Biology subjects

Holden, S.

Publications and source records attributed to Holden, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Harnessing within-cultivar variation to identify hidden genetic resistance using single plant-omics

Fusarium graminearum is a fungal pathogen that causes scab or head blight in small grain cereals and threatens global cereal production. Disease progression varies widely among individual plants of the same cultivar, reflecting both genetic and environmental heterogeneity. This complicates the identification of early host responses, because each individual plant in a population is at a slightly different phase of disease progression. Here we apply single plant-transcriptomics to a population of 121 malt-barley exposed to F. graminearum, enabling us to reconstruct a temporal sequence of regulatory processes during early infection. We identified several disease-resistance associated genetic variants that are already endemic in this high-yielding cultivar, suggesting potential as breeding targets. These variants were within proteins involved in ROS-burst production, a lectin-kinase PRR, and enzymes with DON-detoxification activity. Single plant-transcriptomics offers a novel strategy for characterising early plant-pathogen interactions, turning intra-population heterogeneity from an experimental barrier into an asset.

plant biology↗

bioRxiv2026

Watkins wheat landraces: a treasure of stripe rust resistance alleles identified using multi-model association analyses

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), remains a major global constraint to wheat production. Rapid pathogen evolution, exemplified by the recent breakdown of Yr15 in Europe, underscores the need to identify diverse and durable resistance loci. The A.E. Watkins landrace collection represents a globally diverse pre-breeding resource with substantial untapped variation for stripe rust resistance. In this study, 297 Watkins landraces were evaluated against six diverse Pst isolates (representing six races and three North American lineages) and subjected to genome-wide association analysis using high-density whole-genome resequencing data. Continuous phenotypic variation was observed across isolates, with several accessions displaying stable resistance across all lineages. A total of 87 QTLs were identified across all 21 wheat chromosomes. Ten loci co-localized with designated or cloned Yr genes, including Yr84, Yr85, Yrq1, Yr71, Yr60, Yr62, Yr50, Yr68, Yr34, and Lr34/Yr18/Sr57. An additional 34 loci overlapped previously reported stripe rust QTL, whereas the majority did not coincide with known loci, suggesting potential novel resistance regions. Eighteen QTLs were supported by multiple isolates, and fourteen showed supports across statistical models, indicating robust genomic signals. Several Watkins accessions carried favorable alleles that co-localized with multiple Yr-aligned loci, identifying promising donor candidates for validation and pre-breeding. Key MessageGenome-wide association mapping of 297 Watkins wheat landraces across diverse stripe rust races & genetic lineages identified 87 QTL, including 10 formally designated Yr genes and 46 novel loci, highlighting Watkins landraces as valuable pre-breeding donors for novel all-stage stripe rust resistance.

plant biology↗

bioRxiv2025

micromorph: a Python toolkit for measurement of microbial morphology

Detection of morphological phenotypes from light microscopy is a key part of microbiology. Despite advances in automated morphological analysis, accurate measurements still often require significant user input. To address this, we have developed micromorph, a Python package to measure bacterial morphological properties from multiple types of light microscopy data. micromorph is available as a Python package with a documented API, or as napari plugin, thus appealing to a range of users regardless of their coding proficiency. The micromorph API allows easy integration into custom analysis pipelines. We demonstrate a range of scenarios in which micromorph demonstrates excellent performance and introduce a new approach for reliably measuring the widths and lengths of quasi-circular cells.

microbiology↗

bioRxiv2025

Wheat and barley stripe rust pan-genome facilitates discovery of the predominant North American lineage's origin in somatic hybridization

