Cell fusion is a fundamental process essential for the development and proliferation of eukaryotic organisms. In the ascomycete fungus Neurospora crassa, germinating spores undergo chemotropic interactions and fusion to merge into a supracellular unit, which gives rise to the mycelial colony. Within mature colonies, hyphal branches fuse to form anastomoses between leading hyphae, enhancing the overall connectivity of the mycelium. Both germling and hyphal fusion rely on the same molecular machinery. The MAP kinase MAK-2 and the fungal-specific protein SO have been identified as key regulators of these processes, and their alternating recruitment to the plasma membrane at interacting cell tips suggests a dialog-like cell communication mechanism involving dynamic switches between signal sending and receiving. However, the mechanisms that trigger the onset of this intercellular communication are still not understood. This study identifies EOP-1 as an interaction partner of the SO protein and functionally characterizes its role in cell communication and fusion. Deletion of the eop-1 gene abolished germling fusion and chemotropic interactions, while live-cell imaging showed EOP-1 oscillating at interacting cell tips, coinciding with SO recruitment. Intriguingly, EOP-1 displayed a similar dynamic, oscillatory tip recruitment also in isolated, non-interacting germlings, setting it apart from previously characterized fusion factors in N. crassa. This observation suggests for the first time that spore germlings of N. crassa exhibit fusion related cell-autonomous oscillatory behavior and implicates EOP-1 in initiating intercellular communication. The oscillatory recruitment pattern of EOP-1 was dependent on the presence of SO, MAK-1, MAK-2, BEM1 and HAM-14 in the cell. Loss of EOP-1 strongly reduced MAK-1 phosphorylation, placing EOP-1 upstream of MAK-1 pathway activation. This work offers new insight into how genetically and developmentally identical cells initiate and coordinate their communication and mutual attraction.

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 of serious 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 investigated 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 hyphae with the polyene drug, amphotericin B, or the saponin tomatine, GFP-Pef1 became distributed as punctate spots at the membrane. In a similar manner, loss of calcineurin A (Cna1), but not of its transcription factor, Crz1, caused this punctate localization pattern of GFP-Pef1. While deletion of PEF1 slightly impaired yeast cell growth rate, filamentation was not affected. Strikingly, pef1Δ hyphae could not maintain plasma membrane integrity in serum, as also seen in the cna1Δ mutant, and exhibited attenuated virulence in an insect larvae infection model. Together, these observations suggest that Pef1 localizes to sites of membrane perturbation to maintain cell integrity, including sites of dynamic polarized growth, septum formation, and fungicide-induced membrane disruption.

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 ΔSmdoc1, ΔSmdoc2 single-deletion strains and the double deletion mutant ΔSmdoc1ΔSmdoc2 in S. macrospora. Deletion phenotypes were paradoxical: single knockouts (ΔSmdoc1 or ΔSmdoc2) were nearly sterile, and sexual development was impaired, yet the double mutant (ΔSmdoc1ΔSmdoc2) exhibited wild-type fertility and development, demonstrating non-redundant and mutually antagonistic roles. 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 structures near 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.

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. ### Competing Interest Statement JJJvdH is member of the Scientific Advisory Board of NAICONS Srl., Milano, Italy and consults for Corteva Agriscience, Indianapolis, IN, USA. All other authors declare to have no competing interests. TKI Agri & Food, https://ror.org/036br4307, LWV21.291, TU-2021-20

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 ΔSmdoc1, ΔSmdoc2 single deletion strains and the double deletion mutant ΔSmdoc1ΔSmdoc2 in S. macrospora . Deletion phenotypes were paradoxical: single knockouts (ΔSmdoc1 or ΔSmdoc2) were nearly sterile and sexual development was impaired, yet the double mutant (ΔSmdoc1ΔSmdoc2) exhibited wild-type fertility and development, demonstrating non-redundant and mutually antagonistic roles. Similarly, we demonstrated an impairment of the N. crassa Δ 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. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, PO 523/10-1 project number 538832008, INST 186/1230-1 FUGG, INST 186/1465-1

