Biology subjects

Fletcher, K.

Publications and source records attributed to Fletcher, K..

11 recordsLinked to original sources

bioRxiv2026

Applying integrated population models to guide conservation planning: Western Capercaillie in Scotland

O_LIMany conservation interventions prove ineffective because they lack a rigorous evidence-base. Multifactorial drivers and data limitations often hinder anticipatory planning and are difficult to tackle with standard methods. Predictive modelling approaches that integrate diverse data sources with biological hypotheses can bridge this gap by clarifying the drivers of decline and evaluating management options under uncertainty. C_LIO_LI We developed a Bayesian integrated population model (IPM) applicable to lekking species, combining incomplete data from different life stages and seasons while accounting for observation error. IPMs are widely recognised for reducing bias relative to single-data-stream analyses, yet their application in conservation planning remains limited. C_LIO_LIWe applied the model to the threatened Western Capercaillie population in Scotland, which has been declining since at least the 1980s despite decades of conservation efforts, a typical case requiring urgent, evidence-based management. By fitting the model to 30 years of biased and incomplete monitoring data, we investigated associations between demographic processes and their potential drivers, including weather variables, fence collision mortality and a proxy of predation pressure. We used model predictions to evaluate the joint effectiveness of proposed management actions aimed at improving vital rates, including fence management and diversionary feeding. C_LIO_LIData integration improved population estimate precision by 17-49% relative to standalone national survey estimates, confirming that the decline continued from 1990-2023. Reproductive rate was related to the pattern of April warming, negatively affected by pre-breeding precipitation and positively by vole abundance, the latter consistent with the alternative prey hypothesis. Model predictions indicated that combining diversionary feeding with fence removal produced the most favourable conservation outcomes, but growth remained limited and uncertainty included possible continued decline. C_LIO_LI5. This modelling approach forms a key component of Scotlands Capercaillie Emergency Plan 2025-2030, guiding management by assessing population-level responses and predicting conservation intervention outcomes. In future, the model could support formal adaptive management through iterative evaluation of interventions as new data become available. Beyond this case study, our integrated approach offers a transferable framework for managing multifactorial declines in other threatened species under uncertainty. C_LI

ecology↗

bioRxiv2025

Stress-induced switch in small extracellular vesicle secretion: from constitutive 'torn bag mechanism' to exocytosis

The biogenesis of small extracellular vesicles (sEVs) is only partially understood. Our recent findings provide evidence that a newly described sEV secretion pathway, the amphiectosome release and the "torn bag mechanism", is present in all tested cell lines and in mouse liver and kidney. Surprisingly, in in situ fixed steady-state cells, transmission electron microscopy did not reveal the classical exosome secretion route, the sEV release via exocytosis of multivesicular endosomes (MVEs). In the current study, we investigated which parameters influence the activation of the two distinct sEV release mechanisms. Our results show that under stress conditions (such as Ca{superscript 2} ionophore-induced membrane stress or metabolic stress-induced by serum starvation), exocytosis of MVEs is activated, while this process is absent in steady-state conditions. By silencing ATG5 (a key regulator of autophagy) and RAB27a (essential small GTPase for MVE exocytosis), we selectively modulated these two mechanisms. Amphiectosome release depended on both autophagy and ATG5, while exocytosis of MVE was autophagy-independent but RAB27a-dependent. Our findings suggest that sEV release via the "torn bag mechanism" is a general and essential secretion pathway in non-stressed, steady-state mammalian cells, while stress conditions induce the sEV release via MVE exocytosis.

cell biology↗

bioRxiv2025

Metagenomics reveals fibre fermentation and AMR pathways in red grouse (Lagopus scotica) microbiota

BackgroundThe avian caecal microbiota plays a vital role in host nutrition, enabling non-digestible, fibrous material to be converted into compounds that can be absorbed and used as an energy source by the host. The diet of adult red grouse (Lagopus scotica) is dominated by heather (Calluna vulgaris), which is particularly high in fibre. It is therefore likely that the caecal microbiota plays a key role in enabling grouse to thrive on this diet. In this study, we present the first characterisation of the caecal microbiota of red grouse using modern sequencing methods. ResultsWe performed metagenomic sequencing on caecal content samples from fifteen red grouse from three upland estates in Scotland. From this data, we constructed and characterised twelve high-quality, species-level metagenome assembled genomes (MAGs). Eleven of these MAGs could not be assigned a taxonomic label at species level, indicating that they may be novel species. MAGs belonged to diverse taxa (5 phyla) and several encoded genes and pathways for the digestion of fibres, including cellulose, hemi-cellulose, xylooligosaccharides and pectin. Several MAGs also contained antimicrobial resistance genes, predominantly related to vancomycin resistance. ConclusionsThis study is the first to reconstruct commensal microbial genomes from red grouse. The caeca contain diverse, often novel, microbial taxa capable of fermenting various fibres, potentially aiding in the digestion of the red grouses high-fibre diet. Further research is necessary to explore how these bacteria support red grouse nutrition and health.

