Biology subjects

Fleissner, A.

Publications and source records attributed to Fleissner, A..

10 recordsLinked to original sources

bioRxiv2026

Cheating (re)shapes pathogen virulence and antifungal resistance

Filamentous fungi grow as fused, multinucleate networks that share secreted public goods vs private goods. We asked whether this sharing enables "cheater" nuclei to increase in frequency by exploiting goods produced by other nuclei, and whether such social conflict shapes virulence and antifungal resistance. We tested this in the gray mold pathogen Botrytis cinerea by contrasting an extracellular detoxification trait, a public good (enzymatic hydrolysis of the tomato saponin alpha-tomatine) with an intracellular antibiotic resistance trait, a private good (hygromycin phosphotransferase). In pairwise competitions, tomatinase-deficient nuclei gained advantage when rare against a constitutive producer, both in vitro and in planta, even though producers drive lesion expansion. An ordinary differential equation model fitted to the competition outcomes identified antibiotic gradients as the key driver of frequency-dependent selection and predicted stable coexistence of producer and non-producer nuclei across multinucleate bottlenecks. Cheating within fungal syncytia can therefore decouple virulence from reproduction and buffer the selection on antifungal resistance.

genetics↗

bioRxiv2025

When flowers turn red: Pathogen induced flavonoid and anthocyanin biosynthesis in Chrysanthemum seticuspe confers resistanceto Botrytis cinerea

Botrytis cinerea is a major threat to ornamental crops, yet floral defence responses remain poorly understood. In Chrysanthemum seticuspe, we found that flower petals, unlike leaves, mount a localized resistance response resulting in red spots appearing at fungal penetration sites. We observed an intensification of the coloured response as infections progressed. To investigate the basis of this phenotype, we performed a time-course paired transcriptomic and metabolomic analysis on mock-inoculated vs B. cinerea-inoculated petals and leaves. Infection triggered strong transcriptional reprogramming in petals, with clear induction of phenylpropanoid and flavonoid/anthocyanin pathway genes and candidate regulators, consistent with the visible pigmentation. Metabolite profiles reflected this response, showing time-dependent accumulation of infection induced flavonoids such as quercetin, tilianin, and their derivatives, as well as cyanidin-based anthocyanins in infected petals. Integrating both omics datasets with MEANtools highlighted an anthocyanin-associated transcript-metabolite module, including a module putatively involved in the synthesis of polyyne-type phytoalexins. Antifungal assays demonstrated that selected flavonoids and cyanidin derivatives inhibit B. cinerea in a dose-dependent manner, supporting a direct antifungal role of these compounds. Altogether, our results show that C. seticuspe petals deploy a spatially confined, multi-layered chemical defence in which pathogen-induced flavonoids and anthocyanins operate as active components of resistance against a necrotrophic pathogen. We anticipate that future paired omics analyses in combination with spatial omics and bioactivity assays will yield insights into the role of specialised defence molecules in response to biotic and abiotic stresses. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/695701v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@e5dbeborg.highwire.dtl.DTLVardef@c94bd1org.highwire.dtl.DTLVardef@190c030org.highwire.dtl.DTLVardef@39a7b5_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2025

Capture of the SmSTRIPAK proxiome identifies the greenbeard proteins SmDOC1/2 as regulators of the MAK2 pathway to control sexual development in Sordaria macrospora

Hyphal fusion and sexual development in filamentous fungi rely on coordinated signaling of numerous conserved nodes such as the striatin interacting phosphatase and kinase (STRIPAK) complex or the pheromone response (PR) MAP kinase cascade (MIK2, MEK2, MAK2, HAM5). Here we used the homothallic ascomycete Sordaria macrospora (Sm) to screen for putative protein interactors of the SmSTRIPAK complex. Using the STRIPAK complex interactor 1 (SCI1) subunit of the complex as bait, we enriched and identified canonical SmSTRIPAK components and a determinant of communication (DOC) protein. The DOC proteins were previously described in the closely related and heterothallic species Neurospora crassa, functioning in allorecognition of germlings and hyphal fusions. We generated {Delta}Smdoc1, {Delta}Smdoc2 single deletion strains and the double deletion mutant {Delta}Smdoc1{Delta}Smdoc2 in S. macrospora. Deletion phenotypes were paradoxical: single knockouts ({Delta}Smdoc1 or {Delta}Smdoc2) were nearly sterile and sexual development was impaired, yet the double mutant ({Delta}Smdoc1{Delta}Smdoc2) exhibited wild-type fertility and development, demonstrating non-redundant and mutually antagonistic roles. Similarly, we demonstrated an impairment of the N. crassa {Delta}doc-2 mutant in sexual development. Using gene tagging at the native locus, we performed TurboID-based proximity mapping with SmDOC1 and SmDOC2 as bait proteins. This proximity mapping demonstrated close ties of SmDOC1/2 to components of the PR MAP kinase pathway and revealed mutual SmDOC1 - SmDOC2 proximity. Yeast Two-Hybrid experiments with SmDOC1 confirmed the direct interaction with the MAP kinases MEK2 and MAK2. Fluorescence microscopy revealed that SmDOC1-TagRFP-T localized to ring-like structures around septal pores. Our results demonstrate that the DOC system is not restricted to heterothallic N. crassa, but also plays an essential role in the development of fruiting bodies in the homothallic fungus S. macrospora. These findings suggest the DOC1/2 proteins as a novel system that integrates STRIPAK and PR pathways, providing a possible mechanistic explanation for their non-additive deletion strain phenotypes.

