Assembly and comparative genome analysis of a Patagonian Aureobasidium pullulans isolate reveals unexpected intraspecific variation.

Aureobasidium pullulans is a yeast-like fungus with remarkable phenotypic plasticity widely studied for its importance for the pharmaceutical and food industries. So far, genomic studies with strains from all over the world suggest they constitute a genetically unstructured population, with no association by habitat. However, the mechanisms by which this genome supports so many phenotypic permutations are still poorly understood. Recent works have shown the importance of sequencing yeast genomes from extreme environments to increase the repertoire of phenotypic diversity of unconventional yeasts. In this study, we present the genomic draft of A. pullulans strain from a Patagonian yeast diversity hotspot, re-evaluate its taxonomic classification based on taxogenomic approaches, and annotate its genome with high-depth transcriptomic data. Our analysis suggests this isolate could be considered a novel variant at an early stage of the speciation process. The discovery of divergent strains in a genomically homogeneous group, such as A. pullulans, can be valuable in understanding the evolution of the species. The identification and characterization of new variants will not only allow finding unique traits of biotechnological importance, but also optimize the choice of strains whose phenotypes will be characterized, providing new elements to explore questions about plasticity and adaptation.

Natural Variation in Diauxic Shift between Patagonian Saccharomyces eubayanus Strains.

The study of natural variation can untap novel alleles with immense value for biotechnological applications. Saccharomyces eubayanus Patagonian isolates exhibit differences in the diauxic shift between glucose and maltose, representing a suitable model to study their natural genetic variation for novel strains for brewing. However, little is known about the genetic variants and chromatin regulators responsible for these differences. Here, we show how genome-wide chromatin accessibility and gene expression differences underlie distinct diauxic shift profiles in S. eubayanus. We identified two strains with a rapid diauxic shift between glucose and maltose (CL467.1 and CBS12357) and one strain with a remarkably low fermentation efficiency and longer lag phase during diauxic shift (QC18). This is associated in the QC18 strain with lower transcriptional activity and chromatin accessibility of specific genes of maltose metabolism and higher expression levels of glucose transporters. These differences are governed by the HAP complex, which differentially regulates gene expression depending on the genetic background. We found in the QC18 strain a contrasting phenotype to those phenotypes described in S. cerevisiae, where hap4Δ, hap5Δ, and cin5Δ knockouts significantly improved the QC18 growth rate in the glucose-maltose shift. The most profound effects were found between CIN5 allelic variants, suggesting that Cin5p could strongly activate a repressor of the diauxic shift in the QC18 strain but not necessarily in the other strains. The differences between strains could originate from the tree host from which the strains were obtained, which might determine the sugar source preference and the brewing potential of the strain. IMPORTANCE The diauxic shift has been studied in budding yeast under laboratory conditions; however, few studies have addressed the diauxic shift between carbon sources under fermentative conditions. Here, we study the transcriptional and chromatin structure differences that explain the natural variation in fermentative capacity and efficiency during diauxic shift of natural isolates of S. eubayanus. Our results show how natural genetic variants in transcription factors impact sugar consumption preferences between strains. These variants have different effects depending on the genetic background, with a contrasting phenotype to those phenotypes previously described in S. cerevisiae. Our study shows how relatively simple genetic/molecular modifications/editing in the lab can facilitate the study of natural variations of microorganisms for the brewing industry.

Genomic and proteomic analysis of Tausonia pullulans reveals a key role for a GH15 glucoamylase in starch hydrolysis.

