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At least 91 records · Page 5

Laminarin stimulates single cell rates of sulfate reduction whereas oxygen inhibits transcriptomic activity in coastal marine sediment

Abstract The chemical cycles carried out by bacteria and archaea living in coastal sediments are vital aspects of benthic ecology. These ecosystems are subject to physical disruption, which may allow for increased respiration and complex carbon consumption—impacting chemical cycling in this environment often thought to be a terminal place of deposition. We use the redox-enzyme sensitive probe RedoxSensor Green to measure rates of electron transfer physiology in individual sulfate reducer cells residing in anoxic sediment, subjected to transient exposure of oxygen and laminarin. We use index fluorescence activated cell sorting and single cell genomics sequencing to link those measurements to genomes of respiring cells. We measure per-cell sulfate reduction rates in marine sediments (0.01–4.7 fmol SO42− cell−1 h−1) and determine that cells within the Chloroflexota phylum are the most active in respiration. Chloroflexota respiration activity is also stimulated with the addition of laminarin, even in marine sediments already rich in organic matter. Evaluating metatranscriptomic data alongside this respiration-based technique, Chloroflexota genomes encode laminarinases indicating a likely ability to degrade laminarin. We also provide evidence that abundant Patescibacteria cells do not use electron transport pathways for energy, and instead likely carry out fermentation of polysaccharides. There is a decoupling of respiration-related activity rates from transcription, as respiration rates increase while transcription decreases with oxygen exposure. Overall, we reveal an active community of respiring Chloroflexota that cycles sulfate at potential rates of 23–40 nmol h−1 per cm3 sediment in incubation settings, and non-respiratory Patescibacteria that can cycle complex polysaccharides.

Lindsay, Melody R.

Transcriptomic and functional analyses uncover a conserved effector driving genotype-dependent virulence in the Sphaerulina musiva-Populus trichocarpa interaction

The introduction of invasive microbes compromises the structure, biodiversity, and function of naïve ecosystems. Sphaerulina musiva, a hemibiotrophic pathogen that causes leaf spot and stem cankers in Populus species, exemplifies an invasive fungal pathogen spread by human activities. However, the genetic mechanisms of pathogenicity and virulence are poorly understood, impeding mitigation strategies. We utilized RNA sequencing to identify fungal effectors linked to stem canker formation, informing the development of future strategies for effective disease management. Our analysis revealed 70 genes differentially expressed at 2 weeks and 110 genes at 3 weeks between inoculated trees and controls. Notably, the gene with the highest expression at 2 weeks and the second highest at 3 weeks was homologous to Extracellular protein 2 (Ecp2). Complementary genome-wide association studies linked sequence polymorphisms in this locus to phenotypic variation in disease severity. Infiltration of S. musiva Ecp2 into Populus trichocarpa leaves induced necrosis in susceptible genotypes. Gene disruption using a CRISPR-Cas9 RNP system resulted in a genotype-dependent reduction of stem canker and disease severity. Tracing the evolutionary history of this effector across the fungal kingdom, we uncovered clade-specific gene-family expansions and orthologs in new species. These findings raise questions about the function and adaptive significance of these gene families in fungal lifestyles. Our study provides the first tractable target for breeding resistant poplar genotypes, addressing the challenges of managing S. musiva and uncovering mechanisms that drive its virulence, and provides deeper insights into the evolutionary dynamics of a conserved small-secreted protein with a diversity of functions.

Sondreli, Kelsey L [Oregon State University]

Transcriptomic data sets examining several stress responses in Zymomonas mobilis strains

We used RNA-seq to compare gene expression from Zymomonas mobilis ZM4 grown under various conditions: aerobic ± paraquat, anaerobic ± hydrogen peroxide, or an iron chelator. We analyzed two mutant strains lacking predicted transcription factors (ZMO_0442 and ZMO_1411) grown under aerobic or anaerobic conditions. We report the RNA-seq data from these experiments.

Bacterial Stress Response

Long-read sequencing transcriptome quantification with lr-kallisto

RNA abundance quantification has become routine and affordable thanks to high-throughput “short-read” technologies that provide accurate molecule counts at the gene level. Similarly accurate and affordable quantification of definitive full-length, transcript isoforms has remained a stubborn challenge, despite its obvious biological significance across a wide range of problems. “Long-read” sequencing platforms now produce data-types that can, in principle, drive routine definitive isoform quantification. However some particulars of contemporary long-read datatypes, together with isoform complexity and genetic variation, present bioinformatic challenges. We show here, using ONT data, that fast and accurate quantification of long-read data is possible and that it is improved by exome capture. To perform quantifications we developed lr-kallisto, which adapts the kallisto bulk and single-cell RNA-seq quantification methods for long-read technologies.

Loving, Rebekah K. (ORCID:0000000187250376)

Building a FAIR data ecosystem for incorporating single-cell transcriptomics data into agricultural genome to phenome research