microbiology↗

bioRxiv2024

A "torn bag mechanism" of small extracellular vesicle release via limiting membrane rupture of en bloc released amphisomes (amphiectosomes)

Recent studies showed an unexpected complexity of extracellular vesicle (EV) biogenesis pathways. We previously found evidence that human colorectal cancer cells in vivo release large multivesicular body-like structures en bloc. Here, we tested whether this large extracellular vesicle type is unique to colorectal cancer cells. We found that all cell types we studied (including different cell lines and cells in their original tissue environment) released multivesicular large EVs (MV-lEVs). We also demonstrated that upon spontaneous rupture of the limiting membrane of the MV-lEVs, their intraluminal vesicles (ILVs) escaped to the extracellular environment by a "torn bag mechanism". We proved that the MV-lEVs were released by ectocytosis of amphisomes (hence, we termed them amphiectosomes). Both ILVs of amphiectosomes and small EVs separated from conditioned media were either exclusively CD63 or LC3B positive. According to our model, upon fusion of multivesicular bodies with autophagosomes, fragments of the autophagosomal inner membrane curl up to form LC3B positive ILVs of amphisomes, while CD63 positive small EVs are of multivesicular body origin. Our data suggest a novel common release mechanism for small EVs, distinct from the exocytosis of multivesicular bodies or amphisomes, as well as the small ectosome release pathway.

cell biology↗

bioRxiv2022

The genome of the oomycete Peronosclerospora sorghi, a cosmopolitan pathogen of maize and sorghum, is inflated with dispersed pseudogenes

Several species in the oomycete genus Peronosclerospora cause downy mildew on maize and can result in significant yield losses in Asia. Biosurveillance of these pathogens is a high priority to prevent epidemics on maize in the US and consequent damage to the US economy. The unresolved taxonomy and dearth of molecular resources for Peronosclerospora spp. hinder these efforts. P. sorghi is a pathogen of sorghum and maize with a global distribution, for which limited diversity has been detected in the southern USA. We characterized the genome, transcriptome, and mitogenome of an isolate, representing the US pathotype 6. The highly homozygous genome was assembled using 10x Genomics linked reads and scaffolded using Hi-C into 13 chromosomes. The total assembled length was 319.6 Mb--larger than any other oomycete previously assembled. The mitogenome was 38 kb, similar in size to other oomycetes, although it had a unique gene order. Nearly 20,000 genes were annotated in the nuclear genome, more than described for other downy mildew causing oomycetes. The 13 chromosomes of P. sorghi were highly syntenic with the 17 chromosomes of Peronospora effusa with conserved centromeric regions and distinct chromosomal fusions. The increased assembly size and gene count of P. sorghi is due to extensive retrotransposition, resulting in putative pseudogenization. Ancestral genes had higher transcript abundance and were enriched for differential expression. This study provides foundational resources for analysis of Peronosclerospora and comparisons to other oomycete genera. Further genomic studies of global Peronosclerospora spp. will determine the suitability of the mitogenome, ancestral genes, and putative pseudogenes for marker development and taxonomic relationships.

genomics↗

bioRxiv2021

Ancestral chromosomes for the Peronosporaceae inferred from a telomere-to-telomere genome assembly of Peronospora effusa.