Basidiomycetous yeasts remain an almost unexplored source of enzymes with great potential in several industries. Tausonia pullulans (Tremellomycetes) is a psychrotolerant yeast with several extracellular enzymatic activities reported, although the responsible genes are not known. We performed the genomic sequencing, assembly and annotation of T. pullulans strain CRUB 1754 (Perito Moreno glacier, Argentina), a gene survey of carbohydrate-active enzymes (CAZymes), and analyzed its secretome by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) after growth in glucose (GLU) or starch (STA) as main carbon sources. T. pullulans has 7210 predicted genes, 3.6% being CAZymes. When compared to other Tremellomycetes, it contains a high number of CAZy domains, and in particular higher quantities of glucoamylases (GH15), pectinolytic enzymes (GH28) and lignocellulose decay enzymes (GH7). When the secretome of T. pullulans was analyzed experimentally after growth in starch or glucose, 98 proteins were identified. The 60% of total spectral counts belonged to GHs, oxidoreductases and to other CAZymes. A 65 kDa glucoamylase of family GH15 (TpGA1) showed the highest fold change (tenfold increase in starch). This enzyme contains a conserved active site and showed extensive N-glycosylation. This study increases the knowledge on the extracellular hydrolytic enzymes of basidiomycetous yeasts and, in particular, establishes T. pullulans as a potential source of carbohydrate-active enzymes. KEY POINTS: • Tausonia pullulans genome harbors a high number of genes coding for CAZymes. • Among CAZy domains/families, the glycoside hydrolases are the most abundant. • Secretome analysis in glucose or starch as main C sources identified 98 proteins. • A 65 kDa GH15 glucoamylase showed the highest fold increase upon culture in starch.

Regulatory mechanisms behind the phenotypic plasticity associated with Setaria italica water deficit tolerance.

Drought is one of the main environmental stresses that negatively impacts vegetative and reproductive yield. Water deficit responses are determined by the duration and intensity of the stress, which, together with plant genotype, will define the chances of plant survival. The metabolic adjustments in response to water deficit are complex and involve gene expression modulation regulated by DNA-binding proteins and epigenetic modifications. This last mechanism may also regulate the activity of transposable elements, which in turn impact the expression of nearby loci. Setaria italica plants submitted to five water deficit regimes were analyzed through a phenotypical approach, including growth, physiological, RNA-seq and sRNA-seq analyses. The results showed a progressive reduction in yield as a function of water deficit intensity associated with signaling pathway modulation and metabolic adjustments. We identified a group of loci that were consistently associated with drought responses, some of which were related to water deficit perception, signaling and regulation. Finally, an analysis of the transcriptome and sRNAome allowed us to identify genes putatively regulated by TE- and sRNA-related mechanisms and an intriguing positive correlation between transcript levels and sRNA accumulation in gene body regions. These findings shed light on the processes that allow S. italica to overcome drought and survive under water restrictive conditions.

Phytochrome-Mediated Light Perception Affects Fruit Development and Ripening Through Epigenetic Mechanisms.

Phytochrome (PHY)-mediated light and temperature perception has been increasingly implicated as important regulator of fruit development, ripening, and nutritional quality. Fruit ripening is also critically regulated by chromatin remodeling via DNA demethylation, though the molecular basis connecting epigenetic modifications in fruits and environmental cues remains largely unknown. Here, to unravel whether the PHY-dependent regulation of fruit development involves epigenetic mechanisms, an integrative analysis of the methylome, transcriptome and sRNAome of tomato fruits from phyA single and phyB1B2 double mutants was performed in immature green (IG) and breaker (BK) stages. The transcriptome analysis showed that PHY-mediated light perception regulates more genes in BK than in the early stages of fruit development (IG) and that PHYB1B2 has a more substantial impact than PHYA in the fruit transcriptome, in both analyzed stages. The global profile of methylated cytosines revealed that both PHYA and PHYB1B2 affect the global methylome, but PHYB1B2 has a greater impact on ripening-associated methylation reprogramming across gene-rich genomic regions in tomato fruits. Remarkably, promoters of master ripening-associated transcription factors (TF) (RIN, NOR, CNR, and AP2a) and key carotenoid biosynthetic genes (PSY1, PDS, ZISO, and ZDS) remained highly methylated in phyB1B2 from the IG to BK stage. The positional distribution and enrichment of TF binding sites were analyzed over the promoter region of the phyB1B2 DEGs, exposing an overrepresentation of binding sites for RIN as well as the PHY-downstream effectors PIFs and HY5/HYH. Moreover, phyA and phyB1B2 mutants showed a positive correlation between the methylation level of sRNA cluster-targeted genome regions in gene bodies and mRNA levels. The experimental evidence indicates that PHYB1B2 signal transduction is mediated by a gene expression network involving chromatin organization factors (DNA methylases/demethylases, histone-modifying enzymes, and remodeling factors) and transcriptional regulators leading to altered mRNA profile of ripening-associated genes. This new level of understanding provides insights into the orchestration of epigenetic mechanisms in response to environmental cues affecting agronomical traits.

