Biology subjects

Karlsson, M.

Publications and source records attributed to Karlsson, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Functional and transcriptomic analyses in Neurospora crassa reveal the crucial role of N-glycoprotein deglycosylation process in fungal homeostasis.

N-glycosylation is an essential post-translational modification required for proper protein folding, stability, trafficking, and secretion in eukaryotes. In such organisms, an efficient endoplasmic reticulum (ER) quality control, such as the ER-associated degradation (ERAD) pathway, is critical for maintaining cellular homeostasis. During ERAD, terminally misfolded glycoproteins undergo N-deglycosylation prior to proteasomal degradation, a process typically mediated by peptide N-glycanase (PNGase). However, in the filamentous fungi, the PNGase seems to be catalytically inactive, indicating evolutionary divergence from the canonical PNGase pathway. Filamentous fungi also encode endo-{beta}-N-acetylglucosaminidases (ENGases), particularly members of glycoside hydrolase family 18 (GH18), which may compensate for the loss of canonical PNGase activity. Here, we investigated the roles of the cytosolic GH18 ENGase and a putative acidic PNGase in N. crassa using transcriptomic and functional approaches. Our results demonstrate that the cytosolic GH18 ENGase is an active deglycosylating enzyme likely associated with the ERAD pathway, whereas no deglycosylation activity was detected for the acidic PNGase. Deletion of the ENGase severely compromises tolerance to diverse stress conditions and induces substantial transcriptomic reprogramming, including upregulation of a GH20 exo-{beta}-N-acetylhexosaminidase under ER stress. These findings identify cytosolic ENGase as a key component of fungal proteostasis and suggest that N. crassa activates alternative compensatory mechanisms to maintain protein quality control when canonical deglycosylation pathways are impaired.

microbiology↗

bioRxiv2026

High-channel-count neural recording and stimulation platform with 5,376 simultaneous recording channels

Advancing neural interfaces requires large-scale, high-density recording technologies capable of capturing full-spectrum neural activity across cortical and subcortical regions. Here, we present a scalable approach to integrate neural electrodes with advanced application-specific integrated circuits (ASICs). Specifically, we custom-designed an ASIC with 5,376 simultaneous channels, each sampling at 20 kS/s and enabling >1.3 Gb/s total data streaming throughput. The ASIC incorporates in-pixel amplification, time-division multiplexed ADCs, and on-chip stimulation capabilities, ensuring precise signal acquisition with minimal power consumption while maintaining a low noise level of 5.5 {micro}Vrms. We further developed an interconnect strategy using gold bump bonding, which allows for high-density integration of the flexible probe and rigid chip. We demonstrate the capacity of this platform through the integration with a flexible ECoG array. The resulting device allows for the high-resolution mapping of in vivo field potentials on the cortical surfaces of rat brains, supported by the precise localization of evoked sensory activities. These results prove an effective approach towards highly integrated neural interfaces with applications in brain-computer interfaces, neuroprosthetics, and large-scale functional brain mapping.

bioengineering↗

bioRxiv2026

Homothallic or heterothallic? A genomic investigation into the sexual capabilities of the ascomycete fungus Clonostachys rosea

Modes of reproduction and sexual strategies strongly influence the genetic diversity and evolutionary potential of a species. The ascomycete fungus Clonostachys rosea is reported to be homothallic (sexually self-fertile), although a rapid decay of genome-wide linkage disequilibrium is also reported, something that is not in line with an obligate homothallic mode of reproduction. To investigate this phenomenon, we identified the mating-type (MAT1) locus in 66 genome-sequenced C. rosea strains under the hypothesis that each strain contains genes from both MAT1 idiomorphs. Eleven strains indeed contained both MAT1-1 and MAT1-2 genes, suggesting homothallism. However, most strains harboured either MAT1-1 or MAT1-2 genes and co-existed in North America, Europe and China, suggesting heterothallism. The MAT1 locus of heterothallic strains was highly conserved, and the linkage disequilibrium half decay distance was 1050 bp, suggesting sexual outcrossing. The presence of conserved MAT1-1 or MAT1-2 idiomorphs in strains of other Clonostachys species shows that heterothallism is likely the ancestral state. A phylogenetic analysis of 2800 single-copy orthologous genes revealed that homothallic and heterothallic strains separated in two well-supported clades, indicating a single lineage of homothallic C. rosea, likely originating in South America, followed by intercontinental dispersal. Homothallic C. rosea strains displayed higher nucleotide diversity than heterothallic strains, indicating a lack of outcrossing. This unique case of both homothallic and heterothallic lineages within the same species provides an opportunity to study the genomic consequences of selfing in very closely related strains.

