Biology subjects

Klingl, A.

Publications and source records attributed to Klingl, A..

17 recordsLinked to original sources

bioRxiv2026

Phagocytosis without mitochondria is a lineage trait of planctomycetal bacteria of prey

Phagocytosis is considered energetically out of reach for prokaryotes. Mitochondria-first models of eukaryogenesis therefore suggest a non-phagocytic archaeal host. Yet two recently described planctomycetes lack mitochondria and engulf entire bacteria. Whether this mode of predation is an oddity of two isolates or the hallmark of a bacterial lineage remained unresolved. Here, we report eight additional predatory Planctomycetota from three aquatic habitats. The ten cultured strains now span two classes of the phylum Planctomycetota,"Candidatus Uabimicrobiia" and one newly proposed class. Using engineered fluorescent prey, we show that nine strains internalize prey into fully sealed compartments whereas one digests its prey externally, a non-engulfing predator for comparison. The strains additionally divide by two distinct modes, however, not correlating with their feeding behavior. Sequence- and structure-based searches detect no eukaryotic phagocytosis protein conserved across the engulfing strains. The actin homologue previously proposed to mediate uptake occurs only in three engulfing strains. Routine engulfment by aerobic bacteria without mitochondria thus unsettles a premise of mitochondria-first models and reopens the question of which cell plans could have hosted the mitochondrial endosymbiont.

microbiology↗

bioRxiv2026

Pathogen cell wall degrading enzymes facilitate extracellular vesicles to deliver RNA into plants

Extracellular vesicles (EVs) can deliver RNA and proteins into host cells to manipulate immunity; but how EVs transverse the cell wall is unknown. Using the fungal pathogen Botrytis cinerea that induces cross-kingdom RNA interference in plants, we uncovered that EV-mediated RNA delivery was dependent on cell wall degrading enzymes. Through fluorescence and transmission electron microscopy, and molecular genetic techniques, we demonstrate that EV-associated proteins compromise the plant cell wall, thereby facilitating RNA delivery. Notably, cell wall degrading enzymes are commonly associated with EVs across plant-colonizing bacterial, fungal and oomycete species, indicating a conserved role in EV transport across cell walls. These findings uncover a formerly unknown mechanism by which cell wall degrading enzymes facilitate EVs to transverse the cell wall for cargo delivery in cross-kingdom communication.

plant biology↗

bioRxiv2026

Gene Supplementation of MYO7A or activation of Myo7b for treatment of Usher syndrome 1B

Mutations in MYO7A result in the most severe subtype of Usher syndrome, the leading genetic cause of deafblindness. The large size of MYO7A requires dual adeno-associated virus (AAV) vectors for gene transfer or alternative methods to treat retinal defects. Here, we evaluated two treatment approaches: i) Supplementation of the human MYO7A gene via dual mRNA trans-splicing AAVs, and ii) CRISPR/Cas-mediated activation of the related murine Myo7b gene. Upon MYO7A supplementation, the transgenic MYO7A transcript and protein were expressed and correctly localized in retinal pigment epithelial (RPE) and photoreceptors of mice, pigs, and human retinal organoids. In RPE-and photoreceptor-specific Myo7a knockout mice, we could restore MYO7A expression and localization of melanosomes in RPE cells to wild-type levels. Myo7b activation led to partial restoration of melanosome localization, and the localization of MYO7B protein was largely comparable to MYO7A. These findings indicate that both approaches are in principle suitable for the therapy of Usher syndrome.

molecular biology↗

bioRxiv2026

Sequence adaptations in the intracellular domain of Symbiosis receptor-like kinase (SymRK) promoted infection thread progression in root nodule primordia

