Duplication and neofunctionalization of a horizontally transferred xyloglucanase as a facet of the Red Queen coevolutionary dynamic.

Oomycete protists share phenotypic similarities with fungi, including the ability to cause plant diseases, but branch in a distant region of the tree of life. It has been suggested that multiple horizontal gene transfers (HGTs) from fungi-to-oomycetes contributed to the evolution of plant-pathogenic traits. These HGTs are predicted to include secreted proteins that degrade plant cell walls, a barrier to pathogen invasion and a rich source of carbohydrates. Using a combination of phylogenomics and functional assays, we investigate the diversification of a horizontally transferred xyloglucanase gene family in the model oomycete species Phytophthora sojae. Our analyses detect 11 xyloglucanase paralogs retained in P. sojae. Using heterologous expression in yeast, we show consistent evidence that eight of these paralogs have xyloglucanase function, including variants with distinct protein characteristics, such as a long-disordered C-terminal extension that can increase xyloglucanase activity. The functional variants analyzed subtend a phylogenetic node close to the fungi-to-oomycete transfer, suggesting the horizontally transferred gene was a bona fide xyloglucanase. Expression of three xyloglucanase paralogs in Nicotiana benthamiana triggers high-reactive oxygen species (ROS) generation, while others inhibit ROS responses to bacterial immunogens, demonstrating that the paralogs differentially stimulate pattern-triggered immunity. Mass spectrometry of detectable enzymatic products demonstrates that some paralogs catalyze the production of variant breakdown profiles, suggesting that secretion of variant xyloglucanases increases efficiency of xyloglucan breakdown as well as diversifying the damage-associated molecular patterns released. We suggest that this pattern of neofunctionalization and the variant host responses represent an aspect of the Red Queen host-pathogen coevolutionary dynamic.

Emergent RNA-RNA interactions can promote stability in a facultative phototrophic endosymbiosis.

Eukaryote-eukaryote endosymbiosis was responsible for the spread of chloroplast (plastid) organelles. Stability is required for the metabolic and genetic integration that drives the establishment of new organelles, yet the mechanisms that act to stabilize emergent endosymbioses-between two fundamentally selfish biological organisms-are unclear. Theory suggests that enforcement mechanisms, which punish misbehavior, may act to stabilize such interactions by resolving conflict. However, how such mechanisms can emerge in a facultative endosymbiosis has yet to be explored. Here, we propose that endosymbiont-host RNA-RNA interactions, arising from digestion of the endosymbiont population, can result in a cost to host growth for breakdown of the endosymbiosis. Using the model facultative endosymbiosis between Paramecium bursaria and Chlorella spp., we demonstrate that this mechanism is dependent on the host RNA-interference (RNAi) system. We reveal through small RNA (sRNA) sequencing that endosymbiont-derived messenger RNA (mRNA) released upon endosymbiont digestion can be processed by the host RNAi system into 23-nt sRNA. We predict multiple regions of shared sequence identity between endosymbiont and host mRNA, and demonstrate through delivery of synthetic endosymbiont sRNA that exposure to these regions can knock down expression of complementary host genes, resulting in a cost to host growth. This process of host gene knockdown in response to endosymbiont-derived RNA processing by host RNAi factors, which we term "RNAi collisions," represents a mechanism that can promote stability in a facultative eukaryote-eukaryote endosymbiosis. Specifically, by imposing a cost for breakdown of the endosymbiosis, endosymbiont-host RNA-RNA interactions may drive maintenance of the symbiosis across fluctuating ecological conditions.

Use of Cold-Stored Whole Blood is Associated With Improved Mortality in Hemostatic Resuscitation of Major Bleeding: A Multicenter Study.