evolutionary biology↗

bioRxiv2025

ChipNMR: Hyperpolarized NMR for non-invasive metabolic flux analysis in perfused microfluidic chips

Dissolution dynamic nuclear polarization NMR Spectroscopy (dDNP-NMR) has become a transformative tool for metabolic studies by significantly enhancing signal sensitivity more than three orders of magnitude compared to traditional NMR. However, NMR detection probes are optimized for round narrow glass tubes typically 5 mm in diameter, which impose constraints on their utility for metabolic studies of adhernt cells. Here, we present a novel NMR probe head integrated with a custom microfluidic chip that facilitates real-time monitoring of hyperpolarized substrate conversion from adhernt cells. This system enables metabolic flux analysis in a controlled, in vitro environment, as demonstrated by tracking the conversion of [1-13C] pyruvate to [1-13C] lactate in HeLa cells over 48 hours. To the best of our knowledge, this is the first demonstration of cell metabolism from an adhering monolayer of mammalian cells in combination with hyperpolarized NMR. The custom microfluidic chip design is modular and adaptable allowing expansion to dual-chamber chips, demonstrating its potential in applications for more complex cellular environments, such as Organ-on-a-Chip systems.

biochemistry↗

bioRxiv2025

Developing ERAF-AI: An Early-stage ResearchAssessment Framework for Artificial Intelligence

Today, most research evaluation frameworks are designed to assess mature projects with well-defined data and clearly articulated outcomes. Yet, few, if any, are equipped to evaluate the promise of early-stage biotechnology research, which is inherently characterized by limited evidence, high uncertainty, and evolving objectives. These early-stage projects require nuanced assessments that can adapt to incomplete information, project maturity, and shifting research questions. Furthermore, these challenges are compounded by the difficulty of systematically scaling evaluations with the increasing volume of research projects. As a step toward addressing this gap, we introduce the biotechnology-oriented Early-Stage Research Assessment Framework for Artificial Intelligence (ERAF-AI), a systematic approach to evaluate research at Technology Readiness Levels (TRLs) 1 to 3 - research maturity levels where ideas are more conceptual and only preliminary evidence exists to indicate potential viability. By leveraging AI-driven methodologies and platforms such as the Coordination.Network, ERAF-AI ensures transparent, scalable, and context-sensitive evaluations that integrate research maturity classification, adaptive scoring, and strategic decision-making. Importantly, ERAF-AI aligns criteria with the unique demands of early-stage research, guiding evaluation through the 4P framework (Promote, Pause, Pivot, Perish) to inform next steps. As an initial demonstration of its potential, we apply ERAF-AI to a high-impact early-stage project, providing actionable insights and measurable improvement over conventional practices. Although ERAF-AI shows significant promise in improving the prioritization of early-stage research, further refinement, and validation across a wider range of disciplines and datasets is required to refine its scalability and adaptability. Overall, we expect this framework to serve as a valuable tool for empowering researchers to make informed decisions and to prioritize high-potential initiatives in the face of uncertainty and limited data.

bioinformatics↗

bioRxiv2024

Evolution of fast-growing piscivorous herring in the young Baltic Sea

The circumstances under which species diversify to genetically distinct lineages is a fundamental question in biology1. Atlantic herring is an extremely abundant zooplanktivorous species that is subdivided into multiple ecotypes that differ regarding spawning time and genetic adaption to local environmental conditions such as temperature, salinity, and light conditions2,3. Here we show using whole genome analysis that multiple populations of piscivorous (fish-eating) herring have evolved sympatrically after the colonization of the brackish Baltic Sea within the last 8,000 years postglaciation. The piscivorous ecotype grows faster, and is much larger and less abundant than the zooplanktivorous Baltic herring. Lesions of the gill rakers in the piscivorous ecotype indicated incomplete adaptation to a fish diet. This niche expansion of herring in the young Baltic Sea with its paucity of piscivorous species suggests that empty niche space is more important than geographic isolation for the evolution of biodiversity.

evolutionary biology↗

bioRxiv2024

Reproducibility of 4D Flow MRI-based Personalized Cardiovascular Models; Inter-sequence, Intra-observer, and Inter-observer variability