Chromatin topology defines estradiol-primed progesterone receptor and PAX2 binding in endometrial cancer cells.

Estrogen (E2) and Progesterone (Pg), via their specific receptors (ERalpha and PR), are major determinants in the development and progression of endometrial carcinomas, However, their precise mechanism of action and the role of other transcription factors involved are not entirely clear. Using Ishikawa endometrial cancer cells, we report that E2 treatment exposes a set of progestin-dependent PR binding sites which include both E2 and progestin target genes. ChIP-seq results from hormone-treated cells revealed a non-random distribution of PAX2 binding in the vicinity of these estrogen-promoted PR sites. Altered expression of hormone regulated genes in PAX2 knockdown cells suggests a role for PAX2 in fine-tuning ERalpha and PR interplay in transcriptional regulation. Analysis of long-range interactions by Hi-C coupled with ATAC-seq data showed that these regions, that we call 'progestin control regions' (PgCRs), exhibited an open chromatin state even before hormone exposure and were non-randomly associated with regulated genes. Nearly 20% of genes potentially influenced by PgCRs were found to be altered during progression of endometrial cancer. Our findings suggest that endometrial response to progestins in differentiated endometrial tumor cells results in part from binding of PR together with PAX2 to accessible chromatin regions. What maintains these regions open remains to be studied.

Nucleo-cytoplasmic shuttling of splicing factor SRSF1 is required for development and cilia function.

Shuttling RNA-binding proteins coordinate nuclear and cytoplasmic steps of gene expression. The SR family proteins regulate RNA splicing in the nucleus and a subset of them, including SRSF1, shuttles between the nucleus and cytoplasm affecting post-splicing processes. However, the physiological significance of this remains unclear. Here, we used genome editing to knock-in a nuclear retention signal (NRS) in Srsf1 to create a mouse model harboring an SRSF1 protein that is retained exclusively in the nucleus. Srsf1NRS/NRS mutants displayed small body size, hydrocephalus, and immotile sperm, all traits associated with ciliary defects. We observed reduced translation of a subset of mRNAs and decreased abundance of proteins involved in multiciliogenesis, with disruption of ciliary ultrastructure and motility in cells and tissues derived from this mouse model. These results demonstrate that SRSF1 shuttling is used to reprogram gene expression networks in the context of high cellular demands, as observed here, during motile ciliogenesis.

Hanseniaspora smithiae sp. nov., a Novel Apiculate Yeast Species From Patagonian Forests That Lacks the Typical Genomic Domestication Signatures for Fermentative Environments.

During a survey of Nothofagus trees and their parasitic fungi in Andean Patagonia (Argentina), genetically distinct strains of Hanseniaspora were obtained from the sugar-containing stromata of parasitic Cyttaria spp. Phylogenetic analyses based on the single-gene sequences (encoding rRNA and actin) or on conserved, single-copy, orthologous genes from genome sequence assemblies revealed that these strains represent a new species closely related to Hanseniaspora valbyensis. Additionally, delimitation of this novel species was supported by genetic distance calculations using overall genome relatedness indices (OGRI) between the novel taxon and its closest relatives. To better understand the mode of speciation in Hanseniaspora, we examined genes that were retained or lost in the novel species in comparison to its closest relatives. These analyses show that, during diversification, this novel species and its closest relatives, H. valbyensis and Hanseniaspora jakobsenii, lost mitochondrial and other genes involved in the generation of precursor metabolites and energy, which could explain their slower growth and higher ethanol yields under aerobic conditions. Similarly, Hanseniaspora mollemarum lost the ability to sporulate, along with genes that are involved in meiosis and mating. Based on these findings, a formal description of the novel yeast species Hanseniaspora smithiae sp. nov. is proposed, with CRUB 1602 H as the holotype.

