discuss single-cell gene expression analysis of protists in culture and what can be learnt from these approaches. Finally, we envision the application of single-cell transcriptomics to protist communities to interrogate not only community components, but also the gene expression signatures of distinct cellular and physiological states, as well as the transcriptional dynamics of interspecific interactions. Overall, we argue that single-cell transcriptomics can significantly contribute to our understanding of the biology of microbial eukaryotes. This article is part of a discussion meeting issue 'Single cell ecology'.

Complete Genome Sequence of Sulfitobacter sp. Strain D7, a Virulent Bacterium Isolated from an Emiliania huxleyi Algal Bloom in the North Atlantic.

A Rhodobacterales bacterium, Sulfitobacter sp. strain D7, was isolated from an Emiliania huxleyi bloom in the North Atlantic and has been shown to act as a pathogen and induce cell death of E. huxleyi during lab coculturing. We report here its complete genome sequence comprising one chromosome and five low-copy-number plasmids.

Bacterial virulence against an oceanic bloom-forming phytoplankter is mediated by algal DMSP.

Emiliania huxleyi is a bloom-forming microalga that affects the global sulfur cycle by producing large amounts of dimethylsulfoniopropionate (DMSP) and its volatile metabolic product dimethyl sulfide. Top-down regulation of E. huxleyi blooms has been attributed to viruses and grazers; however, the possible involvement of algicidal bacteria in bloom demise has remained elusive. We demonstrate that a Roseobacter strain, Sulfitobacter D7, that we isolated from a North Atlantic E. huxleyi bloom, exhibited algicidal effects against E. huxleyi upon coculturing. Both the alga and the bacterium were found to co-occur during a natural E. huxleyi bloom, therefore establishing this host-pathogen system as an attractive, ecologically relevant model for studying algal-bacterial interactions in the oceans. During interaction, Sulfitobacter D7 consumed and metabolized algal DMSP to produce high amounts of methanethiol, an alternative product of DMSP catabolism. We revealed a unique strain-specific response, in which E. huxleyi strains that exuded higher amounts of DMSP were more susceptible to Sulfitobacter D7 infection. Intriguingly, exogenous application of DMSP enhanced bacterial virulence and induced susceptibility in an algal strain typically resistant to the bacterial pathogen. This enhanced virulence was highly specific to DMSP compared to addition of propionate and glycerol which had no effect on bacterial virulence. We propose a novel function for DMSP, in addition to its central role in mutualistic interactions among marine organisms, as a mediator of bacterial virulence that may regulate E. huxleyi blooms.

Late Mitochondrial Origin Is an Artifact.

The origin of mitochondria was a crucial event in eukaryote evolution. A recent report claimed to provide evidence, based on branch length variation in phylogenetic trees, that the mitochondrion came late in eukaryotic evolution. Here, we reinvestigate their claim with a reanalysis of the published data. We show that the analyses underpinning a late mitochondrial origin suffer from multiple fatal flaws founded in inappropriate statistical methods and analyses, in addition to erroneous interpretations.

A natural barrier to lateral gene transfer from prokaryotes to eukaryotes revealed from genomes: the 70 % rule.

BACKGROUND: The literature harbors many claims for lateral gene transfer (LGT) from prokaryotes to eukaryotes. Such claims are typically founded in analyses of genome sequences. It is undisputed that many genes entered the eukaryotic lineage via the origin of mitochondria and the origin of plastids. Claims for lineage-specific LGT to eukaryotes outside the context of organelle origins and claims of continuous LGT to eukaryotic lineages are more problematic. If eukaryotes acquire genes from prokaryotes continuously during evolution, then sequenced eukaryote genomes should harbor evidence for recent LGT, like prokaryotic genomes do. RESULTS: Here we devise an approach to investigate 30,358 eukaryotic sequences in the context of 1,035,375 prokaryotic homologs among 2585 phylogenetic trees containing homologs from prokaryotes and eukaryotes. Prokaryote genomes reflect a continuous process of gene acquisition and inheritance, with abundant recent acquisitions showing 80-100 % amino acid sequence identity to their phylogenetic sister-group homologs from other phyla. By contrast, eukaryote genomes show no evidence for either continuous or recent gene acquisitions from prokaryotes. We find that, in general, genes in eukaryotic genomes that share ≥70 % amino acid identity to prokaryotic homologs are genome-specific; that is, they are not found outside individual genome assemblies. CONCLUSIONS: Our analyses indicate that eukaryotes do not acquire genes through continual LGT like prokaryotes do. We propose a 70 % rule: Coding sequences in eukaryotic genomes that share more than 70 % amino acid sequence identity to prokaryotic homologs are most likely assembly or annotation artifacts. The findings further uncover that the role of differential loss in eukaryote genome evolution has been vastly underestimated.

Endosymbiotic origin and differential loss of eukaryotic genes.