Wheat and barley stripe rust, caused by Puccinia striiformis formae speciales tritici (Pst) and hordei (Psh) respectively is a perennial and endemic threat to those crops worldwide. Outside the Himalayan region the fungus has not been observed reproducing sexually, despite being characterised extensively over the past 100 years by researchers in many countries. Scientific literature divides P. striiformis into lineages termed PstS0, PstS1, up to PstS18. Here we describe the first North American P. striiformis pan-genome, comprising 24 Pst and 3 Psh genomes from North America including five phased assemblies using chromosome conformation capture to separate two haploid genomes. We identify the origin of the predominant North American Pst lineage; PstS18, as a somatic hybridization event between PstS1 and another unknown lineage. As well as identifying gene families which distinguish haplotypes on a trans-lineage and trans-species level, we also show ongoing gene family loss and expansion in clonal lineages after their emergence.

microbiology↗

bioRxiv2025

Epigenetic and Transcriptomic Alterations Precede Amyloidosis in the Hippocampus of the Alzheimer's Disease AppNL-G-F Knock in Mouse Model

Detecting and understanding the early stages of Alzheimers disease (AD) is essential for uncovering initial mechanisms of neuropathology and devising effective interventions. In this study, we leveraged the humanized AppNL-G-F mouse which exhibits early-onset amyloid pathology with a predictable timeline, to investigate molecular changes in the hippocampus and blood before the onset of severe neuropathology and independent of aging. Employing a multi-omics approach, we identified alterations in chromatin accessibility, gene expression, and DNA methylation associated with early amyloidosis. Chromatin accessibility changes were prominent in excitatory neurons during early pathology, with a later shift to inhibitory neurons, potentially reflecting compensatory mechanisms to mitigate excitatory neuron dysregulation. Despite broadly comparable hippocampal cell composition, transcriptomic comparisons between wild-type and AppNL-G-F mice revealed major gene expression differences, particularly in pathways related to mitochondrial function and protein biosynthesis, preceding severe amyloid plaque deposition. In later stages, upregulation of immune and neuroinflammatory pathways was observed, aligning with established neuroinflammatory processes in AD. Additionally, we identified extensive DNA methylation differences in both the blood and hippocampus of AppNL-G-Fmice during early and late stages of pathology. Many differentially methylated regions in the blood, even at early pathology stages, were associated with cis-regulatory elements in the brain and were located near differentially expressed genes in the hippocampus. These regions were enriched in pathways associated with brain function, including neuron development and synaptic processes, highlighting a connection between blood methylation patterns and brain activity. This finding suggests the potential use of blood DNA methylation as a biomarker for the early detection of amyloidosis. Notably, we identified five candidate biomarker genes, including Rbfox1 and Camta1, with epigenetic dysregulation detectable in both the brain and blood prior to severe amyloid accumulation. Our study, leveraging a unique AD mouse model and a multi-omics approach, highlights epigenetic signatures of AD before the onset of clinical symptoms, providing a foundation for future research into early diagnosis and therapeutic strategies, as well as potential blood biomarkers.

molecular biology↗

bioRxiv2024

Systemic exposure to COVID-19 virus-like particles modulates firing patterns of cortical neurons in the living mouse brain

Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) causes a systemic infection that affects the central nervous system. We used virus-like particles (VLPs) to explore how exposure to the SARS-CoV-2 proteins affects brain activity patterns in wild-type (WT) mice and in mice that express the wild-type human tau protein (htau mice). VLP exposure elicited dose-dependent changes in corticosterone and distinct chemokine levels. Longitudinal two-photon microscopy recordings of primary somatosensory and motor cortex neurons that express the jGCaMP7s calcium sensor tracked modifications of neuronal activity patterns following exposure to VLPs. There was a substantial short-term increase in stimulus-evoked activity metrics in both WT and htau VLP-injected mice, while htau mice showed also increased spontaneous activity metrics and increase activity in the vehicle-injected group. Over the following weeks, activity metrics in WT mice subsided, but remained above baseline levels. For htau mice, activity metrics either remain elevated or decreased to lower levels than baseline. Overall, our data suggest that exposure to the SARS-CoV-2 VLPs leads to strong short-term disruption of cortical activity patterns in mice with long-term residual effects. The htau mice, which have a more vulnerable genetic background, exhibited more severe pathobiology that may lead to more adverse outcomes.