Subject-specific parameters in lumped hemodynamic models of the cardiovascular system can be estimated using data from experimental measurements, but the parameter estimation may be hampered by the variability in the input data. In this study, we investigate the influence of inter-sequence, intra-observer, and inter-observer variability in input parameters on estimation of subject-specific model parameters using a previously developed approach for model-based analysis of data from 4D Flow MRI acquisitions and cuff pressure measurements. The parameters describe left ventricular time-varying elastance and aortic compliance. Parameter reproducibility with respect to variability in the MRI input measurements was assessed in a group of ten healthy subjects. The subject-specific parameters had coefficient of variations between 2.5% and 34.9% in the intra- and inter-observer analysis. In comparing parameters estimated using data from the two MRI sequences, the coefficients of variation ranged between 3.6% and 41%. The diastolic time constant of the left ventricle and the compliance of the ascending aorta were the parameters with the lowest and the highest variability, respectively. In conclusion, the modeling approach allows for estimating left ventricular elastance parameters and aortic compliance from non-invasive measurements with good to moderate reproducibility concerning intra-user, inter-user, and inter-sequence variability.

systems biology↗

bioRxiv2024

Plant genotype-specific modulation of Clonostachys rosea-mediated biocontrol of septoria tritici blotch disease on wheat

BackgroundBeneficial microorganisms can act as biological control agents (BCAs) by directly targeting pathogens or indirectly by enhancing the plants defense mechanisms against pathogens. However, efficiencies with which plants benefit from BCAs vary, potentially because of genetic variation in plants for plant-BCA compatibility. The aim of this study was to explore the genetic variation in winter wheat for modulation of Clonostachys rosea-mediated biocontrol of septoria tritici blotch disease caused by the fungal pathogen Zymoseptoria tritici. ResultsIn total, 202 winter wheat genotypes, including landraces and old cultivars grown from 1900 onwards in the Scandinavian countries, were tested under greenhouse-controlled conditions. Foliar spray applications of the pathogen and the fungal BCA in two treatments, i.e., Z. tritici (Zt) alone and Z. tritici along with C. rosea (ZtCr) were used to assess the disease progress over time. The absence and presence of C. rosea in Zt and ZtCr, respectively, allowed the dissection of variation for plant disease resistance and biocontrol efficacy. The study showed significant phenotypic variation among plant genotypes for disease progression in both Zt and ZtCr treatments. Moreover, disease progress for individual plant genotypes differed significantly between the two treatments, indicating a plant genotype-dependent variation in biocontrol efficacy. For the phenotypic variation in disease progress and biocontrol efficacy, a genome-wide association study using a 20K single-nucleotide polymorphism (SNP) marker array was also performed. In total, five distinct SNP markers associated with disease resistance and four SNP markers associated with C. rosea biocontrol efficacy were identified. ConclusionsThis work serves as a foundation to further characterize the genetic basis of plant-BCA interactions, facilitating opportunities for concurrent breeding for disease resistance and biocontrol efficacy.

plant biology↗

bioRxiv2024

SREBP-mediated gene expression regulation is essential for the intrinsic fungicide tolerance and antagonism in the fungal biocontrol agent Clonostachys rosea

Sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors known to regulate sterol biosynthesis and homeostasis in fungi. For this reason they have a role in several biological processes, including virulence, fungicide tolerance, hypoxia adaptation, lipid and carbohydrate metabolisms, and iron homeostasis. While the biological function of SREBPs in yeast and filamentous fungal species pathogenic to humans and plants is known, their role in fungal biocontrol agents (BCAs) is still elusive. This study aimed to investigate the biological and regulatory function of SREBPs in the BCA Clonostachys rosea, with a focus on their role in fungicide tolerance, hypoxia adaptation and antagonisms. The C. rosea genome contains two genes (sre1 and sre2) coding for SREBPs and one gene each coding for Insulin induced gene (INSIG) and SREBP cleavage-activating protein (SCAP), required for SREBP-mediated ergosterol biosynthesis in fungi. Deletion of sre1 resulted in mutants with pleiotropic effects, including the reduced ability to grow on media supplemented with proline (active ingredient prothioconazole) and cantus (active ingredient boscalid) fungicides, hypoxia mimicking agent CoCl2, cell wall stressor SDS, and increased growth rate on medium supplemented with caffeine, compared with C. rosea wild type (WT). In addition, the antagonistic ability against the fungal hosts Botrytis cinerea and Rhizoctonia solani was affected when sre1 was deleted. However, no significant difference between sre2 deletion strains and C. rosea WT was found for any of the tested phenotypes. To investigate the regulatory role of SRE1, the transcriptome of C. rosea WT and a sre1 deletion strain was analyzed. The transcriptome analysis identified differentially expressed genes in the sre1 deletion strain associated with carbohydrate and lipid metabolism, respiration, iron homeostasis, and xenobiotic tolerance. Moreover, genes coding for polyketide synthases and chitinases with a proven antimicrobial role were downregulated in the mutant, corroborating the reduced antagonism phenotypes. In summary, this work sheds light on the regulation role of transcription factor SRE1 while also exploring its effect on regulating the antagonistic activity and fungicide resistance of C. rosea, giving us helpful knowledge to design applications of this organism in IPM strategies.