OBJECTIVE: The aim of this study was to identify a mortality benefit with the use of whole blood (WB) as part of the resuscitation of bleeding trauma patients. BACKGROUND: Blood component therapy (BCT) is the current standard for resuscitating trauma patients, with WB emerging as the blood product of choice. We hypothesized that the use of WB versus BCT alone would result in decreased mortality. METHODS: We performed a 14-center, prospective observational study of trauma patients who received WB versus BCT during their resuscitation. We applied a generalized linear mixed-effects model with a random effect and controlled for age, sex, mechanism of injury (MOI), and injury severity score. All patients who received blood as part of their initial resuscitation were included. Primary outcome was mortality and secondary outcomes included acute kidney injury, deep vein thrombosis/pulmonary embolism, pulmonary complications, and bleeding complications. RESULTS: A total of 1623 [WB: 1180 (74%), BCT: 443(27%)] patients who sustained penetrating (53%) or blunt (47%) injury were included. Patients who received WB had a higher shock index (0.98 vs 0.83), more comorbidities, and more blunt MOI (all P <0.05). After controlling for center, age, sex, MOI, and injury severity score, we found no differences in the rates of acute kidney injury, deep vein thrombosis/pulmonary embolism or pulmonary complications. WB patients were 9% less likely to experience bleeding complications and were 48% less likely to die than BCT patients ( P <0.0001). CONCLUSIONS: Compared with BCT, the use of WB was associated with a 48% reduction in mortality in trauma patients. Our study supports the use of WB use in the resuscitation of trauma patients.

Environment-dependent fitness gains can be driven by horizontal gene transfer of transporter-encoding genes.

Many microbes acquire metabolites in a "feeding" process where complex polymers are broken down in the environment to their subunits. The subsequent uptake of soluble metabolites by a cell, sometimes called osmotrophy, is facilitated by transporter proteins. As such, the diversification of osmotrophic microorganisms is closely tied to the diversification of transporter functions. Horizontal gene transfer (HGT) has been suggested to produce genetic variation that can lead to adaptation, allowing lineages to acquire traits and expand niche ranges. Transporter genes often encode single-gene phenotypes and tend to have low protein-protein interaction complexity and, as such, are potential candidates for HGT. Here we test the idea that HGT has underpinned the expansion of metabolic potential and substrate utilization via transfer of transporter-encoding genes. Using phylogenomics, we identify seven cases of transporter-gene HGT between fungal phyla, and investigate compatibility, localization, function, and fitness consequences when these genes are expressed in Saccharomyces cerevisiae Using this approach, we demonstrate that the transporters identified can alter how fungi utilize a range of metabolites, including peptides, polyols, and sugars. We then show, for one model gene, that transporter gene acquisition by HGT can significantly alter the fitness landscape of S. cerevisiae We therefore provide evidence that transporter HGT occurs between fungi, alters how fungi can acquire metabolites, and can drive gain in fitness. We propose a "transporter-gene acquisition ratchet," where transporter repertoires are continually augmented by duplication, HGT, and differential loss, collectively acting to overwrite, fine-tune, and diversify the complement of transporters present in a genome.

A distinct lineage of giant viruses brings a rhodopsin photosystem to unicellular marine predators.

Giant viruses are remarkable for their large genomes, often rivaling those of small bacteria, and for having genes thought exclusive to cellular life. Most isolated to date infect nonmarine protists, leaving their strategies and prevalence in marine environments largely unknown. Using eukaryotic single-cell metagenomics in the Pacific, we discovered a Mimiviridae lineage of giant viruses, which infects choanoflagellates, widespread protistan predators related to metazoans. The ChoanoVirus genomes are the largest yet from pelagic ecosystems, with 442 of 862 predicted proteins lacking known homologs. They are enriched in enzymes for modifying organic compounds, including degradation of chitin, an abundant polysaccharide in oceans, and they encode 3 divergent type-1 rhodopsins (VirR) with distinct evolutionary histories from those that capture sunlight in cellular organisms. One (VirRDTS) is similar to the only other putative rhodopsin from a virus (PgV) with a known host (a marine alga). Unlike the algal virus, ChoanoViruses encode the entire pigment biosynthesis pathway and cleavage enzyme for producing the required chromophore, retinal. We demonstrate that the rhodopsin shared by ChoanoViruses and PgV binds retinal and pumps protons. Moreover, our 1.65-Å resolved VirRDTS crystal structure and mutational analyses exposed differences from previously characterized type-1 rhodopsins, all of which come from cellular organisms. Multiple VirR types are present in metagenomes from across surface oceans, where they are correlated with and nearly as abundant as a canonical marker gene from Mimiviridae Our findings indicate that light-dependent energy transfer systems are likely common components of giant viruses of photosynthetic and phagotrophic unicellular marine eukaryotes.