The uptake of nitrogen-fixing bacteria into living plant cells and the intracellular accommodation of arbuscular mycorrhiza (AM) fungi requires the plasma membrane-localised Symbiosis Receptor-like Kinase (SymRK). AM is widespread across terrestrial vascular plant lineages, while the nitrogen-fixing root nodule symbiosis (RNS) is restricted to one clade within the eurosids. This distribution led to the concept that SymRK was adopted during evolution to mediate RNS. Comparative analyses revealed that SymRK orthologs from the eurosid clade support RNS while SymRK from the phylogenetically distant species Solanum lycopersicum (tomato) does not. To dissect the molecular basis for this different functionality, we carried out complementation analyses of the Lotus japonicus symrk-3 mutant which is unable to form AM or RNS. Domains swap chimera from the tomato and L. japonicus SymRK orthologs revealed that the intracellular domain of L. japonicus SymRK is necessary and for cortical infection thread (IT) and symbiosome development at 21 days post inoculation. Notably, this signalling specificity could be overcome by ectopic expression of tomato SymRK, pointing to altered protein dosage as a potential determinant of function. Consistent with this idea, SINA family E3 ubiquitin ligases interacted with and ubiquitinylated L. japonicus SymRK, but not tomato SymRK. In yeast two hybrid analysis, the interaction of SymRK with SINA2 and SINA4 depended on the C-terminal intrinsically disordered tail region of L. japonicus SymRK. We conclude that the SymRK intracellular domain evolved interaction capabilities with SINA E3 ligases which correlates with its ability to support RNS.

plant biology↗

bioRxiv2026

The 2D and 3D ultrastructure of symbiosomes and associated vesicular structures in Lotus japonicus root nodule symbiosis

In root nodule symbiosis, symbiosome compartments accommodate nitrogen-fixing rhizobia inside the plant cell. Differentiated into bacteroids, the rhizobia are surrounded by a peribacteroid space and a plant-derived peribacteroid membrane, which separates them from the plant cytoplasm but allows signal and nutrient exchange between host and microbe. The morphological features of symbiosomes are primarily determined by ultrastructural single focal plane imaging, with limited information about spatial details. This study combines 2D and 3D imaging, using transmission electron microscopy and focused ion beam scanning electron microscopy as complementary techniques to analyse the symbiosome ultrastructure and organisation in Lotus japonicus wild-type plants. The 3D model of a mature colonised root nodule cell region demonstrates a dense, puzzle-like arrangement of symbiosomes relative to one another and adjacent plant organelles. The symbiosome shape and size depends on the orientation and number of bacteroids within the compartment and features connective tubular structures. Furthermore, vesicular structures, some likely of bacterial origin, were present at the interface. The study presents a multi-angled analysis of symbiosome-related structures, highlighting their volumes, spatial distribution, and pronounced compactness. Interface associated vesicles, protrusions and connective structures hint towards a dynamic and flexible system that contributes to the plant-microbe crosstalk.

plant biology↗

bioRxiv2025

Expression levels of the Band-7 protein FLOTILLIN modulate salt tolerance, growth and development in the moss Physcomitrium patens

The Band-7 proteins, known as FLOTILLINs (FLOT), are present at the plasma membranes of most land plants. They function in clathrin-independent endocytosis and contribute to nodule formation following symbiotic infections. This study reveals that the single FLOT variant in Physcomitrium patens is located at the thylakoid membranes in chloroplasts, serving an unanticipated function. Phenotypic analysis of knockout and overexpression lines demonstrates that PpFLOT overexpression significantly impairs the high salinity tolerance of P. patens. Additionally, liquid protonema cultures of PpFLOT-OEX lines exhibited a distinct color change due to necrotic events and developed brachycyte-like cells. These changes correlate with the strength of PpFLOT expression and do not occur when these lines are cultivated on solid medium. Our study found that PpFLOT-OEX lines display increased chlorophyll and H2O2 production. We also discovered that PpFLOT is regulated by ABA and light, and its high expression can potentially affect retrograde signaling. Metabolomics and proteomics analyses revealed changes in the pigment and lipid composition as well as differentially accumulated proteins in PpFLOT mutant lines. We also observed changes in the expression of ion-transport related genes, accumulation of lipids crucial during pathogen defense, and differentially accumulated proteins taking part in multiple metabolomic pathways. Consequently, our study suggests a novel role for chloroplastic PpFLOT in plant terrestrialization, as it is putatively involved in Ca2+ and reactive oxygen species (ROS) signaling in response to abiotic and biotic stress, along with the light-dependent regulation of chlorophyll biosynthesis.