neuroscience↗

bioRxiv2024

Genomes of Poaceae sisters reveal key metabolic innovations preceding the emergence of grasses

The grass family (Poaceae, Poales) holds immense economic and ecological significance, exhibiting unique metabolic traits, including dual starch and lignin biosynthetic pathways. To investigate when and how the metabolic innovations known in grasses evolved, we sequenced the genomes of four Poales species, including Joinvillea ascendens and Ecdeiocolea monostachya representing the sister clade to Poaceae. The rho whole genome duplication ({rho}WGD) in the ancestral lineage for all grasses contributed to the gene family expansions underlying cytosolic starch biosynthesis, whereas an earlier tandem duplication of phenylalanine ammonia lyase (PAL) gave rise to phenylalanine/tyrosine ammonia lyase (PTAL) responsible for the dual lignin biosynthesis. Integrated functional genomic and biochemical analyses of grass relatives further revealed the molecular basis of key metabolic innovations predating the evolution of grasses. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/622220v2_ufig1.gif" ALT="Figure 1"> View larger version (108K): org.highwire.dtl.DTLVardef@117dd71org.highwire.dtl.DTLVardef@1cb5daborg.highwire.dtl.DTLVardef@72a331org.highwire.dtl.DTLVardef@38124e_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2023

Complement-mediated killing of bacteria by mechanical destabilization of the cell envelope

Complement proteins eliminate Gram-negative bacteria in serum via the formation of membrane attack complex (MAC) pores in the outer membrane. However, it remains unclear how outer membrane poration leads to inner membrane permeation and cell lysis. Using atomic force microscopy (AFM) on living Escherichia coli (E. coli), we probed MAC-induced changes in the cell envelope and correlated these with subsequent cell death. Initially, bacteria survived despite the formation of hundreds of MACs randomly distributed over the cell surface. This was followed by larger-scale disruption of the outer membrane, including propagating defects and fractures, and by an overall swelling and stiffening of the bacterial surface, which precede inner membrane permeation. We conclude that bacterial cell lysis is only an indirect effect of MAC formation; outer membrane poration leads to mechanical destabilization of the cell envelope, reducing its ability to contain the turgor pressure, causing inner membrane permeation and cell death. This represents a previously unknown route to bacterial cell death that could be exploited by novel antibiotic treatments.

microbiology↗

bioRxiv2023

Origin and evolution of the bread wheat D genome

Bread wheat (Triticum aestivum) is a globally dominant crop and major source of calories and proteins for the human diet. Compared to its wild ancestors, modern bread wheat shows lower genetic diversity caused by polyploidisation, domestication, and breeding bottlenecks1,2. Wild wheat relatives represent genetic reservoirs, harbouring diversity and beneficial alleles that have not been incorporated into bread wheat. Here, we establish and analyse pangenome resources for Tauschs goatgrass, Aegilops tauschii, the donor of the bread wheat D genome. This new pangenome facilitated the cloning of a disease resistance gene and haplotype analysis across a complex disease resistance locus, allowing us to discern alleles from paralogous gene copies. We also reveal the complex genetic composition and history of the bread wheat D genome, involving previously unreported contributions from genetically and geographically discrete Ae. tauschii subpopulations. Together, our results reveal the complex history of the bread wheat D genome and demonstrate the potential of wild relatives in crop improvement.

genomics↗

bioRxiv2023

Transertion and cell geometry organize the Escherichia coli nucleoid during rapid growth