Deciphering the transcriptomic regulation of heat stress responses in Nothofagus pumilio.

Global warming is predicted to exert negative impacts on plant growth due to the damaging effect of high temperatures on plant physiology. Revealing the genetic architecture underlying the heat stress response is therefore crucial for the development of conservation strategies, and for breeding heat-resistant plant genotypes. Here we investigated the transcriptional changes induced by heat in Nothofagus pumilio, an emblematic tree species of the sub-Antarctic forests of South America. Through the performance of RNA-seq of leaves of plants exposed to 20°C (control) or 34°C (heat shock), we generated the first transcriptomic resource for the species. We also studied the changes in protein-coding transcripts expression in response to heat. We found 5,214 contigs differentially expressed between temperatures. The heat treatment resulted in a down-regulation of genes related to photosynthesis and carbon metabolism, whereas secondary metabolism, protein re-folding and response to stress were up-regulated. Moreover, several transcription factor families like WRKY or ERF were promoted by heat, alongside spliceosome machinery and hormone signaling pathways. Through a comparative analysis of gene regulation in response to heat in Arabidopsis thaliana, Populus tomentosa and N. pumilio we provide evidence of the existence of shared molecular features of heat stress responses across angiosperms, and identify genes of potential biotechnological application.

High-throughput meta-analysis and validation of differentially expressed genes as potential biomarkers of ionizing radiation-response.

BACKGROUND AND PURPOSE: The high-throughput analysis of gene expression in ionizing radiation (IR)-exposed human peripheral white blood cells (WBC) has emerged as a novel method for biodosimetry markers detection. We aimed to detect IR-exposure differential expressed genes (DEGs) as potential predictive biomarkers for biodosimetry and radioinduced-response. MATERIALS AND METHODS: We performed a meta-analysis of raw data from public microarrays of ex vivo low linear energy transfer-irradiated human peripheral WBC. Functional enrichment and transcription factors (TF) detection from resulting DEGs were assessed. Six selected DEGs among studies were validated by qRT-PCR on mRNA from human peripheral blood samples from nine healthy human donors 24 h after ex vivo X-rays-irradiation. RESULTS: We identified 275 DEGs after IR-exposure (parameters: |lfc| ≥ 0.7, q value <0.05), enriched in processes such as regulation after IR-exposure, DNA damage checkpoint, signal transduction by p53 and mitotic cell cycle checkpoint. Among these DEGs, DRAM1, NUDT15, PCNA, PLK2 and TIGAR were selected for qRT-PCR validation. Their expression levels significantly increased at 1-4 Gy respect to non-irradiated controls. Particularly, PCNA increased dose dependently. Curiously, TCF4 (Entrez Gene: 6925), detected as overrepresented TF in the radioinduced DEGs set, significantly decreased post-irradiation. CONCLUSION: These six DEGs show potential to be proposed as candidates for IR-exposure biomarkers, considering their observed molecular radioinduced-response. Among them, TCF4, bioinformatically detected, was validated herein as an IR-responsive gene.

Unique genomic traits for cold adaptation in Naganishia vishniacii, a polyextremophile yeast isolated from Antarctica.

Cold environments impose challenges to organisms. Polyextremophile microorganisms can survive in these conditions thanks to an array of counteracting mechanisms. Naganishia vishniacii, a yeast species hitherto only isolated from McMurdo Dry Valleys, Antarctica, is an example of a polyextremophile. Here we present the first draft genomic sequence of N. vishniacii. Using comparative genomics, we unraveled unique characteristics of cold associated adaptations. 336 putative genes (total: 6183) encoding solute transfers and chaperones, among others, were absent in sister species. Among genes shared by N. vishniacii and its closest related species we found orthologs encompassing possible evidence of positive selection (dN/dS > 1). Genes associated with photoprotection were found in agreement with high solar irradiation exposure. Also genes coding for desaturases and genomic features associated with cold tolerance (i.e. trehalose synthesis and lipid metabolism) were explored. Finally, biases in amino acid usage (namely an enrichment of glutamine and a trend in proline reduction) were observed, possibly conferring increased protein flexibility. To the best of our knowledge, such a combination of mechanisms for cold tolerance has not been previously reported in fungi, making N. vishniacii a unique model for the study of the genetic basis and evolution of cold adaptation strategies.

mRNA spindle localization and mitotic translational regulation by CPEB1 and CPEB4.