plant biology↗

bioRxiv2025

Plant cells tolerate high rates of organellar mistranslation

Bacteria can trigger protein mistranslation to survive stress conditions 1. Mitochondria and plastids evolved from bacteria and therefore also use prokaryotic-type expression machineries to synthesize proteins. However, fungi and animal mitochondria are highly sensitive to mistranslation, which for instance manifests in lethal mitochondrial cardiomyopathy disorder 2. The response in plant cells is unknown. Glutaminyl-transferRNAs (Gln-tRNAGln) of bacteria, mitochondria, and plastids are synthesized indirectly 3,4. Initially, tRNAGln gets charged with glutamate. Subsequently, Gln is produced through trans-amidation by the aminoacyl-tRNA amido-transferase complex GatCAB. Consequentially, affected GatCAB activity results in pools of misloaded Glu-tRNAGln. Here we show that Arabidopsis mutants with decreased GatCAB level provide global insights into organellar mistranslation in plants. Proteomics revealed Gln-to-Glu misincorporation in plastid- and mitochondrially-expressed protein complexes with only modest abundance changes in mutant plants. Plastids appear more lenient to mistranslation as they exhibit much higher Gln-to-Glu misincorporation. Through efficient compensatory mechanisms, mutant plants display surprisingly subtle phenotypes. However, their acclimation to temperature stress differs. Interestingly, wild-type plants under similar stress also have altered Gln-to- Glu misincorporation. Our study shows that the response towards organellar mistranslation varies among eukaryotes. In plants, this knowledge can be used to improve stress tolerance.

plant biology↗

bioRxiv2024

Subcellular plant carbohydrate metabolism under elevated temperature

In many plant species, exposure to a changing environmental temperature regime induces an acclimation response which ultimately increases a plants thermotolerance. Under elevated temperature, membrane systems need remodelling to counteract de-stabilising thermodynamic effects. This also affects photosynthesis and carbohydrate metabolism due to heat affected protein functions, enzyme activities and transport processes across membrane systems. In the present study, a combination of electrolyte leakage assays and chlorophyll fluorescence measurements was applied to quantify heat tolerance before and after heat acclimation of Arabidopsis thaliana at different temperature regimes. Subcellular carbohydrate concentrations were determined in a combined approach of non-aqueous fractionation and 3D reconstruction of mesophyll cells and subcellular compartments using serial block-face scanning electron microscopy. Across temperature regimes between 32 {degrees}C and 38 {degrees}C, 7 days heat acclimation at 34 {degrees}C was found to most efficiently increase tissue heat tolerance. Under such conditions, cytosolic sucrose concentrations were stabilised by a shift of sucrose cleavage rates into the vacuolar compartment while invertase-driven cytosolic sucrose cleavage was found to be efficiently quenched by fructose and glucose acting as competitive and non-competitive inhibitors, respectively. Finally, this study provides strong evidence for a sucrose concentration gradient from the cytosol into the vacuole which might directly affect the physiological role and direction of proton gradient-driven sugar transport across the tonoplast.

plant biology↗

bioRxiv2024

On the annotation of Paleo- and Mesoproterozoic microfossils as fungi

How old is the crown group Fungi? Inferences from phylogenetic and fossil-based studies provided far-apart age estimates ranging between 0.75 to 2.7 billion years old. One important criterion for interpreting Paleo- and Mesoproterozoic microfossils as Fungi is their uncanny morphological resemblance with extant fungi. Here, we demonstrate that bacteria exposed to environmental conditions similar to the paleoenvironmental settings where these presumed fungi lived can spontaneously transform into their protoplasts. These protoplasts exhibit morphologies corresponding to those of presumed fungal microfossils. These observations, together with microfossil chemical composition, pose a serious challenge to interpreting Paleo- and Mesoproterozoic microfossils as fungi. Based on these results, we reiterate that morphology is not a reliable indicator of the phylogeny of microfossils older than 2.5Ga.

