Biology subjects

Schaefer, P.

Publications and source records attributed to Schaefer, P..

12 recordsLinked to original sources

bioRxiv2026

Volatile organic compounds of L. enzymogenes LEC suppress the growth of fungal and oomycete pathogens and modify the cell physiology of Rhizoctonia solani

Lysobacter enzymogenes LEC is a plant disease-suppressive bacterium. To obtain a more comprehensive understanding of its biocontrol activity and to identify further potential application methods, we investigated the function of its volatile organic compounds (VOCs). VOC activity was assessed in vitro using split-plate assays with eight different fungal and oomycete isolates and in planta against Pseudoperonospora cubensis on cucumber plants. The volatilomes of LEC and the less active L. enzymogenes DSM2043 were analysed by GC-MS analyses. Further, the effects of VOCs on cell physiology of Rhizoctonia solani were analyzed by fluorescence and transmission electron microscopy. The strongest inhibition of LEC VOCs was observed for Phytophthora infestans (94%) and Phoma lingam (83%), while inhibitions were lower for Rhizoctonia solani (42%) and Alternaria spp. (24% and 29%) and not detectable for two Fusarium isolates. VOC exposure also significantly reduced downy mildew caused by P. cubensis on cucumber plants (39%), although this effect was locally restricted to the leaf directly above the VOC source. In R. solani, VOC exposure reduced nuclei abundance and increased lipid accumulation. GC MS analyses revealed distinct volatile profiles between LEC and L. enzymogenes DSM2043 and identified ketones, such as 2-dodecanone and undecanone as candidate bioactive compounds, which is in agreement with previous findings. Our results demonstrate broad-spectrum VOC-mediated biocontrol activity, and thereby provide a more comprehensive understanding of its biocontrol activity and highlight new ways for potential applications of LEC.

microbiology↗

bioRxiv2026

IDENTIFICATION OF HUMAN PHOTORECEPTORS SUITABLE FOR CELL REPLACEMENT STUDIES IN A PRECLINICAL ACHROMATOPSIA MODEL

Cell replacement represents a potential treatment modality for retinal disorders characterized by photoreceptor loss. However, photoreceptor replacement approaches have not been clinically established. To take this forward, the main goal of this study was to systematically compare human photoreceptors of different ages and identify those that enable functional integration into the degenerative retina. Donor cells were isolated from iPSC-derived retinal organoids generated by a GMP-compliant protocol at differentiation days 120, 150, or 200 and transplanted subretinally into cone photoreceptor function loss 1 (Cpfl1) recipients, an inherited mouse model of cone degeneration. While younger photoreceptors showed slightly improved transplantation outcomes, donor photoreceptors of all culture stages displayed long-term survival, cone identity, structural integration into the host retina, and tight interactions with host Muller glia, including formation of a continuous outer limiting membrane. Transplanted photoreceptors showed signs of advanced maturation, including correct polarization with generation of apical inner- and outer segments, while basal synapses were formed with host bipolar cells. Electrophysiological assessment of host retinal ganglion cells revealed light-evoked responses in transplant-containing regions, providing evidence for functional incorporation of human photoreceptors into the mouse neuro-retinal circuitry. Thus, GMP-compliant human iPSC-derived photoreceptors are stable over a wide range of differentiation stages and constitute a robust cell source for retinal transplantation and functional repair. The findings provide important prerequisites for the development of standardized procedures towards clinical translation of photoreceptor replacement in the retina. Significant statementCell transplantation represents a potential therapy for retinal dystrophies. However, conditions allowing stable functional integration and maturation of donor photoreceptors have not been defined. We systematically compared GMP-compliant iPSC-derived photoreceptors of different culture times after transplantation into a retinal degeneration mouse model. Photoreceptors of all ages showed structural integration and advanced maturation in close interaction with host Muller glia and bipolar cells, allowing restoration of light-driven responses. Thus human photoreceptors isolated from retinal organoids represent a robust source for clinical development of cell replacement in the diseased retina.

neuroscience↗

bioRxiv2025

Cross-kingdom degradomics identifies natural plant small RNAs with disease protecting activity