Single-cell genomics unveils a canonical origin of the diverse mitochondrial genomes of euglenozoans.

BACKGROUND: The supergroup Euglenozoa unites heterotrophic flagellates from three major clades, kinetoplastids, diplonemids, and euglenids, each of which exhibits extremely divergent mitochondrial characteristics. Mitochondrial genomes (mtDNAs) of euglenids comprise multiple linear chromosomes carrying single genes, whereas mitochondrial chromosomes are circular non-catenated in diplonemids, but circular and catenated in kinetoplastids. In diplonemids and kinetoplastids, mitochondrial mRNAs require extensive and diverse editing and/or trans-splicing to produce mature transcripts. All known euglenozoan mtDNAs exhibit extremely short mitochondrial small (rns) and large (rnl) subunit rRNA genes, and absence of tRNA genes. How these features evolved from an ancestral bacteria-like circular mitochondrial genome remains unanswered. RESULTS: We sequenced and assembled 20 euglenozoan single-cell amplified genomes (SAGs). In our phylogenetic and phylogenomic analyses, three SAGs were placed within kinetoplastids, 14 within diplonemids, one (EU2) within euglenids, and two SAGs with nearly identical small subunit rRNA gene (18S) sequences (EU17/18) branched as either a basal lineage of euglenids, or as a sister to all euglenozoans. Near-complete mitochondrial genomes were identified in EU2 and EU17/18. Surprisingly, both EU2 and EU17/18 mitochondrial contigs contained multiple genes and one tRNA gene. Furthermore, EU17/18 mtDNA possessed several features unique among euglenozoans including full-length rns and rnl genes, six mitoribosomal genes, and nad11, all likely on a single chromosome. CONCLUSIONS: Our data strongly suggest that EU17/18 is an early-branching euglenozoan with numerous ancestral mitochondrial features. Collectively these data contribute to untangling the early evolution of euglenozoan mitochondria.

Sequenceserver: A Modern Graphical User Interface for Custom BLAST Databases.

Comparing newly obtained and previously known nucleotide and amino-acid sequences underpins modern biological research. BLAST is a well-established tool for such comparisons but is challenging to use on new data sets. We combined a user-centric design philosophy with sustainable software development approaches to create Sequenceserver, a tool for running BLAST and visually inspecting BLAST results for biological interpretation. Sequenceserver uses simple algorithms to prevent potential analysis errors and provides flexible text-based and visual outputs to support researcher productivity. Our software can be rapidly installed for use by individuals or on shared servers.

Cryptic infection of a broad taxonomic and geographic diversity of tadpoles by Perkinsea protists.

The decline of amphibian populations, particularly frogs, is often cited as an example in support of the claim that Earth is undergoing its sixth mass extinction event. Amphibians seem to be particularly sensitive to emerging diseases (e.g., fungal and viral pathogens), yet the diversity and geographic distribution of infectious agents are only starting to be investigated. Recent work has linked a previously undescribed protist with mass-mortality events in the United States, in which infected frog tadpoles have an abnormally enlarged yellowish liver filled with protist cells of a presumed parasite. Phylogenetic analyses revealed that this infectious agent was affiliated with the Perkinsea: a parasitic group within the alveolates exemplified by Perkinsus sp., a "marine" protist responsible for mass-mortality events in commercial shellfish populations. Using small subunit (SSU) ribosomal DNA (rDNA) sequencing, we developed a targeted PCR protocol for preferentially sampling a clade of the Perkinsea. We tested this protocol on freshwater environmental DNA, revealing a wide diversity of Perkinsea lineages in these environments. Then, we used the same protocol to test for Perkinsea-like lineages in livers of 182 tadpoles from multiple families of frogs. We identified a distinct Perkinsea clade, encompassing a low level of SSU rDNA variation different from the lineage previously associated with tadpole mass-mortality events. Members of this clade were present in 38 tadpoles sampled from 14 distinct genera/phylogroups, from five countries across three continents. These data provide, to our knowledge, the first evidence that Perkinsea-like protists infect tadpoles across a wide taxonomic range of frogs in tropical and temperate environments, including oceanic islands.