Cell-cell fusion in plants and fungi requires localized cell wall dissolution at the contact site to allow direct plasma membrane contact and subsequent membrane merger. Since cell wall removal carries the risk of cell rupture, the process must be tightly regulated to permit localized fusion pore formation while preserving cellular integrity. While the molecular events guiding cell-cell signaling leading to contact between fusing fungal cells have begun to unfold, the post-contact mechanisms stabilizing the forming fusion pore remain largely unknown. Here, we identify the chitin synthase regulator CSR-3 as a molecular factor promoting stable pore formation during somatic fusion in the fungal cell fusion model Neurospora crassa. CSR-3 specifically accumulates at the contact zones of fusing cells and contributes to fusion fidelity by preventing membrane rupture and lysis, particularly under calcium-limited conditions. Loss of CSR-3 leads to elevated fusion-induced lysis, a phenotype rescued by osmotic stabilization, suggesting a cell wall defect. Beyond fusion, CSR-3 is involved in septum formation, septal pore plugging, conidiation, and the response to biotic and abiotic cell wall stress. These observations support a broader role for CSR-3 in chitin-mediated cell wall remodeling. Our data indicate that CSR-3 dynamics at fusion sites depend on the MAP kinase MAK-1, implicating cell wall integrity signaling in post-contact fusion events. Consistent with this finding, phospho-mimetic analysis suggests a regulatory role for CSR-3 phosphorylation. Co-localization and genetic analyses identify the chitin synthase CHS-2 as a likely downstream target of CSR-3, with both proteins functioning in the same pathway. Together, our findings reveal that CSR-3 coordinates cell wall remodeling during cell fusion and stress responses, uncovering a crucial regulatory layer that safeguards fungal cellular integrity during dynamic developmental processes. Our observations support a model in which cell wall biosynthesis plays a critical role in cell wall remodeling during fusion pore formation.

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. ### Competing Interest Statement F.C. is a co-inventor on a patent, Citraconic acid and derivatives thereof for use as a medicament, U.S. Patent Application No. 18/556,649. The other authors declare that they have no conflicts of interest with the content of this article. * ACOD1 : cis -Aconitate Decarboxylase (protein) AT : Adipose Tissue ATCC : American Type Culture Collection Auh : AU RNA Binding Methylglutaconyl-CoA hydratase (gene) AUH : Methylglutaconyl-CoA hydratase (protein), also known as 3-MGH B12 : Vitamin B12 (Cobalamin) BAT : Brown Adipose Tissue BCAA : Branched-chain amino acid BMDM : Bone Marrow-Derived Macrophages 12C : Carbon-12 13C : Carbon-13 CA : Citraconate CDC : CoA-dependent conversion CIC : CoA-independent conversion CiMa : Citramalate CoA : Coenzyme A CpG ODN : Cytosine-phosphorothioate-guanine oligodeoxynucleotides Ctr : Control DMEM : Dulbecco’s Modified Eagle’s Medium FBS : Fetal Bovine Serum FELASA : Federation of European Laboratory Animal Science Associations GC-MS : Gas Chromatography–Mass Spectrometry GC-MS/MS : Gas Chromatography-Tandem Mass Spectrometry gWAT : Gonadal White Adipose Tissue 2HG : 2-Hydroxyglutarate 3HG : 3-Hydroxyglutarate 2HMS : 2-Hydroxymethylsuccinate hMDM : Human Monocyte-Derived Macrophages IFNγ : Interferon-gamma Irg1 : Immunoresponsive gene 1 (gene) IRG1 : Immune-Responsive Gene 1 Protein ita : Itaconate KO : Knockout LC-MS/MS : Liquid Chromatography-Tandem Mass Spectrometry LPS : Lipopolysaccharide 3MeMa : 3-Methylmalate mesa : Mesaconate MGCA1 : 3-methylglutaconic aciduria type 1 MMA : Methylmalonate MTBSTFA : N-tert-Butyldimethylsilyl-N-methyltrifluoracetamid MUT : Methylmalonyl-CoA mutase (protein) m/z : Mass-to-charge ratio NaDC3, SLC13A3 : Sodium-dependent dicarboxylate transporter 3 (protein) NMR : Nuclear Magnetic Resonance PBMC : Peripheral Blood Mononuclear Cells rPFO : Recombinant perfringolysin RPMI : Roswell Park Memorial Institute medium RT : Retention time SA : Standard addition SCS : Succinyl-CoA synthetase (protein) scWAT : Subcutaneous White Adipose Tissue s.e.m. : Standard error of mean siRNA : Small interfering RNA Slc13a3 : Solute carrier family 13 member 3 (gene) Suclg1 : Succinate-CoA ligase GDP/ADP-forming subunit alpha (gene) Sugct : Succinyl-CoA:glutarate CoA-transferase (gene) SUGCT : Succinyl-CoA:glutarate CoA-transferase (protein) TCA : Tricarboxylic acid cycle tBDMS : tert-Butyldimethylsilylchlorid TIC : Total ion chromatogram TLR : Toll-like receptor U : Uniformly [U-13C]substrate : Uniformly labeled 13C carbon tracer WAT : White Adipose Tissue WT : Wild-Type