RNA interference (RNAi) is a highly specific process regulating genes transcriptionally or post-transcriptionally via silencing. small regulatory RNAs (sRNAs) provide the specificity of post-transcriptional silencing by binding to complementary mRNAs leading to their degradation or translational repression. sRNAs are promising non-chemical control agents against plant pests and diseases. Their application requires, however, a deep understanding of the determinants of functional specificity and efficiency. In nature, interacting plants and pathogens follow the manipulative strategy of cross-kingdom RNAi (ckRNAi) based on the targeted transfer of sRNAs. In this study, we employed a genome-wide approach to explore the diversity of natural ck-sRNAs exchanged in the pathogenic interaction of Brachypodium distachyon with the fungal pathogen Fusarium graminearum. By deep-sequencing of the cross-kingdom sRNAome, transcriptome and degradome 258 ckRNAi-mediating sRNAs were discovered. In a simulation-based approach, specific sequence characteristics allowed categorizing sRNAs into functionality classes. Subsequent genome annotation analyses revealed the organization of plant ck-sRNAs in long non-coding RNAs. Moreover, comparative sequence analyses revealed an evolution of ck-sRNAs towards invariant mRNA target regions. Functional analyses with sRNA candidates confirmed an antipathogenic activity at exceptionally low application doses. It indicates natural sRNA as untapped resource and potential blueprint for the development of highly specific and effective plant-derived bioprotectants.

plant biology↗

bioRxiv2025

High Precision Quantification of small RNA Slicing Activity - Native Index Ligation-based Targeted Degradome Sequencing (NIL-TDS)

RNA interference (RNAi) is an effective and precise regulatory mechanism in eukaryotes in which small RNAs mediate endonucleolytic slicing of complementary target mRNAs. Despite the potential of RNAi for human therapeutics and crop bio-protection, analytical platforms to quantitatively validate the slicing activities of small RNAs remain limited. Here, we present NIL-TDS, a cost-effective method that combines RNA ligase-mediated PCR with Nanopore Sequencing for direct, rapid, and high-resolution detection and quantification of sRNA-mediated slicing events. Using NIL-TDS, we quantitatively detected minute changes in Ath-mir400 mediated slicing of PPR1 in heat and salt stressed Arabidopsis plants. We further demonstrate the broader applicability of NIL-TDS by detecting rare slicing events in a mammalian system. Of relevance for malignancy of certain cancers, and tumor progression and metastasis, NIL-TDS further confirmed miR-196-HOXB8 interactions in lung cancer cells and discovered a novel miR-7162-HOXA10-AS slicing site. These findings demonstrate the sensitivity of NIL-TDS for uncovering small RNA mediated regulatory mechanisms of gene expression and disease progression in eukaryotes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/679503v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@adef35org.highwire.dtl.DTLVardef@1339570org.highwire.dtl.DTLVardef@1980652org.highwire.dtl.DTLVardef@1c732ef_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Fertilization impacts microbiomes along the grassland trophic chain

Agricultural grasslands are often managed intensively, influencing soil properties and microbial communities. This can result in significant community changes and challenges to health and function at different levels along the trophic chain. This study investigates how fertilization affects microbial communities in grassland ecosystems in multiple connected trophic compartments. Shifts in microbial composition occurred in response to fertilization with effects being host-dependent and changes being more pronounced in belowground compartments. Strong interactions between trophic levels facilitated transmission of bacterial genera from soil and roots to higher trophic levels. Pig slurry- derived microbes were found in all compartments, but their low prevalence suggests an indirect effect of fertilisation, primarily due to changes in nutrient availability. Our findings emphasize the importance of considering both individual compartments and trophic interactions in ecosystems to fully understand the effects of anthropogenic disturbances on environmental and human health.