microbiology↗

bioRxiv2023

Quantitative target engagement of RIPK1 in human whole blood via the cellular thermal shift assay for potential pre-clinical and clinical applications

The cellular thermal shift assay (CETSA(R)) is a target engagement method widely used for preclinical characterization of small molecule compounds. CETSA(R) has been used for semi-quantitative readouts in whole blood with PBMC isolation, and quantitative, plate-based readouts using cell lines. However, there has been no quantitative evaluation of CETSA(R) in unprocessed human whole blood, which is preferred for clinical applications. Here we report two separate assay formats - Alpha CETSA(R) and MSD CETSA(R) - that require less than 100 L of whole blood per sample without PBMC isolation. We chose RIPK1 as a proof-of-concept target and, by measuring engagement of seven different inhibitors, demonstrate high assay sensitivity and robustness. These quantitative CETSA(R) platforms enable possible applications in preclinical pharmacokinetic-pharmacodynamic studies, and direct target engagement with small molecules in clinical trials.

molecular biology↗

bioRxiv2023

Dicer-mediated RNA silencing is the key regulatory mechanism in the biocontrol fungus Clonostachys rosea-wheat interactions

The intricate molecular interplay between beneficial fungi and plants is vital to plant growth promotion and induced defense response. This study explored the role of DCL-mediated RNA silencing in the interaction between the biocontrol fungus Clonostachys rosea and wheat roots. We investigated the impact of DCL (Dicer-like) gene deletions in C. rosea on its root colonization ability. Our results revealed that the deletion of dcl2 significantly enhanced C. rosea biomass on wheat roots, indicating a pivotal role of DCL2 in root colonization. Transcriptome sequencing of C. rosea and wheat during their interactions unveiled extensive gene expression changes. In wheat, genes related to stress responses were upregulated during C. rosea interactions, while genes associated with plant cell wall modification and metabolic processes were downregulated, suggesting complex regulatory responses and a trade-off between defense mechanisms and growth promotion. Deletion of C. rosea dcl1 and dcl2 altered the transcriptomic responses of wheat roots during interactions. Wheat genes associated with stress responses were downregulated during interactions with DCL deletion strains. In contrast, genes involved in metabolic processes and growth were upregulated, emphasizing the cross-kingdom regulatory role of C. rosea small RNAs (sRNAs). We identified 18 wheat miRNAs responsive to C. rosea interactions. Furthermore, we identified 24 endogenous and six cross-kingdom potential gene targets for seven and five differentially expressed miRNAs, supported by their inverse gene expression pattern. In C. rosea, we found a large transcriptional reprogramming of genes during interaction with wheat roots. The upregulated genes were associated with carbohydrate and polysaccharide catabolic processes, membrane transporters and effectors. Conversely, downregulated genes were mainly associated with transition metal ion transport and homeostasis processes. The deletion of dcl1 and dcl2 had significant effects on gene expression. A higher number of genes upregulated in WT during the interaction were restored in DCL deletion mutants, suggesting DCL-mediated gene expression regulation. Furthermore, we identified 21 differentially expressed micro-RNA-like RNAs (milRNAs) in C. rosea; nine were DCL-dependent. They had putative gene targets in C. rosea, including transcription factors, effectors, transporters, and enzymes involved in specialized metabolite production. Cross-kingdom RNA silencing was also observed, with seven DCL-dependent C. rosea milRNAs potentially targeting 29 genes in wheat. These findings provide valuable insights into the molecular mechanisms underlying the beneficial interaction between fungi and plant roots. In addition, the study shed light on the role of sRNA-mediated gene regulation in the C. rosea-wheat interaction, with potential implications for sustainable agriculture and biocontrol strategies.