We report the first telomere-to-telomere genome assembly for an oomycete. This assembly has extensive synteny with less complete genome assemblies of other oomycetes and will therefore serve as a reference genome for this taxon. Downy mildew disease of spinach, caused by the oomycete Peronospora effusa, causes major losses to spinach production. The 17 chromosomes of P. effusa were assembled telomere-to-telomere using Pacific Biosciences High Fidelity reads. Sixteen chromosomes are complete and gapless; Chromosome 15 contains one gap bridging the nucleolus organizer region. Putative centromeres were identified on all chromosomes. This new assembly enables a re-evaluation of the genomic composition of Peronospora spp.; the assembly was almost double the size and contained more repeat sequences than previously reported for any Peronospora spp. Genome fragments consistently under-represented in six previously reported assemblies of P. effusa typically encoded repeats. Some genes annotated as encoding effectors were organized into multigene clusters on several chromosomes. At least two effector-encoding genes were annotated on every chromosome. The intergenic distances between annotated genes were consistent with the two-speed genome hypothesis, with some effectors located in gene-sparse regions. The near-gapless assembly revealed apparent horizontal gene transfer from Ascomycete fungi. Gene order was highly conserved between P. effusa and the genetically oriented assembly of the oomycete Bremia lactucae. High levels of synteny were also detected with Phytophthora sojae. Many oomycete species may have similar chromosome organization; therefore, this genome assembly provides the foundation for genomic analyses of diverse oomycetes.

genomics↗

bioRxiv2021

Variance of allele balance calculated from low coverage sequencing data infers departure from a diploid state.

MotivationPolyploidy and heterokaryosis are common and consequential genetic phenomena that increase the number of haplotypes in an organism and complicate whole-genome sequence analysis. Allele balance has been used to infer polyploidy and heterokaryosis in diverse organisms using read sets sequenced to greater than 50x whole-genome coverage. However, Sequencing to adequate depth is costly if applied to multiple individuals or large genomes. ResultsWe developed VCFvariance.pl to utilize the variance of allele balance to infer polyploidy and/or heterokaryosis at low sequence coverage. This analysis requires as little as 10x whole-genome coverage and reduces the allele balance profile down to a single value, which can be used to determine if an individual has two or more haplotypes. This approach was validated on simulated, synthetic, and authentic read sets from an oomycete, fungus, and plant. The approach was deployed to ascertain the genome status of multiple isolates of Bremia lactucae and Phytophthora infestans. Availability and implementationVCFvariance.pl is a Perl script available at https://github.com/kfletcher88/VCFvariance.

bioinformatics↗

bioRxiv2021

GABARAP membrane conjugation sequesters the FLCN-FNIP tumor suppressor complex to activate TFEB and lysosomal biogenesis

Adaptive changes in lysosomal capacity are driven by the transcription factors TFEB and TFE3 in response to increased autophagic flux and endolysosomal stress, yet the molecular details of their activation are unclear. LC3 and GABARAP members of the ATG8 protein family are required for selective autophagy and sensing perturbation within the endolysosomal system. Here we show that during single membrane ATG8 conjugation (SMAC), Parkin-dependent mitophagy, and Salmonella-induced xenophagy, the membrane conjugation of GABARAP, but not LC3, is required for activation of TFEB/TFE3 to control lysosomal homeostasis and capacity. GABARAP directly binds to a novel LC3-interacting motif (LIR) in the FLCN/FNIP tumor suppressor complex with picomolar affinity and regulates its relocalization to these GABARAP-conjugated membrane compartments. This disrupts the regulation of RagC/D by the FLCN/FNIP GAP complex, resulting in impaired mTOR-dependent phosphorylation of TFEB without changing mTOR activity towards other substrates. Thus, the GABARAP-FLCN/FNIP-TFEB axis serves as a universal molecular sensor that coordinates lysosomal homeostasis with perturbations and cargo flux within the autophagy-lysosomal network.

cell biology↗

bioRxiv2020

AFLAP: Assembly-Free Linkage Analysis Pipeline using k-mers from whole genome sequencing data

BackgroundGenetic maps are an important resource for validation of genome assemblies, trait discovery, and breeding. Next generation sequencing has enabled production of high-density genetic maps constructed with 10,000s of markers. Most current approaches require a genome assembly to identify markers. Our Assembly Free Linkage Analysis Pipeline (AFLAP) removes this requirement by using uniquely segregating k-mers as markers to rapidly construct a genotype table and perform subsequent linkage analysis. This avoids potential biases including preferential read alignment and variant calling. ResultsThe performance of AFLAP was determined in simulations and contrasted to a conventional workflow. We tested AFLAP using 100 F2 individuals of Arabidopsis thaliana, sequenced to low coverage. Genetic maps generated using k-mers contained over 130,000 markers that were concordant with the genomic assembly. The utility of AFLAP was then demonstrated by generating an accurate genetic map using genotyping-by-sequencing data of 235 recombinant inbred lines of Lactuca spp. AFLAP was then applied to 83 F1 individuals of the oomycete Bremia lactucae, sequenced to >5x coverage. The genetic map contained over 90,000 markers ordered in 19 large linkage groups. This genetic map was used to fragment, order, orient, and scaffold the genome, resulting in a much-improved reference assembly. ConclusionsAFLAP can be used to generate high density linkage maps and improve genome assemblies of any organism when a mapping population is available using whole genome sequencing or genotyping-by-sequencing data. Genetic maps produced for B. lactucae were accurately aligned to the genome and guided significant improvements of the reference assembly.