evolutionary biology↗

bioRxiv2022

Evolution of lineage-specific trafficking proteins and a novel post-Golgi trafficking pathway in Apicomplexa

The Organelle Paralogy Hypothesis (OPH) posits a mechanism to explain the evolution of non-endosymbiotically derived organelles, predicting that lineage-specific pathways organelles should result when identity-encoding membrane trafficking components duplicate and co-evolve. Here we investigate the presence of such lineage-specific membrane-trafficking machinery paralogs in the globally important lineage of parasites, the Apicomplexa. Using a new phylogenetic workflow, we are able to identify 18 novel paralogs of known membrane-trafficking machinery, the emergence of several of which correlate with the presence of new endomembrane organelles in apicomplexans or their larger lineage. Gene coregulation analysis of a large set of membrane-trafficking proteins in Toxoplasma both corroborate known molecular cell biological interactions between characterized machinery and suggest involvement of many of these new components into established pathways for biogenesis of or trafficking to the microneme and rhoptry invasion organelles. Moreover, focused molecular parasitological analysis of the apicomplexan Arf-like small GTPases, and the ArlX3 protein specifically, revealed a novel post-Golgi trafficking pathways involved in delivery of proteins to micronemes and rhoptries, with knock down demonstrating reduced invasion capacity. The totality of our data has identified an unforeseen post-Golgi trafficking pathway in apicomplexans and is consistent with the OPH mechanism acting to produce novel endomembrane pathways or organelles at various evolutionary stages across the Alveolate lineage. Significance statementThe mechanism of non-endosymbiotic organelle evolution has been relatively poorly explored and yet is relevant to many eukaryotic compartments, including the endomembrane system. The Organelle Paralogy Hypothesis predicts novel lineage-specific paralogs evolutionarily concurrent with emergence of new endomembrane organelles or pathways. By investigating this phenomenon in the apicomplexan parasites and their relatives, we identify and profile over a dozen new trafficking factors, several correlating with emergence of lineage-specific organelles. Cell biological study of one such factor demonstrates the existence of a novel post-Golgi trafficking pathway for components to the invasion organelles in the parasite Toxoplasma gondii. This work reveals how non-endosymbiotic organelle evolution has shaped cellular novelty in this lineage, relevant both to global health and fundamental evolutionary biology.

microbiology↗

bioRxiv2022

Stable and ancient endocytic structures navigate the complex pellicle of apicomplexan parasites

Apicomplexan parasites have an immense impact on humanity, but their basic cellular processes are often poorly understood. The sites of endocytosis, the conservation of this process with other eukaryotes, and its functions across Apicomplexa are major unanswered questions. Yet endocytosis in Plasmodium is implicated in antimalarial drug failure. Using the apicomplexan model Toxoplasma, we identified the molecular composition and behavior of unusual, fixed endocytic structures. Here, stable complexes of endocytic proteins differ markedly from the dynamic assembly/disassembly of these machineries in other eukaryotes. Moreover, conserved molecular adaptation of this structure is seen in Apicomplexa, including the kelch-domain protein K13 central to malarial drug-resistance. We determine that an essential function of endocytosis in Toxoplasma is plasma membrane homeostasis, rather than parasite nutrition, and that these specialized endocytic structures originated early in infrakingdom Alveolata, likely in response to the complex cell pellicle that defines this medically and ecologically important ancient eukaryotic lineage.