Chloroplasts arose from cyanobacteria, mitochondria arose from proteobacteria. Both organelles have conserved their prokaryotic biochemistry, but their genomes are reduced, and most organelle proteins are encoded in the nucleus. Endosymbiotic theory posits that bacterial genes in eukaryotic genomes entered the eukaryotic lineage via organelle ancestors. It predicts episodic influx of prokaryotic genes into the eukaryotic lineage, with acquisition corresponding to endosymbiotic events. Eukaryotic genome sequences, however, increasingly implicate lateral gene transfer, both from prokaryotes to eukaryotes and among eukaryotes, as a source of gene content variation in eukaryotic genomes, which predicts continuous, lineage-specific acquisition of prokaryotic genes in divergent eukaryotic groups. Here we discriminate between these two alternatives by clustering and phylogenetic analysis of eukaryotic gene families having prokaryotic homologues. Our results indicate (1) that gene transfer from bacteria to eukaryotes is episodic, as revealed by gene distributions, and coincides with major evolutionary transitions at the origin of chloroplasts and mitochondria; (2) that gene inheritance in eukaryotes is vertical, as revealed by extensive topological comparison, sparse gene distributions stemming from differential loss; and (3) that continuous, lineage-specific lateral gene transfer, although it sometimes occurs, does not contribute to long-term gene content evolution in eukaryotic genomes.

Endosymbiotic gene transfer from prokaryotic pangenomes: Inherited chimerism in eukaryotes.

Endosymbiotic theory in eukaryotic-cell evolution rests upon a foundation of three cornerstone partners--the plastid (a cyanobacterium), the mitochondrion (a proteobacterium), and its host (an archaeon)--and carries a corollary that, over time, the majority of genes once present in the organelle genomes were relinquished to the chromosomes of the host (endosymbiotic gene transfer). However, notwithstanding eukaryote-specific gene inventions, single-gene phylogenies have never traced eukaryotic genes to three single prokaryotic sources, an issue that hinges crucially upon factors influencing phylogenetic inference. In the age of genomes, single-gene trees, once used to test the predictions of endosymbiotic theory, now spawn new theories that stand to eventually replace endosymbiotic theory with descriptive, gene tree-based variants featuring supernumerary symbionts: prokaryotic partners distinct from the cornerstone trio and whose existence is inferred solely from single-gene trees. We reason that the endosymbiotic ancestors of mitochondria and chloroplasts brought into the eukaryotic--and plant and algal--lineage a genome-sized sample of genes from the proteobacterial and cyanobacterial pangenomes of their respective day and that, even if molecular phylogeny were artifact-free, sampling prokaryotic pangenomes through endosymbiotic gene transfer would lead to inherited chimerism. Recombination in prokaryotes (transduction, conjugation, transformation) differs from recombination in eukaryotes (sex). Prokaryotic recombination leads to pangenomes, and eukaryotic recombination leads to vertical inheritance. Viewed from the perspective of endosymbiotic theory, the critical transition at the eukaryote origin that allowed escape from Muller's ratchet--the origin of eukaryotic recombination, or sex--might have required surprisingly little evolutionary innovation.

Endosymbiotic theory for organelle origins.

Endosymbiotic theory goes back over 100 years. It explains the similarity of chloroplasts and mitochondria to free-living prokaryotes by suggesting that the organelles arose from prokaryotes through (endo)symbiosis. Gene trees provide important evidence in favour of symbiotic theory at a coarse-grained level, but the finer we get into the details of branches in trees containing dozens or hundreds of taxa, the more equivocal evidence for endosymbiotic events sometimes becomes. It seems that either the interpretation of some endosymbiotic events are wrong, or something is wrong with the interpretations of some gene trees having many leaves. There is a need for evidence that is independent of gene trees and that can help outline the course of symbiosis in eukaryote evolution. Protein import is the strongest evidence we have for the single origin of chloroplasts and mitochondria. It is probably also the strongest evidence we have to sort out the number and nature of secondary endosymbiotic events that have occurred in evolution involving the red plastid lineage. If we relax our interpretation of individual gene trees, endosymbiotic theory can tell us a lot.

Molecular evolution of the actin-like MreB protein gene family in wall-less bacteria.

The mreB gene family encodes actin-like proteins that determine cell shape by directing cell wall synthesis and often exists in one to three copies in the genomes of non-spherical bacteria. Intriguingly, while most wall-less bacteria do not have this gene, five to seven mreB homologs are found in Spiroplasma and Haloplasma, which are both characterized by cell contractility. To investigate the molecular evolution of this gene family in wall-less bacteria, we sampled the available genome sequences from these two genera and other related lineages for comparative analysis. The gene phylogenies indicated that the mreB homologs in Haloplasma are more closely related to those in Firmicutes, whereas those in Spiroplasma form a separate clade. This finding suggests that the gene family expansions in these two lineages are the results of independent ancient duplications. Moreover, the Spiroplasma mreB homologs can be classified into five clades, of which the genomic positions are largely conserved. The inference of gene gains and losses suggests that there has been an overall trend to retain only one homolog from each of the five mreB clades in the evolutionary history of Spiroplasma.