AO_SCPLOWBSTRACTC_SCPLOWBacterial chromosomes are spatiotemporally organized and sensitive to environmental changes. However, the mechanisms underlying chromosome configuration and reorganization are currently not fully understood. Using 3D single-molecule localization microscopy and live-cell imaging, we show that the Escherichia coli nucleoid adopts a condensed, membrane-associated configuration during rapid growth. To study the influence of different biosynthetic processes on nucleoid morphology and positioning, we recorded multi-colour super-resolution images during drug treatment. After developing analysis routines for confocal and super-resolution images, we captured highly resolved snapshots which revealed the complete loss of the membrane-bound state of the nucleoid within 10 minutes of halting transcription and translation. This indicates an active role of transertion (coupled transcription, translation and membrane insertion) in nucleoid organization. In contrast, cell wall synthesis inhibition only affects nucleoid organization during morphological changes. Further, we provide evidence that the E. coli nucleoid spatially correlates with MreB in unperturbed E. coli cells, while this correlation diminishes in cells with changed cell geometry or upon inhibition of protein biosynthesis. Replication inhibition experiments, as well as multi-drug treatments highlight the role of entropic effects and transcription in nucleoid condensation and positioning. In summary, we provide experimental evidence for transertion as a principal organiser of the bacterial nucleoid, and show that an altered metabolic state and antibiotic treatment both lead to major changes in the degree of transertion and overall spatial organization of the nucleoid. Our high-resolution characterization reveals dynamics of antibiotic action and provides tools to quantify bacterial chromosome organization. This does not only provide valuable insights into the role of transertion, but can also be applied to study other cell-biological processes.

microbiology↗

bioRxiv2023

A one-track model for spatiotemporal coordination of Bacillus subtilis septal cell wall synthesis

Bacterial cell division requires synthesis of a septal peptidoglycan (sPG) wall across the middle of the cell. This is accomplished by the divisome synthesis complex in coordination with numerous other division proteins--such as the essential tubulin homolog FtsZ--but the molecular mechanism of its spatiotemporal regulation remains unclear. Here, we investigate the dynamics of sPG synthesis in the model Gram-positive bacterium Bacillus subtilis using live-cell single-molecule imaging of the divisome transpeptidase PBP2B. In contrast to previous models for division, we show that there is a single population of processively-moving PBP2B molecules whose motion is driven by peptidoglycan synthesis and is not associated with FtsZ treadmilling. However, although the motions of PBP2B and FtsZ are asynchronous, we demonstrate that processive PBP2B motion is partially dependent on FtsZ treadmilling. Additionally, we provide evidence that the divisome synthesis complex is multimeric. Our results support a new model for division in B. subtilis where a multimeric synthesis complex follows a single track dependent on sPG synthesis whose activity and dynamics are asynchronous with FtsZ treadmilling.

microbiology↗

bioRxiv2023

Molecular motor tug-of-war regulates elongasome cell wall synthesis dynamics in Bacillus subtilis

Most rod-shaped bacteria elongate by inserting new cell wall material into the inner surface of the cell sidewall. This is performed by class A penicillin binding proteins (PBPs) and a highly conserved protein complex, the elongasome, which moves processively around the cell circumference and inserts long glycan strands that act as barrel-hoop-like reinforcing structures, thereby giving rise to a rod-shaped cell. However, it remains unclear how elongasome synthesis dynamics and termination events are regulated to determine the length of these critical cell-reinforcing structures. To address this, we developed a method to track individual elongasome complexes around the entire circumference of Bacillus subtilis cells for minutes-long periods using single molecule fluorescence microscopy. We found that the B. subtilis elongasome is highly processive and that processive synthesis events are frequently terminated by rapid reversal or extended pauses. We found that cellular levels of RodA regulate elongasome processivity, reversal and pausing. Our single molecule data, together with stochastic simulations, show that elongasome dynamics and processivity are regulated by molecular motor tug-of-war competition between several, likely two, oppositely oriented peptidoglycan synthesis complexes associated with the MreB filament. Our data, thus, demonstrate that molecular motor tug-of-war is a key regulator of elongasome dynamics in B. subtilis, which likely also regulates the cell shape via modulation of elongasome processivity.

microbiology↗

bioRxiv2023

Self-organisation of mortal filaments: the role of FtsZ treadmilling in bacterial division ring formation