Forests world-wide are under escalating threat from emerging and invasive fungal and oomycete pathogens, driven by globalization and shifting climate dynamics. Effective strategies to manage the current scale and rate of changes in forest health remain hindered by our limited ability to study the underlying mechanisms of pathogen-host and pathogen-microbiome interactions, especially at a molecular and cellular level, compared to general plant pathology, where experimental and model systems exist. Such models facilitate the integration of diverse methodologies from a broader base of the research community, allowing for a more holistic and deeper examination of complex research questions. Here, we propose a framework for the development of such model systems also for forest pathology. This goal is more feasible than ever, thanks to rapid technological advancements, increasing open data availability and a globally interconnected research community. These factors create a unique opportunity to integrate ecosystem-focused research in forest pathology with a unified model organism strategy. Achieving this goal will require a dedicated community effort in the coming years, as such model systems are not discovered but built.

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.

Diplodia sapinea (Fr.) Fuckel is a widespread fungal pathogen affecting conifers worldwide. Infections can lead to severe symptoms, such as shoot blight, canker, tree death, or blue stain in harvested wood, especially in Pinus species. Its impact on forest health is currently intensified, likely due to climate change, posing an increasing threat to global ecosystems and forestry. Despite extensive and successful research on this pathogen system, fundamental questions about its biology and plant-associated lifestyle remain unanswered. Addressing these questions will necessitate the development of additional experimental tools, including protocols for molecular genetics and cell biology approaches. In this study, we continue to address this need by establishing an Agrobacterium-mediated genetic transformation protocol for D. sapinea, enabling targeted mutagenesis and heterologous gene expression. We utilized this methodology to localize the histone H2B by tagging it with the fluorescent protein mCherry. Additionally, we established a time- and space-efficient laboratory-scale infection assay using two-week-old Pinus sylvestris seedlings. Integrating these tools in a proof-of-concept study enabled the visualization of D. sapinea in planta growth through the fluorescently labeled reporter strain.

Saponins are plant secondary metabolites comprising glycosylated triterpenoids, steroids or steroidal alkaloids with a broad spectrum of toxicity to microbial pathogens and pest organisms that contribute to basal plant defense to biotic attack. Secretion of glycosyl hydrolases that enzymatically convert saponins into less toxic products was thus far the only mechanism reported to enable fungal pathogens to colonize their saponin-containing host plant(s). We studied the mechanisms that the fungus Botrytis cinerea utilizes to be tolerant to well-characterized, structurally related saponins from tomato and Digitalis purpurea. By gene expression studies, comparative genomics, enzyme assays and testing a large panel of fungal (knockout and complemented) mutants, we unraveled four distinct cellular mechanisms that participate in the mitigation of the toxic activity of these saponins and in virulence on saponin-producing host plants. The enzymatic deglycosylation that we identified is novel and unique to this fungus-saponin combination. The other three tolerance mechanisms operate in the fungal membrane and are mediated by protein families that are widely distributed in the fungal kingdom. We present a spatial and temporal model on how these mechanisms jointly confer tolerance to saponins and discuss the repercussions of these findings for other plant pathogenic fungi, as well as human pathogens.