A Cyclic GMP-Dependent K+ Channel in the Blastocladiomycete Fungus Blastocladiella emersonii.

Phototaxis in flagellated zoospores of the aquatic fungus Blastocladiella emersonii depends on a novel photosensor, Blastocladiella emersonii GC1 (BeGC1), comprising a type I (microbial) rhodopsin fused to a guanylyl cyclase catalytic domain, that produces the conserved second messenger cyclic GMP (cGMP). The rapid and transient increase in cGMP levels during the exposure of zoospores to green light was shown to be necessary for phototaxis and dependent on both rhodopsin function and guanylyl cyclase activity. It is noteworthy that BeGC1 was localized to the zoospore eyespot apparatus, in agreement with its role in the phototactic response. A putative cyclic nucleotide-gated channel (BeCNG1) was also identified in the genome of the fungus and was implicated in flagellar beating via the action of a specific inhibitor (l-cis-diltiazem) that compromised zoospore motility. Here we show that B. emersonii expresses a K(+) channel that is activated by cGMP. The use of specific channel inhibitors confirmed the activation of the channel by cGMP and its K(+) selectivity. These characteristics are consistent with the function of an ion channel encoded by the BeCNG1 gene. Other blastocladiomycete fungi, such as Allomyces macrogynus and Catenaria anguillulae, possess genes encoding a similar K(+) channel and the rhodopsin-guanylyl cyclase fusion protein, while the genes encoding both these proteins are absent in nonflagellated fungi. The presence of these genes as a pair seems to be an exclusive feature of blastocladiomycete fungi. Taken together, these data demonstrate that the B. emersonii cGMP-activated K(+) channel is involved in the control of zoospore motility, most probably participating in the cGMP-signaling pathway for the phototactic response of the fungus.

Molecular diversity and distribution of marine fungi across 130 European environmental samples.

Environmental DNA and culture-based analyses have suggested that fungi are present in low diversity and in low abundance in many marine environments, especially in the upper water column. Here, we use a dual approach involving high-throughput diversity tag sequencing from both DNA and RNA templates and fluorescent cell counts to evaluate the diversity and relative abundance of fungi across marine samples taken from six European near-shore sites. We removed very rare fungal operational taxonomic units (OTUs) selecting only OTUs recovered from multiple samples for a detailed analysis. This approach identified a set of 71 fungal 'OTU clusters' that account for 66% of all the sequences assigned to the Fungi. Phylogenetic analyses demonstrated that this diversity includes a significant number of chytrid-like lineages that had not been previously described, indicating that the marine environment encompasses a number of zoosporic fungi that are new to taxonomic inventories. Using the sequence datasets, we identified cases where fungal OTUs were sampled across multiple geographical sites and between different sampling depths. This was especially clear in one relatively abundant and diverse phylogroup tentatively named Novel Chytrid-Like-Clade 1 (NCLC1). For comparison, a subset of the water column samples was also investigated using fluorescent microscopy to examine the abundance of eukaryotes with chitin cell walls. Comparisons of relative abundance of RNA-derived fungal tag sequences and chitin cell-wall counts demonstrate that fungi constitute a low fraction of the eukaryotic community in these water column samples. Taken together, these results demonstrate the phylogenetic position and environmental distribution of 71 lineages, improving our understanding of the diversity and abundance of fungi in marine environments.

Genome-scale comparative analysis of gene fusions, gene fissions, and the fungal tree of life.

During the course of evolution genes undergo both fusion and fission by which ORFs are joined or separated. These processes can amend gene function and represent an important factor in the evolution of protein interaction networks. Gene fusions have been suggested to be useful characters for identifying evolutionary relationships because they constitute synapomorphies or cladistic characters. To investigate the fidelity of gene-fusion characters, we developed an approach for identifying differentially distributed gene fusions among whole-genome datasets: fdfBLAST. Applying this tool to the Fungi, we identified 63 gene fusions present in two or more genomes. Using a combination of phylogenetic and comparative genomic analyses, we then investigated the evolution of these genes across 115 fungal genomes, testing each gene fusion for evidence of homoplasy, including gene fission, convergence, and horizontal gene transfer. These analyses demonstrated 110 gene-fission events. We then identified a minimum of three mechanisms that drive gene fission: separation, degeneration, and duplication. These data suggest that gene fission plays an important and hitherto underestimated role in gene evolution. Gene fusions therefore are highly labile characters, and their use for polarizing evolutionary relationships, without reference to gene and species phylogenies, is limited. Accounting for these considerable sources of homoplasy, we identified fusion characters that provide support for multiple nodes in the phylogeny of the Fungi, including relationships within the deeply derived flagellum-forming fungi (i.e., the chytrids).