Protein filaments in the cell commonly treadmill - they grow on one end while shrinking on the other, driven by energy consumption. Treadmilling filaments appear to be moving, even though individual proteins remain static. Here, we investigate the role of treadmilling, implemented as dynamic turnover, in the collective filament self-organisation. On the example of the bacterial FtsZ protein, a highly conserved tubulin homologue, we show, in computer simulations and in vitro experiments, that treadmilling drives filament nematic ordering by dissolving misaligned filaments. We demonstrate that ordering via local dissolution allows the system to quickly respond to chemical and geometrical biases in the cell, and is necessary for the formation of the FtsZ ring required for bacterial cell division in living Bacillus subtilis cells. We finally use simulations to quantitatively explain the characteristic dynamics of FtsZ ring formation in vivo. Beyond FtsZ and other cytoskeletal filaments, our study identifies a novel mechanism for nematic ordering via constant birth and death of energy-consuming filaments.

biophysics↗

bioRxiv2023

Uncovering the history of recombination and population structure in western Canadian stripe rust populations through mating-type alleles

The population structure of crop pathogens such as Puccinia striiformis f. sp. tritici (Pst); the cause of wheat stripe rust, is of interest to researchers looking to understand these pathogens on a molecular level, as well as those with an applied focus such as disease epidemiology. Cereal rusts can reproduce sexually or asexually, and the introduction of novel genetic lineages has the potential to cause serious epidemics such as the one caused by Warrior lineage in Europe. In a global context, Pst lineages in Canada were not well-characterized and origin of foreign incursions was not known. We used a whole-genome/transcriptome sequencing approach for the Canadian Pst population to identify lineages in a global context, origin, and evidence for foreign incursion. More importantly, for the first time ever, we identified nine alleles of the homeodomain mating-type locus in the worldwide Pst population and show that previously identified lineages generally exhibit a single pair of these alleles. In addition, we find only two pheromone receptor alleles. We show that the recent population shift from the PstS1 lineage to the PstS1-related lineage is also associated with the introduction of a novel mating-type allele (b-3) to the Canadian population. We also show evidence for high levels of mating-type diversity in samples associated with the Himalayan center of diversity for Pst, including a single Canadian race previously identified as PstPr (probable recombinant) which we identify as a foreign incursion from China circa. 2010. These data provide comprehensive details on the population biology of Canadian Pst diversity and mating-type alleles in the global Pst population which can be utilized in testing several research questions and hypotheses around sexuality and parasexuality in rust fungi.

genomics↗

bioRxiv2022

Recording large-scale, cellular-resolution neuronal activity from freely-moving mice

Current methods for recording large-scale neuronal activity from behaving mice with single-cell resolution require either fixing the mouse head under a microscope or attachment of a recording device to the animals skull. Both of these options significantly affect the animal behavior and hence also the recorded brain activity patterns. Here, we introduce a new method to acquire snapshots of single-cell cortical activity maps from freely-moving mice using a calcium sensor called CaMPARI. CaMPARI has a unique property of irreversibly changing its color from green to red inside active neurons when illuminated with 400nm light. We capitalize on this property to demonstrate cortex-wide activity recording without any head fixation or attachment of a miniaturized device to the mouses head. Multiple cortical regions were recorded while the mouse was performing a battery of behavioral and cognitive tests. We identified task-dependent activity patterns across motor and somatosensory cortices, with significant differences across sub-regions of the motor cortex. This new CaMPARI-based recording method expands the capabilities of recording neuronal activity from freely-moving and behaving mice under minimally-restrictive experimental conditions and provides large-scale volumetric data that are not accessible otherwise.

neuroscience↗

bioRxiv2022

Phylogenetic and phenotypic analysis of five subgroups of Ralstonia solanacearum phylotype IIB sequevar 4 reveals that the "not pathogenic to banana (NPB)" ecotype is polyphyletic.