Abstract Saponins are plant secondary metabolites comprising glycosylated triterpenoids, steroids or steroidal alkaloids with a broad spectrum of toxicity to microbial pathogens and pest organisms that contribute to basal plant defence to biotic attack. Secretion of glycosyl hydrolases that enzymatically convert saponins into less toxic products was thus far the only mechanism reported to enable fungal pathogens to colonize their saponin-containing host plant(s). We studied the mechanisms that the fungus Botrytis cinerea utilizes to be tolerant to well-characterized, structurally related saponins from tomato and Digitalis purpurea. By gene expression studies, comparative genomics, enzyme assays and testing a large panel of fungal (knockout and complemented) mutants, we unraveled four distinct cellular mechanisms that participate in mitigation of the toxic activity of these saponins. The enzymatic deglycosylation is novel and unique to this fungus-saponin combination, while the genes involved in other tolerance mechanisms have orthologs that are widely distributed in the fungal kingdom. We present a spatial and temporal model on how these mechanisms jointly confer tolerance to α- tomatine and we discuss the repercussions for other fungi and different saponins.

Diplodia sapinea is a globally distributed opportunistic fungal pathogen of conifers that causes severe production losses in forestry. The fungus frequently colonizes pine trees as an endophyte without causing visible symptoms but can become pathogenic when the host plant is weakened by stress, such as drought or heat. Forest damage might therefore further increase due to the effects of climate change. The future development of control strategies depends on a better understanding of the fungus’ biology, which requires experimental methods for its investigation in the laboratory. An efficient, standardized protocol for the production and storage of highly viable pycnidiospores was developed, and a spore-based infection method was devised. We compared infection rates of dormant and actively growing, wounded, or nonwounded Scots pine seedlings inoculated with in vitro-produced spores and mycelium from agar-plugs. Spores were a much more efficient inoculum for causing disease symptoms on wounded plants than the conventional agar plug. The application of spores on nonwounded plants lead to high rates of asymptomatic infection, suggesting endophytic fungal development. These methods enable standardized spore infection and virulence assays and promote D. sapinea as a model organism for studying the switch from endophytic to pathogenic life styles of forest pathogens.

Significance This study reveals that a dialogue-like communication mechanism, which mediates cell–cell fusion in filamentous fungi, is a conserved complex trait. It allows the communication and behavioral coordination of cells of distantly related species and mediates their mutual attraction and subsequent physical contact, although interspecies fusion does not occur. Through the activation of this signaling machinery, one species can reprogram the developmental program of the other fungus. These data promote our understanding of microbial communication, illustrate the mechanism of repurposing of existing building blocks in cellular evolution, revive the hypothesis of vegetative fusion as an avenue of horizontal gene transfer in fungi, and establish the idea of developmental reprogramming as a tool for controlling fungi.

The development of ascomycete fungal colonies involves cell-cell fusion at different growth stages. In the model fungus Neurospora crassa, communication of two fusing cells is mediated by an unusual signaling mechanism, in which the two partners take turns in signal sending and receiving. In recent years, the molecular basis of this unusual cellular behavior has started to unfold, indicating the presence of an excitable signaling network. New evidence suggests that this commu-nication system is highly conserved in ascomycete fungi and, unexpectedly, even mediates interspecies interactions. At the same time, intricate allorecognition mechanisms were identi-fied, which prevent the fusion of genetically unlike individuals. These observations suggest that signal specificity during fungal social behavior has not evolved on the level of signals and receptors, but is achieved at downstream checkpoints. Despite growing insight into the molecular mechanisms con-trolling self and non-self fungal interactions, their role in natural environments remains largely unknown.

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.