Horizontal gene transfer facilitated the evolution of plant parasitic mechanisms in the oomycetes.

Horizontal gene transfer (HGT) can radically alter the genomes of microorganisms, providing the capacity to adapt to new lifestyles, environments, and hosts. However, the extent of HGT between eukaryotes is unclear. Using whole-genome, gene-by-gene phylogenetic analysis we demonstrate an extensive pattern of cross-kingdom HGT between fungi and oomycetes. Comparative genomics, including the de novo genome sequence of Hyphochytrium catenoides, a free-living sister of the oomycetes, shows that these transfers largely converge within the radiation of oomycetes that colonize plant tissues. The repertoire of HGTs includes a large number of putatively secreted proteins; for example, 7.6% of the secreted proteome of the sudden oak death parasite Phytophthora ramorum has been acquired from fungi by HGT. Transfers include gene products with the capacity to break down plant cell walls and acquire sugars, nucleic acids, nitrogen, and phosphate sources from the environment. Predicted HGTs also include proteins implicated in resisting plant defense mechanisms and effector proteins for attacking plant cells. These data are consistent with the hypothesis that some oomycetes became successful plant parasites by multiple acquisitions of genes from fungi.

Duplication and neofunctionalization of a horizontally-transferred xyloglucanase as a facet of the red queen co-evolutionary dynamic.

Oomycetes are heterotrophic protists that share phenotypic similarities with fungi, including the ability to cause plant diseases, but branch in a separate and distant region of the eukaryotic tree of life. It has been suggested that multiple horizontal gene transfers (HGTs) from fungi-to-oomycetes contributed to the evolution of plant-pathogenic traits. These HGTs are predicted to include secreted proteins that degrade plant cell walls. This is a key trait in the pathology of many oomycetes, as the plant cell wall represents a primary barrier to pathogen invasion and a rich source of carbohydrates. Many of the HGT gene families identified have undergone multiple rounds of duplication. Using a combination of phylogenomic analysis and functional assays, we investigate the diversification of a horizontally-transferred xyloglucanase gene family in the model oomycete species Phytophthora sojae. Our analyses detect 11 genes retained in P. sojae among a complex pattern of gene duplications and losses. Using a phenotype assay, based on heterologous expression in yeast, we show that eight of these paralogs have xyloglucanase function, including variants with distinct protein characteristics, such as a long-disordered C-terminal extension that can increase xyloglucanase activity. The functional xyloglucanase variants analysed subtend an ancestral node close to the fungi-oomycetes gene transfer, suggesting the horizontally-transferred gene was a bona fide xyloglucanase. Expression of xyloglucanase paralogs in Nicotiana benthamiana triggers distinct patterns of reactive oxygen species (ROS) generation, demonstrating that enzyme variants differentially stimulate pattern-triggered immunity in plants. Mass spectrometry of detectable enzymatic products demonstrates that some paralogs catalyze production of variant breakdown profiles, suggesting that secretion of multiple xyloglucanase variants increases efficiency of xyloglucan breakdown, as well as potentially diversifying the range of Damage-Associated Molecular Patterns (DAMPs) released during pathogen attack. We suggest that such patterns of protein neofunctionalization, and variant host responses, represent an aspect of the Red Queen host-pathogen co-evolutionary dynamic. Significance Statement: The oomycetes are a diverse group of eukaryotic microbes that include some of the most devastating pathogens of plants. Oomycetes perceive, invade, and colonize plants in similar ways to fungi, in part because they acquired the genes to attack and feed on plants from fungi. These genes are predicted to be useful to oomycete plant pathogens because they have undergone multiple rounds of gene duplication. One key enzyme for attacking plant cell wall structures is called xyloglucanase. Xyloglucanase in the oomycetes has undergone multiple rounds of gene duplication, leading to variants including an enzyme with a C-terminal extension that increases activity. Some xyloglucanase variants trigger unique patterns of reactive oxygen species (ROS) in planta, and generate different profiles of cell wall breakdown products - such outcomes could act to mystify and increase the workload of the plant immune system, allowing successful pathogens to proliferate.