Transition through cell cycle phases requires temporal and spatial regulation of gene expression to ensure accurate chromosome duplication and segregation. This regulation involves dynamic reprogramming of gene expression at multiple transcriptional and posttranscriptional levels. In transcriptionally silent oocytes, the CPEB-family of RNAbinding proteins coordinates temporal and spatial translation regulation of stored maternal mRNAs to drive meiotic progression. CPEB1 mediates mRNA localization to the meiotic spindle, which is required to ensure proper chromosome segregation. Temporal translational regulation also takes place in mitosis, where a large repertoire of transcripts are activated or repressed in specific cell cycle phases. However, whether control of localized translation at the spindle is required for mitosis is unclear, as mitotic and acentriolar-meiotic spindles are functionally and structurally different. Furthermore, the large differences in scale-ratio between cell volume and spindle size in oocytes compared to somatic mitotic cells may generate distinct requirements for gene expression compartmentalization in meiosis and mitosis. Here we show that mitotic spindles contain CPE-localized mRNAs and translating ribosomes. Moreover, CPEB1 and CPEB4 localize in the spindles and they may function sequentially in promoting mitotic stage transitions and correct chromosome segregation. Thus, CPEB1 and CPEB4 bind to specific spindle-associated transcripts controlling the expression and/or localization of their encoded factors that, respectively, drive metaphase and anaphase/cytokinesis.

The Untapped Australasian Diversity of Astaxanthin-Producing Yeasts with Biotechnological Potential-Phaffia australis sp. nov. and Phaffia tasmanica sp. nov.

Phaffia is an orange-colored basidiomycetous yeast genus of the order Cystofilobasidiales that contains a single species, P. rhodozyma. This species is the only fungus known to produce the economically relevant carotenoid astaxanthin. Although Phaffia was originally found in the Northern hemisphere, its diversity in the southern part of the globe has been shown to be much greater. Here we analyze the genomes of two Australasian lineages that are markedly distinct from P. rhodozyma. The two divergent lineages were investigated within a comprehensive phylogenomic study of representatives of the Cystofilobasidiales that supported the recognition of two novel Phaffia species, for which we propose the names of P. australis sp. nov. and P. tasmanica sp. nov. Comparative genomics and other analyses confirmed that the two new species have the typical Phaffia hallmark-the six genes necessary for the biosynthesis of astaxanthin could be retrieved from the draft genome sequences, and this carotenoid was detected in culture extracts. In addition, the organization of the mating-type (MAT) loci is similar to that of P. rhodozyma, with synteny throughout most regions. Moreover, cases of trans-specific polymorphism involving pheromone receptor genes and pheromone precursor proteins in the three Phaffia species, together with their shared homothallism, provide additional support for their classification in a single genus.

Canonical ErbB-2 isoform and ErbB-2 variant c located in the nucleus drive triple negative breast cancer growth.

Triple negative breast cancer (TNBC) refers to tumors that do not express clinically significant levels of estrogen and progesterone receptors, and lack membrane overexpression or gene amplification of ErbB-2/HER2, a receptor tyrosine kinase. Transcriptome and proteome heterogeneity of TNBC poses a major challenge to precision medicine. Clinical biomarkers and targeted therapies for this disease remain elusive, so chemotherapy has been the standard of care for early and metastatic TNBC. Our present findings placed ErbB-2 in an unanticipated scenario: the nucleus of TNBC (NErbB-2). Our study on ErbB-2 alternative splicing events, using a PCR-sequencing approach combined with an RNA interference strategy, revealed that TNBC cells express either the canonical (wild-type) ErbB-2, encoded by transcript variant 1, or the non-canonical ErbB-2 isoform c, encoded by alternative variant 3 (RefSeq), or both. These ErbB-2 isoforms function in the nucleus as transcription factors. Evicting both from the nucleus or silencing isoform c only, blocks TN cell and tumor growth. This reveals not only NErbB-2 canonical and alternative isoforms role as targets of therapy in TNBC, but also isoform c dominant oncogenic potential. Furthermore, we validated our findings in the clinic and observed that NErbB-2 correlates with poor prognosis in primary TN tumors, disclosing NErbB-2 as a novel biomarker for TNBC. Our discoveries challenge the present scenario of drug development for personalized BC medicine that focuses on wild-type RefSeq proteins, which conserve the canonical domains and are located in their classical cellular compartments.