genomics↗

bioRxiv2023

Molecular Pixelation: Single cell spatial proteomics by sequencing

The spatial distribution of cell surface proteins govern vital processes of the immune system such as inter-cell communication and mobility. However, tools for studying these at high multiplexing scale, resolution, and throughput needed to drive novel discoveries are lacking. We present Molecular Pixelation, a DNA-sequencing based method for single cell analysis to quantify protein abundance, spatial distribution, and colocalization of targeted proteins using Antibody Oligonucleotide Conjugates (AOCs). Relative locations of AOCs are inferred by sequentially associating these into local neighborhoods using DNA-pixels containing unique pixel identifier (UPI) sequences, forming >1,000 connected spatial zones per single cell in three dimensions. DNA-sequencing reads are computationally arranged into spatial single cell maps for 76 proteins without cell compartmentalization. By studying immune cell dynamics and using spatial statistics on graph representations of the data, previously known and novel patterns of protein spatial polarization and co-localization were found in chemokine-stimulated T-cells.

immunology↗

bioRxiv2023

The role of B cells in immune cell activation in polycystic ovary syndrome

Variations in B cell numbers are associated with polycystic ovary syndrome (PCOS) through unknown mechanisms. Here we demonstrate that B cells are not central mediators of PCOS pathology and that their frequencies are altered as a direct effect of androgen receptor activation. Hyperandrogenic women with PCOS have increased frequencies of age-associated double-negative B memory cells and increased levels of circulating immunoglobulin M (IgM). However, the transfer of serum IgG from women into wild-type female mice induces only an increase in body weight. Furthermore, RAG1 knock-out mice, which lack mature T- and B cells, fail to develop any PCOS-like phenotype. In wild-type mice, co-treatment with flutamide, an androgen receptor antagonist, prevents not only the development of a PCOS-like phenotype but also alterations of B cell frequencies induced by dihydrotestosterone (DHT). Finally, B cell-deficient mice, when exposed to DHT, are not protected from developing a PCOS-like phenotype. These results urge further studies on B cell functions and their effects on autoimmune comorbidities highly prevalent among women with PCOS. SummaryAndrogen receptor activation alters B cell frequencies and functionality as the transfer of human PCOS IgG increase weight in female mice. Lack of B cells does not protect from the development of a PCOS phenotype, suggesting an unrecognized role for B cells in PCOS autoimmune comorbidities. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/525671v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@15802fborg.highwire.dtl.DTLVardef@12bd154org.highwire.dtl.DTLVardef@1bc14a9org.highwire.dtl.DTLVardef@f08d2b_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

Distribution and environmental drivers of fungal denitrifiers in global soils

The microbial process denitrification is the primary source of the greenhouse gas nitrous oxide (N2O) from terrestrial ecosystems. Fungal denitrifiers, unlike many bacteria, lack the N2O reductase and are potential sources of N2O. Still, their diversity, distribution, and environmental determinants in terrestrial ecosystems remain unresolved. We used a phylogenetically informed approach to screen 1 980 soil and rhizosphere metagenomes representing 608 globally distributed sampling sites for the denitrification marker gene nirK, coding for nitrite reductase. We show that fungal denitrifiers are sparse, yet cosmopolitan and dominated by saprotrophs and opportunistic plant pathogens. Few showed biome-specific distribution patterns. However, members of the Fusarium oxysporum species complex, known to produce substantial amounts of N2O, were proportionally more abundant and diverse in the rhizosphere than in other biomes. Fungal denitrifiers were most frequently detected in croplands but were most abundant in forest soils. The overall low abundance of fungal relative to bacterial and archaeal denitrifiers suggests that their role in denitrification and contribution to soil N2O emissions may be less important than previously suggested. Nevertheless, in relative terms, they could play a role in soils characterized by high carbon to nitrogen ratio and low pH, especially in tundra and boreal and temperate coniferous forests. Our results further indicate that plant-pathogen interactions may favor fungal denitrifiers. Thus, increasing global warming with predicted proliferation of pathogens and the fact that many of the fungi with nirK detected in the metagenomes are stress-tolerant cosmopolitans suggest that fungal denitrifier abundance may increase in terrestrial ecosystems.