The history and organization of the Workshop on Population and Speciation Genomics.

With the advent of high-throughput genome sequencing, bioinformatics training has become essential for research in evolutionary biology and related fields. However, individual research groups are often not in the position to teach students about the most up-to-date methodology in the field. To fill this gap, extended bioinformatics courses have been developed by various institutions and provide intense training over the course of two or more weeks. Here, we describe our experience with the organization of a course in one of the longest-running extended bioinformatics series of workshops, the Evomics Workshop on Population and Speciation Genomics that takes place biennially in the UNESCO world heritage town of Ceský Krumlov, Czech Republic. We list the key ingredients that make this workshop successful in our view, explain the routine for workshop organization that we have optimized over the years, and describe the most important lessons that we have learned from it. We report the results of a survey conducted among past workshop participants that quantifies measures of effective teaching and provide examples of how the workshop setting has led to the cross-fertilisation of ideas and ultimately scientific progress. We expect that our account may be useful for other groups aiming to set up their own extended bioinformatics courses.

Whole Blood Resuscitation is Safe in Pediatric Trauma Patients: A Multicenter Study.

INTRODUCTION: Whole blood (WB) resuscitation has been associated with a mortality benefit in trauma patients. Several small series report the safe use of WB in the pediatric trauma population. We performed a subgroup analysis of the pediatric patients from a large prospective multicenter trial comparing patients receiving WB or blood component therapy (BCT) during trauma resuscitation. We hypothesized that WB resuscitation would be safe compared to BCT resuscitation in pediatric trauma patients. METHODS: This study included pediatric trauma patients (0-17 y), from ten level-I trauma centers, who received any blood transfusion during initial resuscitation. Patients were included in the WB group if they received at least one unit of WB during their resuscitation, and the BCT group was composed of patients receiving traditional blood product resuscitation. The primary outcome was in-hospital mortality with secondary outcomes being complications. Multivariate logistic regression was performed to assess for mortality and complications in those treated with WB vs BCT. RESULTS: Ninety patients, with both penetrating and blunt mechanisms of injury (MOI), were enrolled in the study (WB: 62 (69%), BCT: 28 (21%)). Whole blood patients were more likely to be male. There were no differences in age, MOI, shock index, or injury severity score between groups. On logistic regression, there was no difference in complications. Mortality was not different between the groups (P = .983). CONCLUSION: Our data suggest WB resuscitation is safe when compared to BCT resuscitation in the care of critically injured pediatric trauma patients.

A Genome Sequence Assembly of the Phototactic and Optogenetic Model Fungus Blastocladiella emersonii Reveals a Diversified Nucleotide-Cyclase Repertoire.

The chytrid fungus Blastocladiella emersonii produces spores with swimming tails (zoospores); these cells can sense and swim toward light. Interest in this species stems from ongoing efforts to develop B. emersonii as a model for understanding the evolution of phototaxis and the molecular cell biology of the associated optogenetic circuits. Here, we report a highly contiguous genome assembly and gene annotation of the B. emersonii American Type Culture Collection 22665 strain. We integrate a PacBio long-read library with an Illumina paired-end genomic sequence survey leading to an assembly of 21 contigs totaling 34.27 Mb. Using these data, we assess the diversity of sensory system encoding genes. These analyses identify a rich complement of G-protein-coupled receptors, ion transporters, and nucleotide cyclases, all of which have been diversified by domain recombination and tandem duplication. In many cases, these domain combinations have led to the fusion of a protein domain to a transmembrane domain, tying a putative signaling function to the cell membrane. This pattern is consistent with the diversification of the B. emersonii sensory-signaling systems, which likely plays a varied role in the complex life cycle of this fungus.

A diversified and segregated mRNA spliced-leader system in the parasitic Perkinsozoa.