microbiology↗

bioRxiv2024

Ammonia leakage can underpin nitrogen-sharing among soil microbes

AbstractSoil microbial communities host a large number of microbial species that support important ecological functions such as biogeochemical cycling and plant nutrition. The extent and stability of these functions are affected by inter-species interactions among soil microbes, yet the different mechanisms underpinning microbial interactions in the soil are not fully understood. Here, we study the extent of nutrient-based interactions among two model, plant-supporting soil microbes, the fungi Serendipita indica and the bacteria Bacillus subtilis. We find that S. indica is unable to grow with nitrate - a common nitrogen source in the soil - but this inability can be rescued, and growth restored in the presence of B. subtilis. We demonstrate that this effect is due to B. subtilis utilising nitrate and releasing ammonia, which can be used by S. indica. We refer to this type of mechanism as ammonia mediated nitrogen sharing (N-sharing). Using a mathematical model, we demonstrate that the pH dependent equilibrium between ammonia (NH3) and ammonium (NH4+) results in an inherent cellular leakiness, and that reduced amonnium uptake or assimilation rates can result in higher levels of leaked ammonia. In line with this model, a mutant B. subtilis - devoid of ammonia uptake - shows higher S. indica growth support in nitrate media. These findings highlight that ammonia based N-sharing can be a previously under-appreciated mechanism underpinning interaction among soil microbes and could be influenced by microbial or abiotic alteration of pH in microenvironments. Significance statementSoil microbial communities are an important factor in environmental nutrient cycling and sub-sequently plant health. S. indica is a well-studied plant growth promoting soil fungus but its inability to use nitrate, a major component of both agricultural/natural soils and crop fertilisers, may have important implications for agriculture and microbial ecology. We have demonstrated that S. indica is dependant on external sources of nitrogen in nitrate-only environments and these can be produced by B. subtilis, another common soil microbe. We then demonstrate that this nitrogen sharing interaction is likely mediated by leaked ammonia and that ammonia leakage is influenced by environmental pH. Ammonia leakage and sharing represent currently unexplored and potentially vital components of nutrient interactions between microbes in soil communities, with profound implications for microbiome community structure and subsequent consequences for soil biogeochemical cycling and crop health.

microbiology↗

bioRxiv2024

Building a Collaborative DNA Barcode Library for the Diptera of Churchill, Canada: A Resource for Northern Research

Churchill, Manitoba, Canada is a diverse subarctic region that has been the focus of DNA barcoding efforts and research for decades. Despite this effort, there are still taxa in this region that are underrepresented in public databases, particularly hyper diverse insect groups. We present a collaborative large-scale DNA barcode reference library for Diptera based on molecular data and expert taxonomic identification, and we explore habitat occupancy, biogeographic patterns, and molecular evolution. The reference library contains 16786 specimens sampled around Churchill from 2005 to 2011. Selected specimens were identified by morphological means, sequenced for the animal barcode marker, and sorted into 2225 Molecular Operational Taxonomic Units (using Barcode Index Numbers: BINs) representing 68 families and 1216 named species. Eighty-five species shared a BIN with another species, 143 species were split across several BINs, and 990 species were assigned to a unique BIN, including 129 BINs new to the Barcode of Life Data System. The intraspecific and nearest neighbour distances varied across families, but most species can be easily distinguished from their nearest neighbours. By combining molecular data with taxonomic expertise, the barcode library provides species-level information for 12608 specimens, supplying future researchers with detailed taxonomic information and the opportunity to perform barcode-based specimen identifications for large-scale studies through sequence matching. Comparison of the Diptera composition of Churchill to other subarctic regions reflected likely postglacial colonization northwards as well as a Beringian and circumpolar component. This study also provides an initial investigation of molecular evolution in the best-represented species and finds variability in the rate of molecular evolution, opening intriguing avenues for further investigation. This study provides a publicly available, detailed reference library for use in future research, as well as new insights into the current diversity and taxonomic composition of Diptera present among the diverse habitats of Churchill.

molecular biology↗

bioRxiv2024

Gene regulatory network landscape of Group 3/4 medulloblastoma

Cellular heterogeneity in Group 3 and Group 4 medulloblastomas is a major driver of therapeutic intractability. Here, we describe transcription factor-driven mechanisms underlying cellular diversity in these tumors using a comprehensive single-nucleus multi-omics atlas. Our analysis reveals that rather than fixed entities, these tumors exist along a continuum of cell states defined by four molecular identity axes. We show that perturbing transcription factor activity drives cellular plasticity, a key contributor to tumor heterogeneity. Strikingly, modulation of the lineage determinant PAX6 redirects tumor cells along both Group 3- and Group 4-like differentiation trajectories, modeling a bi-lineage medulloblastoma, and reduces aggressiveness of MYC-driven tumor models. Together, our findings reveal how oncogenic signals co-opt cerebellar unipolar brush cell programs to rewire tumor cell states and identify cellular plasticity as a potential targetable vulnerability in medulloblastoma.

cancer biology↗

bioRxiv2023

Efficient scar-free knock-ins of several kilobases by engineered CRISPR/Cas endonucleases