microbiology↗

bioRxiv2022

Larger but younger fish when growth compensates for higher mortality in heated ecosystem

Ectotherms are predicted to "shrink" with global warming, in line with general growth models and the temperature-size rule (TSR), both predicting smaller adult sizes with warming. However, they also predict faster juvenile growth rates and thus larger size-at-age of young organisms. Hence, the result of warming on the size-structure of a population depends on the interplay between how mortality rate, juvenile- and adult growth rates are affected by warming. Here, we use two-decade long time series of biological samples from a unique enclosed bay heated by cooling water from a nearby nuclear power plant to become 5-10 {degrees}C warmer than its reference area. We used growth-increment biochronologies (12658 reconstructed length-at-age estimates from 2426 individuals) to quantify how >20 years of warming has affected body growth and size-at-age and catch data to quantify mortality rates and population size- and age structure of Eurasian perch (Perca fluviatilis). In the heated area, growth rates were faster for all sizes, and hence size-at-age was larger for all ages, compared to the reference area. While mortality rates were also higher (lowering mean age by 0.4 years), the faster growth rates lead to a 2 cm larger mean size in the heated area. Differences in the size-spectrum exponent (describing the proportion of fish by size) were less clear statistically. Our analyses reveal that mortality, in addition to plastic growth and size-responses, is a key factor determining the size structure of populations exposed to warming. Understanding the mechanisms by which warming affects the size- and age structure of populations is critical for predicting the impacts of climate change on ecological functions, interactions, and dynamics.

ecology↗

bioRxiv2022

Comparative small RNA and degradome sequencing provide new insights into antagonistic interactions in the biocontrol fungus Clonostachys rosea

Necrotrophic mycoparasitism is an intricate process involving recognition, physical mycelial contact and killing of host fungi (mycohosts). During such interactions, mycoparasites undergo a complex developmental process involving massive regulatory changes of gene expression to produce a range of chemical compounds and proteins that contribute to the parasitism of the mycohosts. Small-RNAs (sRNAs) are vital components of post-transcriptional gene regulation, although their role in gene expression regulation during mycoparasitism remain understudied. Here, we investigated the role of sRNA-mediated gene regulation in mycoparasitism by performing sRNA and degradome tags sequencing of the mycoparasitic fungus Clonostachys rosea interacting with the plant pathogenic mycohosts Botrytis cinerea and Fusarium graminearum at two time points. The majority of differentially expressed sRNAs were down-regulated during the interactions with the mycohosts compared to a C. rosea self-interaction control, thus allowing de-suppression (up-regulation) of mycohost-responsive genes. Degradome analysis showed a positive correlation between high degradome counts and antisense sRNA mapping and led to the identification of 201 sRNA-mediated gene targets for 282 differentially expressed sRNAs. Analysis of sRNA gene targets revealed that the regulation of genes coding for membrane proteins was a common response against both mycohosts. While the regulation of genes involved in oxidative stress tolerance and cellular metabolic and biosynthetic processes was exclusive against F. graminearum highlighting common and mycohosts-specific gene regulation of C. rosea. By combining these results with transcriptome data collected in similar experimental conditions during a previous study, we expand the understanding of the role of sRNA in regulating interspecific fungal interactions and mycoparasitism. ImportanceSmall-RNAs (sRNAs) are emerging as key players in pathogenic and symbiotic fungus-plant interactions, however, their role in fungal-fungal interactions remains elusive. In this study, we employed the necrotrophic mycoparasite Clonostachys rosea and plant pathogenic mycohots Botrytis cinerea and Fusarium graminearum and investigated the sRNA-mediated gene regulation in mycoparasitic interactions. The combined approach of sRNA and degradome tag sequencing identified 201 sRNA-mediated putative gene targets for 282 differentially expressed sRNAs highlighting the role of sRNA-mediated regulation of mycoparasitism in C. rosea. We also identified 36 known and 13 novel miRNAs and their potential gene targets at endogenous level, and at a cross-species level in B. cinerea and F. graminearum indicating a role of cross-species RNAi in mycoparasitism, representing a novel mechanism in biocontrol interactions. Furthermore, we showed that C. rosea adapts its transcriptional response, and thereby its interaction mechanisms, based on the interaction stages and identity of the mycohost.

microbiology↗

bioRxiv2021

The role of Dicer-dependent RNA interference in regulating cross-species communication during fungus-fungus interactions