Spliced-leader trans-splicing (SLTS) has been described in distantly related eukaryotes and acts to mark mRNAs with a short 5' exon, giving different mRNAs identical 5' sequence-signatures. The function of these systems is obscure. Perkinsozoa encompasses a diversity of parasitic protists that infect bivalves, toxic-tide dinoflagellates, fish and frog tadpoles. Here, we report considerable sequence variation in the SLTS-system across the Perkinsozoa and find that multiple variant SLTS-systems are encoded in parallel in the ecologically important Perkinsozoa parasite Parvilucifera sinerae. These results demonstrate that the transcriptome of P. sinerae is segregated based on the addition of different spliced-leader (SL) exons. This segregation marks different gene categories, suggesting that SL-segregation relates to functional differentiation of the transcriptome. By contrast, both sets of gene categories are present in the single SL-transcript type sampled from Maranthos, implying that the SL-segregation of the Parvilucifera transcriptome is a recent evolutionary innovation. Furthermore, we show that the SLTS-system marks a subsection of the transcriptome with increased mRNA abundance and includes genes that encode the spliceosome system necessary for SLTS-function. Collectively, these data provide a picture of how the SLTS-systems can vary within a major evolutionary group and identify how additional transcriptional-complexity can be achieved through SL-segregation.

Characterization of the RNA-interference pathway as a tool for reverse genetic analysis in the nascent phototrophic endosymbiosis, Paramecium bursaria.

Endosymbiosis was fundamental for the evolution of eukaryotic complexity. Endosymbiotic interactions can be dissected through forward- and reverse-genetic experiments, such as RNA-interference (RNAi). However, distinguishing small (s)RNA pathways in a eukaryote-eukaryote endosymbiotic interaction is challenging. Here, we investigate the repertoire of RNAi pathway protein-encoding genes in the model nascent endosymbiotic system, Paramecium bursaria-Chlorella spp. Using comparative genomics and transcriptomics supported by phylogenetics, we identify essential proteome components of the small interfering (si)RNA, scan (scn)RNA and internal eliminated sequence (ies)RNA pathways. Our analyses reveal that copies of these components have been retained throughout successive whole genome duplication (WGD) events in the Paramecium clade. We validate feeding-induced siRNA-based RNAi in P. bursaria via knock-down of the splicing factor, u2af1, which we show to be crucial to host growth. Finally, using simultaneous knock-down 'paradox' controls to rescue the effect of u2af1 knock-down, we demonstrate that feeding-induced RNAi in P. bursaria is dependent upon a core pathway of host-encoded Dcr1, Piwi and Pds1 components. Our experiments confirm the presence of a functional, host-derived RNAi pathway in P. bursaria that generates 23-nt siRNA, validating the use of the P. bursaria-Chlorella spp. system to investigate the genetic basis of a nascent endosymbiosis.

Comparative genomics reveals new functional insights in uncultured MAST species.

Heterotrophic lineages of stramenopiles exhibit enormous diversity in morphology, lifestyle, and habitat. Among them, the marine stramenopiles (MASTs) represent numerous independent lineages that are only known from environmental sequences retrieved from marine samples. The core energy metabolism characterizing these unicellular eukaryotes is poorly understood. Here, we used single-cell genomics to retrieve, annotate, and compare the genomes of 15 MAST species, obtained by coassembling sequences from 140 individual cells sampled from the marine surface plankton. Functional annotations from their gene repertoires are compatible with all of them being phagocytotic. The unique presence of rhodopsin genes in MAST species, together with their widespread expression in oceanic waters, supports the idea that MASTs may be capable of using sunlight to thrive in the photic ocean. Additional subsets of genes used in phagocytosis, such as proton pumps for vacuole acidification and peptidases for prey digestion, did not reveal particular trends in MAST genomes as compared with nonphagocytotic stramenopiles, except a larger presence and diversity of V-PPase genes. Our analysis reflects the complexity of phagocytosis machinery in microbial eukaryotes, which contrasts with the well-defined set of genes for photosynthesis. These new genomic data provide the essential framework to study ecophysiology of uncultured species and to gain better understanding of the function of rhodopsins and related carotenoids in stramenopiles.