Background: Filamentous fungi are excellent lignocellulose degraders, which they achieve through producing carbohydrate active enzymes (CAZymes). CAZyme production is highly orchestrated and the application of –omics methods such as RNA-Seq has greatly expanded understanding of this important biotechnological process. The thermophilic fungus Thermoascus aurantiacus secretes high amounts of highly active thermostable enzymes that enable saccharifications at higher temperatures; however, the genome-wide response to CAZyme induction is not understood. Results: A fed-batch system with plant biomass-derived sugars D-xylose, L-arabinose and cellobiose established that these sugars induce CAZyme expression in T. aurantiacus . The C5 sugars induced both cellulases and hemicellulases, while cellobiose specifically induced cellulases. A minimal medium formulation was developed to enable RNA-seq studies of T. aurantiacus with these inducers. It was found that D-xylose and L-arabinose strongly induced a wide variety of CAZymes, auxiliary activity (AA) enzymes and carbohydrate esterases (CEs), while cellobiose facilitated lower expression of mostly cellulase genes. Furthermore, putative orthologues of different unfolded protein response genes were up-regulated during the C5 sugar feeding together with genes in the C5 sugar assimilation pathways. Conclusion: This work has identified two additional CAZyme inducers for T. aurantiacus , L-arabinose and cellobiose, along with D-xylose. A combination of biochemical assays and RNA-seq measurements established that C5 sugars induce a suite of cellulases and hemicellulases, providing a path to produce a broad spectrum thermotolerant enzymatic mixture for deconstruction of plant biomass.

Carbohydrate active enzymes (CAZymes) are vital for the lignocellulose-based biorefinery. The development of hypersecreting fungal protein production hosts is therefore a major aim for both academia and industry. However, despite advances in our understanding of their regulation, the number of promising candidate genes for targeted strain engineering remains limited. Here, we resequenced the genome of the classical hypersecreting Neurospora crassa mutant exo-1 and identified the causative point of mutation to reside in the F-box protein-encoding gene, NCU09899. The corresponding deletion strain displayed amylase and invertase activities exceeding those of the carbon catabolite derepressed strain Δcre-1, while glucose repression was still mostly functional in Δexo-1 Surprisingly, RNA sequencing revealed that while plant cell wall degradation genes are broadly misexpressed in Δexo-1, only a small fraction of CAZyme genes and sugar transporters are up-regulated, indicating that EXO-1 affects specific regulatory factors. Aiming to elucidate the underlying mechanism of enzyme hypersecretion, we found the high secretion of amylases and invertase in Δexo-1 to be completely dependent on the transcriptional regulator COL-26. Furthermore, misregulation of COL-26, CRE-1, and cellular carbon and nitrogen metabolism was confirmed by proteomics. Finally, we successfully transferred the hypersecretion trait of the exo-1 disruption by reverse engineering into the industrially deployed fungus Myceliophthora thermophila using CRISPR-Cas9. Our identification of an important F-box protein demonstrates the strength of classical mutants combined with next-generation sequencing to uncover unanticipated candidates for engineering. These data contribute to a more complete understanding of CAZyme regulation and will facilitate targeted engineering of hypersecretion in further organisms of interest.

Having an adequate understanding of the Nature of Science (NOS) is an integral part of scientific literacy. However, NOS is usually not yet explicitly embedded in the science curricula at German universities. To fill this gap, we have introduced NOS elements in the undergraduate course on genetics at the biology department of an Institute of Technology in North-western Germany in summer semester 2018. The strategy used an exclusive-reflective approach by emphasising socio-scientific issues. As Kostas Kampourakis (2016) suggests, our design considers not only general aspects of the NOS concept, but also the family resemblance approach presented by Erduran and Dagher (2014). To evaluate changes in students’ NOS understanding, we did a pre- and post-survey about their NOS understanding following the SUSSI questionnaire designed by Liang et al. (2008). The NOS understanding of the 93 participants shows statistically significant improvement in 14 out of 24 items (58,3%) after the teaching unit, compared to the pre-survey. While the pre-survey shows a larger gap of understanding regarding the relations of environment, theory, and law, the post-test results show significant effects on learning, in particular regarding subjective, social, and cultural influences on science. However, the students’ understanding regarding the relations of environment, theory, and law still remains weak. The findings indicate that some preconceptions were not as amenable to change as others. In particular, the assumed facticity of scientific knowledge seems to be a powerful preconception that is much more firmly fixed than the contextualization of scientific discovery.

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 Epichloë 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.