Novel yeast taxa from the cold: description of Cryolevonia giraudoae sp. nov. and Camptobasidium gelus sp. nov.

Twenty-one psychrophilic yeast isolates related to the Camptobasidiaceae family in the Microbotryomycetes class were obtained from ice collected from cold environments worldwide. A new psychrophilic species from the recently described genus Cryolevonia, Cryolevania giraudoae is proposed to accommodate 18 isolates from Patagonia (Argentina) and Antarctica (holotype CRUB 2086T). In addition, a new psychrophilic species in the genus Camptobasidium is described as Camptobasidium gelus sp. nov. (holotype CBS 8941T), based on three isolates from glacial ice in the Russel glacier (Greenland ice sheet) and Antarctica. The strict psychrophilic profile is the salient feature of both novel species.

ILF3 contributes to the establishment of the antiviral type I interferon program.

Upon detection of viral infections, cells activate the expression of type I interferons (IFNs) and pro-inflammatory cytokines to control viral dissemination. As part of their antiviral response, cells also trigger the translational shutoff response which prevents translation of viral mRNAs and cellular mRNAs in a non-selective manner. Intriguingly, mRNAs encoding for antiviral factors bypass this translational shutoff, suggesting the presence of additional regulatory mechanisms enabling expression of the self-defence genes. Here, we identified the dsRNA binding protein ILF3 as an essential host factor required for efficient translation of the central antiviral cytokine, IFNB1, and a subset of interferon-stimulated genes. By combining polysome profiling and next-generation sequencing, ILF3 was also found to be necessary to establish the dsRNA-induced transcriptional and translational programs. We propose a central role for the host factor ILF3 in enhancing expression of the antiviral defence mRNAs in cellular conditions where cap-dependent translation is compromised.

Molecular bases of responses to abiotic stress in trees.

Trees are constantly exposed to climate fluctuations, which vary with both time and geographic location. Environmental changes that are outside of the physiological favorable range usually negatively affect plant performance and trigger responses to abiotic stress. Long-living trees in particular have evolved a wide spectrum of molecular mechanisms to coordinate growth and development under stressful conditions, thus minimizing fitness costs. The ongoing development of techniques directed at quantifying abiotic stress has significantly increased our knowledge of physiological responses in woody plants. However, it is only within recent years that advances in next-generation sequencing and biochemical approaches have enabled us to begin to understand the complexity of the molecular systems that underlie these responses. Here, we review recent progress in our understanding of the molecular bases of drought and temperature stresses in trees, with a focus on functional, transcriptomic, epigenetic, and population genomic studies. In addition, we highlight topics that will contribute to progress in our understanding of the plastic and adaptive responses of woody plants to drought and temperature in a context of global climate change.

Genomic content of a novel yeast species Hanseniaspora gamundiae sp. nov. from fungal stromata (Cyttaria) associated with a unique fermented beverage in Andean Patagonia, Argentina.