cell biology↗

bioRxiv2025

Itaconate is metabolized to 2-hydroxymethylsuccinate through a CoA-independent degradation pathway in mitochondria

The immunometabolite itaconate modulates cellular metabolism and is converted into structurally similar C5 dicarboxylates that require advanced analytics to decipher their metabolic fate. Here, we employ high-resolution mass spectrometry and tracing approaches and identify 2-hydroxymethylsuccinate (2HMS) as a previously unrecognized C5 dicarboxylate derived from itaconate. 2HMS synthesis occurs during inflammatory responses and upon itaconate treatment, as detected by 13C itaconate tracing. Pathway analysis reveals that methylglutaconyl-CoA hydratase (AUH) drives 2HMS synthesis through a CoA-independent conversion (CIC) pathway. This pathway is distinct from the CoA-dependent conversion (CDC) pathway that generates mesaconate and itaconyl-CoA influencing B12-dependent processes. In vivo inflammation studies reveal that adipose tissue prefers CIC to produce 2HMS and liver favors CDC-mediated mesaconate synthesis, highlighting tissue-specific itaconate degradation routes. This study identifies a new branch of itaconate metabolism, provides an analytical framework to resolve C5 dicarboxylate networks, and links 2HMS to inflammation and mitochondrial metabolism that might be targeted therapeutically.

cell biology↗

bioRxiv2025

PmaI-dependent pH gradients initiate inter- and intraspecies germling fusion in filamentous fungi

Somatic cell fusion among spore germlings is a complex process in filamentous fungi that enables the formation of a syncytial unit and facilitates the exchange of genetic material apart from sexual reproduction. In this study, we investigated the molecular mechanisms underlying germling fusion in the maize pathogen Colletotrichum graminicola. Based on secretomes harvested during the germling fusion process of fusion-competent oval and fusion-deficient falcate conidia, we identified pH as a communication signal. We showed that the plasma membrane localized ATPase PmaI is a probable source for pH gradient formation. Our results also suggest that the a-pheromone receptor CgSte3 mediates sensing of acidic pH gradients, activating downstream the Cell Wall Integrity Mitogen Activated Protein Kinase pathway. We propose a new model for germling interactions, in which pH serves as an interaction initiation signal, resulting in the activation of the cell-dialogue. Our findings have implications for our understanding of fungal communication and pathogenicity, and highlight the importance of pH as a signal molecule in germling fusion.

microbiology↗

bioRxiv2024

The penta-EF-hand protein Pef1 of Candida albicans functions at sites of membrane perturbation to support polarized growth and membrane integrity

The fungal plasma membrane is the target of fungicidal compounds, such as polyenes and saponins, that directly interact with fungus-specific ergosterol to cause deleterious membrane disruption. To counter membrane attack, diverse eukaryotic cells employ Ca2+-binding penta-EF (PEF)-hand proteins, including the human ortholog, ALG-2, to maintain membrane integrity. Candida albicans is a major fungal pathogen in humans, where increasing resistance to current antifungal drugs that target the plasma membrane is a serious cause of concern. Combinatorial treatments that additionally compromise the plasma membrane offer a way forward, but our mechanistic understanding of how fungi respond to direct membrane disruption remains limited. Here, we characterized the PEF-hand ortholog, Pef1, in this polymorphic species. GFP-tagged Pef1 localized at sites of polarized growth in yeast and hyphal cells of C. albicans. On treatment of cells with the polyene drug, amphotericin B, or the saponin, tomatine, GFP-Pef1 appeared as punctate spots at the membrane. In a similar manner, loss of calcineurin, but not of its transcription factor, Crz1, caused a punctate localization pattern of GFP-Pef1, which correlated with the serum sensitivity of the cna1{Delta} mutant. While deletion of PEF1 impaired yeast cell separation, filamentation was not affected. Strikingly, pef1{Delta} hyphae could not maintain plasma membrane integrity in serum. Consistent with this, the mutant exhibited attenuated virulence in an insect larvae infection model. Taken together, these observations suggest that Pef1 localizes to sites of membrane perturbation in order to maintain cell integrity, including sites of dynamic polarized growth and fungicide-induced membrane disruption.

cell biology↗

bioRxiv2021

The landscape of toxic intermediates in the metabolic networks of pathogenic fungi reveals targets for antifungal drugs