The bacterial wilt pathogens in the Ralstonia solanacearum species complex (RSSC) have broad but finite host ranges. Population genetic surveys of RSSC pathogens show that many sequevars (subspecies groups) are predominantly recovered from wilting solanaceous plants. In contrast, strains in the IIB-4 sequevar have been isolated from plants in over a dozen families. Certain IIB-4 lineages have been classified as banana-virulent or "not pathogenic to banana (NPB)". Prior analysis suggested that the NPB lineage has diverged from the banana-virulent IIB-4 strains. To test this model, we analyzed the phenotypes and phylogeny of a diverse collection of 19 IIB-4 isolates. We used Illumina sequencing to assemble draft genomes of 12 new strains. Based on whole genome phylogenetic analysis, these IIB-4 strains clustered into five subclades. We quantified virulence of each strain on tomato, banana, melon, and impatiens plants. Overall, the virulence patterns correlated with phylogeny. Banana virulence was restricted to the 4/4 IIB-4D subclade (N=4/4 strains) and IIB-4E subclade (N=1/2 strains). Subclades IIB-4D and IIB-4E are sister subclades and their closest relative, the IIB-4A-C subclade, lacked virulence on banana. Our data support a revised model in which banana virulence is an innovation within the IIB4D/E subclades. Data SummaryIllumina sequencing and genome assembly data are available as NCBI BioProject PRJNA826884, and Table S1 lists the accession numbers for assemblies in GenBank and raw sequencing data in SRA. To enable future meta-analyses that identify genetic factors that drive host-range, the raw virulence data is included as Table S2.

microbiology↗

bioRxiv2021

A lineage-specific Exo70 is required for receptor kinase-mediated immunity in barley

In the evolution of land plants, the plant immune system has experienced expansion in immune receptor and signaling pathways. Lineage-specific expansions have been observed in diverse gene families that are potentially involved in immunity, but lack causal association. Here, we show that Rps8-mediated resistance in barley to the fungal pathogen Puccinia striiformis f. sp. tritici (wheat stripe rust) is conferred by a genetic module: LRR-RK and Exo70FX12, which are together necessary and sufficient. The Rps8 LRR-RK is the ortholog of rice extracellular immune receptor Xa21 and Exo70FX12 is a member of the Poales-specific Exo70FX clade. The Exo70FX clade emerged after the divergence of the Bromeliaceae and Poaceae, and comprises from 2 to 75 members in sequenced grasses. These results demonstrate the requirement of a lineage-specific Exo70FX12 in Rps8 LRR-RK immunity and suggest that the Exo70FX clade may have evolved a specialized role in receptor kinase signaling.

plant biology↗

bioRxiv2021

DeepBacs: Bacterial image analysis using open-source deep learning approaches

Deep Learning (DL) is rapidly changing the field of microscopy, allowing for efficient analysis of complex data while often out-performing classical algorithms. This revolution has led to a significant effort to create user-friendly tools allowing biomedical researchers with little background in computer sciences to use this technology effectively. Thus far, these approaches have mainly focused on analysing microscopy images from eukaryotic samples and are still underused in microbiology. In this work, we demonstrate how to use a range of state-of-the-art artificial neural-networks particularly suited for the analysis of bacterial microscopy images, using our recently developed ZeroCostDL4Mic platform. We showcase different DL approaches for segmenting bright field and fluorescence images of different bacterial species, use object detection to classify different growth stages in time-lapse imaging data, and carry out DL-assisted phenotypic profiling of antibiotic-treated cells. To also demonstrate the DL capacity to enhance low-phototoxicity live-cell microscopy, we showcase how image denoising can allow researchers to attain high-fidelity data in faster and longer imaging. Finally, artificial labelling of cell membranes and predictions of super-resolution images allow for accurate mapping of cell shape and intracellular targets. To aid in the training of novice users, we provide a purposefully-built database of training and testing data, enabling bacteriologists to quickly explore how to analyse their data through DL. We hope this lays a fertile ground for the efficient application of DL in microbiology and fosters the creation of novel tools for bacterial cell biology and antibiotic research.

microbiology↗