In plants and mammals, non-homologous end-joining is the dominant pathway to repair DNA double strand breaks, making it challenging to generate knock-in events. Using a transient assay in Nicotiana benthamiana we identified two groups of exonucleases, respectively from the Herpes Virus and from the bacteriophage T7 families, hat confer up to 38-fold increase in HDR frequencies when fused to Cas9. We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems. In Arabidopsis thaliana, fusion of Cas9 to a Herpes Virus family exonuclease leads to 10-fold higher frequencies of knock-ins. Our results open perspectives for the routine production of knock-in and gene replacement events in plants. One-Sentence SummaryFusions of CRISPR endonucleases to specific 5'-exonucleases leads to significant increase in scar-free multikilobase knock-ins.

molecular biology↗

bioRxiv2022

Rnf20 shapes the endothelial control of heart morphogenesis and function

During embryogenesis, distinct cardiac cell types form, which shape the structural and functional properties of the heart. How their activity is coordinated is largely unknown. Here we show that Rnf20 is a multifaceted regulator of cardiac morphogenesis and function. On the one hand, Rnf20 controls extracellular matrix dynamics and endothelial-cardiomyocyte crosstalk essential for second heart field development. On the other hand, it safeguards endothelial cell identity and function by maintaining physiological angiocrine signaling and preventing endothelial-to-mesenchymal transition. Endothelial-specific deletion of Rnf20 led to ventricular septal defects, myocardial thinning and cardiac dysfunction as a result of aberrant signaling and excessive extracellular matrix deposition that induced precocious cardiomyocyte binucleation and irregular contractility. Furthermore, we uncovered upstream factors (e.g. Sox9) and multiple angiocrine and extracellular matrix molecules that alter cardiomyocyte functionality upon endothelial Rnf20 loss. In summary, our work identifies a novel, endothelial-specific role of Rnf20 in regulating cardiac morphogenesis and function.

developmental biology↗

bioRxiv2018

Reconstitution of mammalian Cleavage Factor II involved in 3’ processing of mRNA precursors

Cleavage factor II (CF II) is a poorly characterized component of the multi-protein complex catalyzing 3 cleavage and polyadenylation of mammalian mRNA precursors. We have reconstituted CF II as a heterodimer of hPcf11 and hClp1. The heterodimer is active in partially reconstituted cleavage reactions, whereas hClp1 by itself is not. Pcf11 moderately stimulates the RNA 5 kinase activity of hClp1; the kinase activity is dispensable for RNA cleavage. CF II binds RNA with nanomolar affinity. Binding is mediated mostly by the two zinc fingers in the C-terminal region of hPcf11. RNA is bound without pronounced sequence-specificity, but extended G-rich sequences appear to be preferred. We discuss the possibility that CF II contributes to the recognition of cleavage/polyadenylation substrates through interaction with G-rich far-downstream sequence elements.

biochemistry↗

bioRxiv2017

Impact of spatial organization on a novel auxotrophic interaction among soil microbes

A key prerequisite to achieve a deeper understanding of microbial communities and to engineer synthetic ones is to identify the individual metabolic interactions among key species and how these interactions are affected by different environmental factors. Deciphering the physiological basis of species-species and species-environment interactions in spatially organized environment requires reductionist approaches using ecologically and functionally relevant species. To this end, we focus here on a specific defined system to study the metabolic interactions in a spatial context among a plant-beneficial endophytic fungus Serendipita indica, and the soil-dwelling model bacterium Bacillus subtilis. Focusing on the growth dynamics of S. indica under defined conditions, we identified an auxotrophy in this organism for thiamine, which is a key co-factor for essential reactions in the central carbon metabolism. We found that S. indica growth is restored in thiamine-free media, when co-cultured with B. subtilis. The success of this auxotrophic interaction, however, was dependent on the spatial and temporal organization of the system; the beneficial impact of B. subtilis was only visible when its inoculation was separated from that of S. indica either in time or space. These findings describe a key auxotrophic interaction in the soil among organisms that are shown to be important for plant ecosystem functioning, and point to the potential importance of spatial and temporal organization for the success of auxotrophic interactions. These points can be particularly important for engineering of minimal functional synthetic communities as plant-seed treatments and for vertical farming under defined conditions.

systems biology↗