genetics↗

bioRxiv2019

Effector prediction and characterization in the oomycete pathogen Bremia lactucae reveal host-recognized WY domain proteins that lack the canonical RXLR motif

Pathogens infecting plants and animals use a diverse arsenal of effector proteins to suppress the host immune system and promote infection. Identification of effectors in pathogen genomes is foundational to understanding mechanisms of pathogenesis, for monitoring field pathogen populations, and for breeding disease resistance. We identified candidate effectors from the lettuce downy mildew pathogen, Bremia lactucae, using comparative genomics and bioinformatics to search for the WY domain. This conserved structural element is found in Phytophthora effectors and some other oomycete pathogens; it has been implicated in the immune-suppressing function of these effectors as well as their recognition by host resistance proteins. We predicted 54 WY domain containing proteins in isolate SF5 of B. lactucae that have substantial variation in both sequence and domain architecture. These candidate effectors exhibit several characteristics of pathogen effectors, including an N-terminal signal peptide, lineage specificity, and expression during infection. Unexpectedly, only a minority of B. lactucae WY effectors contain the canonical N-terminal RXLR motif, which is a conserved feature in the majority of cytoplasmic effectors reported in Phytophthora spp. Functional analysis effectors containing WY domains revealed eleven out of 21 that triggered necrosis, which is characteristic of the immune response on wild accessions and domesticated lettuce lines containing resistance genes. Only two of the eleven recognized effectors contained a canonical RXLR motif, suggesting that there has been an evolutionary divergence in sequence motifs between genera; this has major consequences for robust effector prediction in oomycete pathogens.\n\nAuthor SummaryThere is a microscopic battle that takes place at the molecular level during infection of plants and animals by pathogens. Some of the weapons that pathogens battle with are known as \"effectors,\" which are secreted proteins that enter host cells to alter physiology and suppress the immune system. Effectors can also be a liability for plant pathogens because plants have evolved ways to recognize these effectors, triggering a defense response leading to localized cell death, which prevents the spread of the pathogen. Here we used computer models to predict effectors from the genome of Bremia lactucae, the causal agent of lettuce downy mildew. Three effectors were demonstrated to suppress the basal immune system of lettuce. Eleven effectors were recognized by one or more resistant lines of lettuce. In addition to contributing to our understanding of the mechanisms of pathogenesis, this study of effectors is useful for breeding disease resistant lettuce, decreasing agricultural reliance on fungicides.

plant biology↗

bioRxiv2019

Genomic signatures of somatic hybrid vigor due to heterokaryosis in the oomycete pathogen, Bremia lactucae.

Lettuce downy mildew caused by Bremia lactucae is the most important disease of lettuce globally. This oomycete pathogen is highly variable and has rapidly overcome resistance genes and fungicides deployed in attempts to control it. The described high-quality genome assembly of B. lactucae provides the foundation for detailed understanding of this economically important pathogen. The biotrophic nature of B. lactucae coupled with high levels of heterozygosity and the recently expanded repeat content made genome assembly challenging. The combined use of multiple read types, including synthetic long reads, single molecule sequences, and Hi-C, resulted in a high-quality, chromosome-scale, consensus assembly of this diploid organism. Phylogenetic analysis supports polyphyly in the downy mildews consistent with the biotrophic mode of pathogenesis evolving more than once in the Peronosporaceae. Flow cytometry plus resequencing of 30 field isolates as well as sexual offspring and asexual derivatives from multinucleate single sporangia demonstrated a high incidence of heterokaryosis in B. lactucae. Heterokaryons have phenotypic differences and increased fitness compared to homokaryotic derivatives. Consequently, B. lactucae exhibits somatic hybrid vigor and selection should be considered as acting on a population of nuclei within coenocytic mycelia. This provides evolutionary flexibility to the pathogen enabling rapid adaptation to different repertoires of host resistance genes and other challenges. The advantages of asexual persistence of heterokaryons may have been one of the drivers of selection that resulted in the loss of uninucleate zoospores in multiple downy mildews.

genomics↗