Dicer-like (DCL) proteins play a vital role in transcriptional and post-transcriptional gene silencing, also known as RNA interference (RNAi), by cleaving double-stranded RNAs or single-stranded RNAs with stem-loop structures into small RNAs. Although DCL-mediated RNAi can regulate interspecific communication between pathogenic/mutualistic organisms and their hosts, its role in parasitic fungus-fungus interactions is yet to be investigated. In this study, we deleted dcl genes in the mycoparasitic fungus Clonostachys rosea and analyzed the transcriptome and secondary metabolome to characterize the regulatory functions of DCL-dependent RNAi in mycoparasitism. Deletion of dcl2 resulted in a mutant with reduced growth rate, pigment production and antagonism towards the plant pathogenic fungus Botrytis cinerea. Moreover, the {Delta}dcl2 mutant displayed a reduced ability to control fusarium foot rot disease on wheat, caused by Fusarium graminearum, and reduced production of 62 secondary metabolites (SM) including yellow-coloured sorbicillinoids. Transcriptome sequencing of the in vitro interaction between the C. rosea {Delta}dcl2 strain and B. cinerea or F. graminearum identified downregulation of genes coding for transcription factors, membrane transporters, hydrolytic enzymes and SM biosynthesis enzymes putatively involved in antagonistic interactions, in comparison with the C. rosea wild type interaction. Sixty-one putative novel microRNA-like RNAs (milRNAs) were identified in C. rosea, and 11 was upregulated in the {Delta}dcl2 mutant. In addition to putative endogenous gene targets, these DCL2-dependent milRNAs were predicted to target B. cinerea and F. graminearum virulence factor genes, which showed an increased expression during interaction with the {Delta}dcl2 mutant incapable of producing the targeting milRNAs. This paper constitutes the first step in elucidating the role of RNAi in mycoparasitism, with important implications for biological control of plant diseases. This study further indicates a possible cross-species regulatory activity of fungal milRNAs, emphasizing a novel role of RNAi in fungal interactions and ecology. Author summaryRNA interference (RNAi) is a conserved cellular mechanism mediated by small RNAs (sRNAs) regulating biological processes through the targeted destruction or modulation of RNA filaments necessary for protein synthesis. Dicer-like endoribonucleases (DCL) play a vital role in the RNAi pathway by generating sRNAs. In this study, we identified two DCL-encoding genes in the mycoparasitic fungus Clonostachys rosea and investigated a role of DCL-mediated RNAi in interference interactions between Clonostachys rosea and the two important fungal pathogens Botrytis cinerea and Fusarium graminearum (here called mycohost). Using transcriptome (sRNA and mRNA) sequencing and secondary metabolome analysis approach, we found that the dcl mutants were not able to produce 11 sRNAs predicted to finetune the regulatory network of genes known to be involved in production of hydrolytic enzymes, antifungal compounds, and membrane transporters needed for antagonistic action of C. rosea. We also found C. rosea sRNAs putatively targeting known virulence factors in the mycohost, indicating RNAi-mediated cross-species communication. Our study expanded the understanding of underlying mechanisms of cross-species communication during interference interactions and showed that DCL-mediated RNAi is an important regulator of parasitic fungus-fungus interactions. The results pose the base for future works studying the role of DCL-based cross-species RNAi in fungal interactions.

genomics↗

bioRxiv2021

Genetic determinants of endophytism in the Arabidopsis root mycobiome

Roots of Arabidopsis thaliana do not engage in symbiotic associations with mycorrhizal fungi but host taxonomically diverse fungal communities that influence health and disease states. We sequenced the genomes of 41 fungal isolates representative of the A. thaliana root mycobiota for comparative analysis with 79 other plant-associated fungi. We report that root mycobiota members evolved from ancestors with diverse lifestyles and retained large repertoires of plant cell wall-degrading enzymes (PCWDEs) and effector-like small secreted proteins. We identified a set of 84 gene families predicting best endophytism, including families encoding PCWDEs acting on xylan (GH10) and cellulose (AA9). These genes also belong to a core transcriptional response induced by phylogenetically-distant mycobiota members in A. thaliana roots. Recolonization experiments with individual fungi indicated that strains with detrimental effects in mono-association with the host not only colonize roots more aggressively than those with beneficial activities but also dominate in natural root samples. We identified and validated the pectin degrading enzyme family PL1_7 as a key component linking aggressiveness of endophytic colonization to plant health.

microbiology↗