The burden of fungal infections for humans, animals and plants is widely underestimated and comprises deadly infections as well as great economic costs. Despite that, antifungal drugs are scarce and emergence of resistance in fungal strains contributes to a high mortality. To overcome this shortage, we propose toxic intermediates and their controlling enzymes in metabolic pathways as a resource for new targets and provide a web-service, FunTox-Networks to explore the landscape of toxic intermediates in the metabolic networks of fungal pathogens. The toxicity of metabolites is predicted by a new random forest regression model and is available for over one hundred fungal species. Further, for major fungal pathogens, metabolic networks from the KEGG database were enriched with data of toxicity and regulatory effort for each enzyme to support identification of targets. We determined several toxic intermediates in fungal-specific pathways like amino acid synthesis, nitrogen and sulfur assimilation, and the glyoxylate bypass. For the latter, we show experimentally that growth of the pathogen Candida albicans is inhibited when the detoxifying enzymes Mls1 and Hbr2 are deleted and toxic glyoxylate accumulates in the cell. Thus, toxic pathway intermediates and their controlling enzymes represent an untapped resource of antifungal targets.

systems biology↗

bioRxiv2020

The Cell Wall Integrity MAPK pathway controls actin cytoskeleton assembly during fungal somatic cell fusion.

Somatic cell fusion in Neurospora crassa depends on two MAP kinases, MAK-1 and MAK-2, but their precise contributions to directed growth have remained unclear. Using analog-sensitive alleles and live-cell imaging, we found that MAK-1 activity is required for RAC-1 activation and polarized actin assembly, while MAK-2 controls the positioning of RAC-1 and actin at the fusion tips. Inhibition of MAK-1 disrupted cell communication, prevented actin focus formation, and blocked membrane recruitment of MAK-2 and SO. In contrast, MAK-2 inhibition caused SO and actin to shift to alternative cortical sites, showing that MAK-2 regulates their spatial orientation rather than activation. Actin disruption with latrunculin A eliminated MAK-2 and SO recruitment, confirming its essential role in their localization. Importantly, MAK-1 controlled actin polymerization only during tropic growth, not during general hyphal extension, and acted upstream of RAC-1. These findings establish complementary roles for MAK-1 and MAK-2 in directing RAC-1-dependent actin dynamics during fungal cell fusion. Summary statementMAK-1 activates the Rho GTPase RAC-1 to drive actin polymerization, while MAK-2 orients this RAC-1/actin complex toward the fusion partner, defining complementary MAPK roles in somatic cell fusion.

cell biology↗

bioRxiv2020

Development of genetic tools for the thermophilic filamentous fungus Thermoascus aurantiacus

BackgroundFungal enzymes are vital for industrial biotechnology, including the conversion of plant biomass to biofuels and bio-based chemicals. In recent years, there is increasing interest in using enzymes from thermophilic fungi, which often have higher reaction rates and thermal tolerance compared to currently used fungal enzymes. The thermophilic filamentous fungus Thermoascus aurantiacus produces large amounts of highly thermostable plant cell wall degrading enzymes. However, no genetic tools have yet been developed for this fungus, which prevents strain engineering efforts. The goal of this study was to develop strain engineering tools such as a transformation system, a CRISPR/Cas9 gene editing system and a sexual crossing protocol to improve enzyme production. ResultsHere we report Agrobacterium tumefaciens-mediated transformation (ATMT) of T. aurantiacus using the hph marker gene, conferring resistance to hygromycin B. The newly developed transformation protocol was optimized and used to integrate an expression cassette of the transcriptional xylanase regulator xlnR, which led to up to 500% increased xylanase activity. Furthermore, a CRISPR/Cas9 gene editing system was established in this fungus, and two different gRNAs were tested to delete the pyrG orthologue with 10% and 35% deletion efficiency, respectively. Lastly, a sexual crossing protocol was established using a hygromycin B- and a 5-fluororotic acid-resistant parent strain. Crossing and isolation of progeny on selective media was completed in a week. ConclusionThe genetic tools developed for T. aurantiacus can now be used individually or in combination to further improve thermostable enzyme production by this fungus.

microbiology↗

bioRxiv2019

Phosphatidic acid produced by phospholipase D is required for hyphal cell-cell fusion and fungal-plant symbiosis

Although lipid signaling has been shown to serve crucial roles in mammals and plants, little is known about this process in filamentous fungi. Here we analyse the contribution of phospholipase D (PLD) and its product phosphatidic acid (PA) in hyphal morphogenesis and growth of Epichloe festucae and Neurospora crassa, and in the establishment of a symbiotic interaction between E. festucae and Lolium perenne. Growth of E. festucae and N. crassa PLD deletion strains in axenic culture, and for E. festucae in association with L. perenne, were analysed by light-, confocal- and electron microscopy. Changes in PA distribution were analysed in E. festucae using a PA biosensor and the impact of these changes on endocytic recycling and superoxide production investigated. We found that E. festucae PldB and the N. crassa ortholog, PLA-7, are required for polarized growth, cell fusion and ascospore development, whereas PldA/PLA-8 are dispensable for these functions. Exogenous addition of PA rescues the cell-fusion phenotype in E. festucae. PldB is also crucial for E. festucae to establish a symbiotic association with L. perenne. This study identifies a new component of the cell-cell communication and cell fusion signaling network that controls hyphal morphogenesis and growth in filamentous fungi.

genetics↗