cell biology↗

bioRxiv2021

On the reproductive mechanism of Gram-negative protocells

Bacterial protoplasts are known to reproduce independently of canonical molecular biological processes. Their reproduction is shown to be mediated entirely by the physicochemical properties of cell constituents. However, the physiochemical properties of the cell constituents are influenced by the environmental conditions like salinity, salt composition, and mechanical stresses experienced by a cell in natural environments. The influence of such environmental conditions on protoplast reproduction is seldom investigated. Here, we studied protoplast reproduction in their native environmental conditions. Contrary to the previous perceptions of protoplasts reproducing in an erratic manner, cells in our study reproduced in a defined sequence of steps. The process of their reproduction can be explained by an interplay between intracellular metabolism, the physicochemical properties of cell constituents, and the nature of cations in the growth media. We observed a minimal leakage of intracellular constituents during protoplast reproduction, suggesting an efficient reproduction. However, the efficiency of reproduction is determined by the environmental conditions. Under favorable environmental conditions, protoplasts reproduce with nearly similar efficiency to cells that possess a cell wall. In short, here we demonstrate the simplest method of cellular reproduction and the influence of environmental conditions on this process.

biophysics↗

bioRxiv2021

On the reproductive mechanisms of Gram-positive protocells

Prokaryotes are hypothesized to have evolved from more primitive protocells. Unlike present-day cells, protocells are thought to have been devoid of complex molecular biological processes. They are believed to have mediated reproduction entirely by biophysical forces under favorable environmental conditions. Despite this proposition, little is known about the actual mechanism of their reproduction. To understand the reproduction process of protocells in their native habitat, here we used a top-down approach to transform bacterial cells into a primitive lipid vesicle-like state. Given that environmental conditions are thought to have played an essential role in mediating protocell reproduction, we then studied these cells under the presumed environmental conditions of Archaean Eon Earth. Even in the absence of functioning biological processes, cells in our study reproduced in a defined sequence of steps, always leading to the formation of viable daughter cells. Their reproduction mechanism can be explained by the interaction between intracellular metabolism, physicochemical properties of cell constituents, and, most importantly, environmental conditions. Given the simplicity of this reproduction mechanism and its suitability to environmental conditions of early Earth, we propose that protocells reproduced by this process. Moreover, this method of reproduction is also in tune with the earlier theoretical propositions on protocells, the results of the top- down approach of building a minimal cell, and the paleontological record of the Achaean Eon. Our study is the first to bridge the gap between non-living systems like lipid vesicles, living cells, and the paleontology of the Archaean Eon.

biophysics↗

bioRxiv2021

On the nature of the earliest known life forms

Microfossils from the Paleoarchean Eon are the oldest known evidence of life. Despite their significance in understanding the history of life on Earth, any interpretation of the nature of these microfossils has been a point of contention among researchers. Decades of back-and-forth arguments led to the consensus that reconstructing the lifecycles of Archaean Eon organisms is the most promising way of understanding the nature of these microfossils. Here, we transformed a Gram-positive bacterium into a primitive lipid vesicle-like state and studied it under environmental conditions prevalent on early Earth. Using this approach, we successfully reconstructed morphologies and life cycles of Archaean microfossils. In addition to reproducing microfossil morphologies, we conducted experiments that spanned years to understand the process of cell degradation and how Archaean cells could have undergone encrustation minerals (in this case, salt), leading to their preservation as fossilized organic carbon in the rock record. These degradation products strongly resemble fossiliferous features from Archaean rock formations. Our observations suggest that microfossils aged between 3.8 to 2.5Ga most likely were liposome-like protocells that have evolved physiological pathways of energy conservation but not the mechanisms to regulate their morphology. Based on these observations, we propose that morphology is not a reliable indicator of taxonomy in these microfossils.

evolutionary biology↗

bioRxiv2021

Biophysical and proteomic analyses suggest functions of Pseudomonas syringae pv tomato DC3000 extracellular vesicles in bacterial growth during plant infection