A novel yeast species was isolated from the sugar-rich stromata of Cyttaria hariotii collected from two different Nothofagus tree species in the Andean forests of Patagonia, Argentina. Phylogenetic analyses of the concatenated sequence of the rRNA gene sequences and the protein-coding genes for actin and translational elongation factor-1α indicated that the novel species belongs to the genus Hanseniaspora. De novo genome assembly of the strain CRUB 1928T yielded a 10.2-Mbp genome assembly predicted to encode 4452 protein-coding genes. The genome sequence data were compared to the genomes of other Hanseniaspora species using three different methods, an alignment-free distance measure, Kr, and two model-based estimations of DNA-DNA homology values, of which all provided indicative values to delineate species of Hanseniaspora. Given its potential role in a rare indigenous alcoholic beverage in which yeasts ferment sugars extracted from the stromata of Cytarria sp., we searched for the genes that may suggest adaptation of novel Hanseniaspora species to fermenting communities. The SSU1-like gene encoding a sulfite efflux pump, which, among Hanseniaspora, is present only in close relatives to the new species, was detected and analyzed, suggesting that this gene might be one factor that characterizes this novel species. We also discuss several candidate genes that likely underlie the physiological traits used for traditional taxonomic identification. Based on these results, a novel yeast species with the name Hanseniaspora gamundiae sp. nov. is proposed with CRUB 1928T (ex-types: ZIM 2545T = NRRL Y-63793T = PYCC 7262T; MycoBank number MB 824091) as the type strain. Furthermore, we propose the transfer of the Kloeckera species, K. hatyaiensis, K. lindneri and K. taiwanica to the genus Hanseniaspora as Hanseniaspora hatyaiensis comb. nov. (MB 828569), Hanseniaspora lindneri comb. nov. (MB 828566) and Hanseniaspora taiwanica comb. nov. (MB 828567).

Comprehensive phylogeny of ray-finned fishes (Actinopterygii) based on transcriptomic and genomic data.

Our understanding of phylogenetic relationships among bony fishes has been transformed by analysis of a small number of genes, but uncertainty remains around critical nodes. Genome-scale inferences so far have sampled a limited number of taxa and genes. Here we leveraged 144 genomes and 159 transcriptomes to investigate fish evolution with an unparalleled scale of data: >0.5 Mb from 1,105 orthologous exon sequences from 303 species, representing 66 out of 72 ray-finned fish orders. We apply phylogenetic tests designed to trace the effect of whole-genome duplication events on gene trees and find paralogy-free loci using a bioinformatics approach. Genome-wide data support the structure of the fish phylogeny, and hypothesis-testing procedures appropriate for phylogenomic datasets using explicit gene genealogy interrogation settle some long-standing uncertainties, such as the branching order at the base of the teleosts and among early euteleosts, and the sister lineage to the acanthomorph and percomorph radiations. Comprehensive fossil calibrations date the origin of all major fish lineages before the end of the Cretaceous.

Tumor Necrosis Factor-Mediated Survival of CD169+ Cells Promotes Immune Activation during Vesicular Stomatitis Virus Infection.

Innate immune activation is essential to mount an effective antiviral response and to prime adaptive immunity. Although a crucial role of CD169+ cells during vesicular stomatitis virus (VSV) infections is increasingly recognized, factors regulating CD169+ cells during viral infections remain unclear. Here, we show that tumor necrosis factor is produced by CD11b+ Ly6C+ Ly6G+ cells following infection with VSV. The absence of TNF or TNF receptor 1 (TNFR1) resulted in reduced numbers of CD169+ cells and in reduced type I interferon (IFN-I) production during VSV infection, with a severe disease outcome. Specifically, TNF triggered RelA translocation into the nuclei of CD169+ cells; this translocation was inhibited when the paracaspase MALT-1 was absent. Consequently, MALT1 deficiency resulted in reduced VSV replication, defective innate immune activation, and development of severe disease. These findings indicate that TNF mediates the maintenance of CD169+ cells and innate and adaptive immune activation during VSV infection.IMPORTANCE Over the last decade, strategically placed CD169+ metallophilic macrophages in the marginal zone of the murine spleen and lymph nodes (LN) have been shown to play a very important role in host defense against viral pathogens. CD169+ macrophages have been shown to activate innate and adaptive immunity via "enforced virus replication," a controlled amplification of virus particles. However, the factors regulating the CD169+ macrophages remain to be studied. In this paper, we show that after vesicular stomatitis virus infection, phagocytes produce tumor necrosis factor (TNF), which signals via TNFR1, and promote enforced virus replication in CD169+ macrophages. Consequently, lack of TNF or TNFR1 resulted in defective immune activation and VSV clearance.