Introduction The agriculture genomics community has numerous data submission standards available, but the standards for describing and storing single-cell (SC, e.g., scRNA- seq) data are comparatively underdeveloped. Methods To bridge this gap, we leveraged recent advancements in human genomics infrastructure, such as the integration of the Human Cell Atlas Data Portal with Terra, a secure, scalable, open-source platform for biomedical researchers to access data, run analysis tools, and collaborate. In parallel, the Single Cell Expression Atlas at EMBL-EBI offers a comprehensive data ingestion portal for high-throughput sequencing datasets, including plants, protists, and animals (including humans). Developing data tools connecting these resources would offer significant advantages to the agricultural genomics community. The FAANG data portal at EMBL-EBI emphasizes delivering rich metadata and highly accurate and reliable annotation of farmed animals but is not computationally linked to either of these resources. Results Herein, we describe a pilot-scale project that determines whether the current FAANG metadata standards for livestock can be used to ingest scRNA-seq datasets into Terra in a manner consistent with HCA Data Portal standards. Importantly, rich scRNA-seq metadata can now be brokered through the FAANG data portal using a semi-automated process, thereby avoiding the need for substantial expert curation. We have further extended the functionality of this tool so that validated and ingested SC files within the HCA Data Portal are transferred to Terra for further analysis. In addition, we verified data ingestion into Terra, hosted on Azure, and demonstrated the use of a workflow to analyze the first ingested porcine scRNA-seq dataset. Additionally, we have also developed prototype tools to visualize the output of scRNA-seq analyses on genome browsers to compare gene expression patterns across tissues and cell populations. This JBrowse tool now features distinct tracks, showcasing PBMC scRNA-seq alongside two bulk RNA-seq experiments. Discussion We intend to further build upon these existing tools to construct a scientist-friendly data resource and analytical ecosystem based on Findable, Accessible, Interoperable, and Reusable (FAIR) SC principles to facilitate SC-level genomic analysis through data ingestion, storage, retrieval, re-use, visualization, and comparative annotation across agricultural species.

Genetics & Heredity

Single-cell and spatiotemporal transcriptomic profiling of brain immune infiltration following Venezuelan equine encephalitis virus infection

Neurotropic alphaviruses such as Venezuelan equine encephalitis virus (VEEV) are critical human pathogens that continually expand to naïve populations and for which there are no licensed vaccines or therapeutics. VEEV is highly infectious via the aerosol route and is a recognized weaponizable biothreat that causes neurological disease in humans. The neuropathology of VEEV has been attributed to an inflammatory immune response in the brain yet the underlying mechanisms and specific immune cell populations involved are not fully elucidated. This study uses single-cell RNA sequencing to produce a comprehensive transcriptional profile of immune cells isolated from the brain over a time course of infection in a mouse model of VEEV. Analyses reveal differentially activated subpopulations of microglia, including a distinct type I interferon-expressing subpopulation. This is followed by the sequential infiltration of myeloid cells and cytotoxic lymphocytes, also comprising subpopulations with unique transcriptional signatures. We identify a subpopulation of myeloid cells that form a distinct localization pattern in the hippocampal region whereas lymphocytes are widely distributed, indicating differential modes of recruitment, including that to specific regions of the brain. Altogether, this study provides a high-resolution analysis of the immune response to VEEV in the brain and highlights potential avenues of investigation for therapeutics that target neuroinflammation in the brain.

59 BASIC BIOLOGICAL SCIENCES

Comparative transcriptomic and phenotypic analysis of monoclonal and polyclonal Populus deltoides genotypes

Populus species are highly valued for bioenergy and bioproducts due to their rapid growth and productivity. Polyclonal plantings, or mixtures of Populus clones, have shown the potential to enhance resource utilization and productivity, likely due to phenotypic differences arising from niche differentiation. In this study, we investigated gene expression and productivity in monoclonal and polyclonal stands of P. deltoides . Phenotypic results showed that polyclonal plots exhibited higher leaf area index (LAI; p < 0.01, 2.96 ± 0.057 m 2 ) and total biomass (p < 0.01, 2.74 ± 0.06) compared to monoclonal plots, indicating superior productivity. RNA sequencing revealed upregulation of key genes such as exocyst subunit exo70 family protein H7 (EXO70H7), NDH-dependent cyclic electron flow 5 (NDF5) , and expansin-like A3 (EXLA3) . We also observed enrichment in phenylalanine metabolism and other secondary metabolic pathways in clone S7C8. Phenotypic results, upregulated genes and enriched biological pathways identified in this study may explain the enhanced productivity, increased nitrate content, and expanded canopy in polyclonal plantings. Overall, this study provides a foundation for future research to enhance forest productivity by linking molecular mechanisms to practical applications in field plantings.

differential gene expression

Evaluation of Correction Methods for NASA GeneLab Transcriptomic Datasets

Conducting space biology experiments aboard the International Space Station, particularly those utilizing complex model organisms like mice, is expensive and difficult due to limited crew availability, hardware, and space. As a result, sample numbers from these studies are low, reducing the statistical power of any one experiment. Aggregating spaceflight datasets serves as a method to increase sample numbers, allowing for novel insights through bioinformatic analysis of ‘omics data from merged datasets. However, aggregating datasets can introduce unwanted variation including 1) differences in sample handling, processing, and sequencing platforms between datasets (technical variation) as well as 2) differences in experimental design between datasets such as sex or age of the model organism used. In the present study, NASA GeneLab-hosted RNAseq datasets from rodent liver tissues were used to evaluate several statistical methods to correct for this unwanted variation through two approaches, reference-based and standard. The following correction algorithms were applied with (reference-based) and/or without (standard) considering Universal Mouse RNA Reference samples: ComBat and ComBat_seq from the SVA package, median polish, empirical Bayes, and ANOVA-based algorithms from the MBatch package, and negative binomial regression normalization in the DESeq2 package. For each approach, after the correction algorithm was applied, differential gene expression (DGE) analysis of flight and ground control samples was performed with the combined data. The robustness of each tool was evaluated using BatchQC, to determine statistical differences between datasets before and after correction, Principal Component Analysis, to evaluate global gene expression in samples before and after correction, and by comparing DGE analysis of individual datasets and combined datasets before and after correction. The results showed that the reference-based approach introduced several additional (and likely artificial) DEGs when compared with the standard approach. Thus, the most robust standard correction will be implemented in the GeneLab Visualization 2.0 platform when datasets are combined.

GeneLab, RNA-seq, Batch Correction