Vesiculation is a process employed by Gram-negative bacteria to release extracellular vesicles (EVs) into the environment. Bacterial EVs contain molecular cargo from the donor bacterium and play important roles in bacterial survival and growth. Here, we describe EV production in plant-pathogenic Pseudomonas syringae pv. tomato DC3000 (Pto DC3000), the causal agent of bacterial speck disease. Cultured Pto DC3000 exhibited EV structures both on the cell surface and in the vicinity of bacterial cells, observed as outer membrane vesicle (OMV) release. We used in-solution trypsin digestion coupled to mass spectrometry to identify 369 proteins enriched in EVs recovered from cultured Pto DC3000. The predicted localization profile of EV proteins supports the production of EVs also in the form of outer-inner-membrane vesicles (OIMVs). EV production varied slightly between bacterial lifestyles and also occurred in planta. The potential contribution of EVs to Pto DC3000 plant infection was assessed using plant treatments and bioinformatic analysis of the EV-enriched proteins. While these results identify immunogenic activities of the EVs, they also point at roles for EVs in bacterial defences and nutrient acquisition by Pto DC3000.

plant biology↗

bioRxiv2020

Genome-informed microscopy reveals infections of uncultivated carbon-fixing archaea by lytic viruses in Earth's crust

The continental subsurface houses a major portion of lifes abundance and diversity, yet little is known about viruses infecting microbes that reside there. Here, we used a combination of metagenomics and genome-informed microscopy to show that highly abundant carbon-fixing organisms of the uncultivated genus Candidatus Altiarchaeum are frequent targets of previously unrecognized viruses in the deep subsurface. Analysis of CRISPR spacer matches displayed resistances of Ca. Altiarchaea against eight predicted viral clades, which showed genomic relatedness across continents but little similarity to previously identified viruses. Based on metagenomic information, we tagged and imaged a putatively viral genome rich in protospacers using fluorescence microscopy. Virus-targeted genomeFISH revealed a lytic lifestyle of the respective virus and challenges previous predictions that lysogeny prevails as the dominant viral lifestyle in the subsurface. CRISPR development over time and imaging of 18 samples from one subsurface ecosystem suggest a sophisticated interplay of viral diversification and adapting CRISPR-mediated resistances of Ca. Altiarchaeum. We conclude that infections of primary producers with lytic viruses followed by cell lysis potentially jump-start heterotrophic carbon cycling in these subsurface ecosystems.

microbiology↗

bioRxiv2019

The International Space Station selects for microorganisms adapted to the extreme environment, but does not induce genomic and physiological changes relevant for human health

The International Space Station (ISS) is a unique, completely confined habitat for the human crew and co-inhabiting microorganisms. Here, we report on the results of the ISS experiment "EXTREMOPHILES". We aimed to exploit the microbial information obtained from three surface and air sampling events aboard the International Space Station during increments 51 and 52 (2017) with respect to: i) microbial sources, diversity and distribution within the ISS, ii) functional capacity of microbiome and microbial isolates, iii) extremotolerance and antibiotics-resistance (compared to ground controls), and iv) microbial behavior towards ISS-relevant materials such as biofilm formation, or potential for degradation. We used wipe samples and analyzed them by amplicon and metagenomics sequencing, cultivation, comparative physiological studies, antibiotic resistance tests, genome analysis of isolates and co-incubation experiments with ISS-relevant materials. The major findings were: i) the ISS microbiome profile is highly similar to ground-based confined indoor environments, ii) the ISS microbiome is subject to fluctuations and indicative for the (functional) location, although a core microbiome was present over time and independent from location, iii) the ISS selects for microorganisms adapted to the extreme environment, but does not necessarily induce genomic and physiological changes which might be relevant for human health, iv) cleanrooms and cargo seems to be a minor source of microbial contamination aboard, and v) microorganisms can attach to and grow on ISS-relevant materials. Biofilm formation might be a threat for spacecraft materials with the potential to induce instrument malfunctioning with consequences for mission success. We conclude that our data do not raise direct reason for concern with respect to crew health, but indicate a potential threat towards biofilm formation and material integrity in moist areas.

microbiology↗