Biology subjects

Harting, R.

Publications and source records attributed to Harting, R..

10 recordsLinked to original sources

bioRxiv2026

Verticillium dahliae ubiquitin-specific proteases coordinate key developmental processes and pathogenicity

Ubiquitin is a posttranslational modifier that is conserved among eukaryotes. Ubiquitination alters stability and folding of cellular proteins. Deubiquitinases (DUBs) reverse ubiquitination and often function as part of protein complexes. There are 32 predicted DUB-encoding genes present in the soil-borne phytopathogenic fungus Verticillium dahliae. Nuclear ubiquitin-specific protease 3 (Usp3) is a member of the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex, whereas Usp1 is predicted to associate with the COP9 signalosome (CSN), which controls specificities of cellular E3 ubiquitin ligase activities. A proteomics approach using biotin capture and identification (BioID) supports that Usp3 regulates gene expression beyond the transcription level. Western experiments showed a dysregulation in ubiquitinated cellular proteins in corresponding deletion strains. Usp3 and Usp1 are both required for fungal development. They regulate microsclerotia formation based on different environmental cues and provide redundant functions in controlling conidiation. Absence of both corresponding genes resulted in significant impairment of conidiospore formation, which is required for fungal propagation within the plant vascular system. This paralysed spreading ability reduced virulence on tomato plants (Solanum lycopersicum). In summary, V. dahliae responds to environmental cues by Usp3- and Usp1-mediated adjustment of gene expression and protein stability. This is important for key developmental processes of the V. dahliae disease cycle and its virulence towards the host plant. Author summaryUbiquitination and deubiquitination of proteins enable cells to rapidly react to environmental cues and adjust protein stabilities and subsequently transcriptomic profiles. Usp3 is a nuclear deubiquitinase subunit of the Spt-Ada-Gcn5 acetyltransferase (SAGA) transcriptional coactivator complex. Usp1 is predicted to be associated with the COP9 signalosome that regulates substrate specificities of the ubiquitin-proteasome system. BioID experiments suggest that other SAGA complex subunits, histone proteins, spliceosomal proteins, ribosomal proteins, a protein that tackles transcriptionally stalled RNAPII, and a protein that degrades mRNA with premature stop codons locate proximal to Usp3 within the cell. Deletion of USP3 led to the dysregulation of protein ubiquitination. A single deletion of USP1 did not significantly change ubiquitination profiles, however, a double deletion of USP1/3 significantly affected the ubiquitin-proteasome system. The altered ubiquitination profile correlated with a dysregulation of key developmental processes. Microsclerotia formation was decoupled from environmental cues in the {Delta}USP3 strain, whereas an additional deletion of USP1 reconnected it in a media-dependent manner. USP3 and USP1 contribute to a common governing process in conidiation, and the defect in spreading of the {Delta}USP1/3 strain is reflected by a significant reduction in plant pathogenicity.

microbiology↗

bioRxiv2025

The adapt-to-nutrient NRPS-like secondary metabolite gene cluster facilitates Verticillium dahliae adaptation to different nutrient environments

Filamentous fungi produce a wide range of secondary metabolites to adapt to changing environments. RNA sequencing revealed that nine biosynthetic gene clusters (BGCs) of the phytopathogenic Verticillium dahliae react to different nutrient environments. The adapt-to-nutrient NRPS-like (ANN) cluster contributes to antibacterial activity and developmental processes important for the early biotrophic life cycle, but is dispensable for virulence on tomato (Solanum lycopersicum). Transcription of the core biosynthetic enzyme-encoding ANN3 is highly induced in nutrient-poor environment. ANN3 is transcriptionally controlled by global and in-cluster transcription factors. ANN3 is activated by early colonisation transcription factors Som1 and Vta2, but repressed by Mtf1, which governs late stages of disease progression. The in-cluster transcription factor Ann1, which represses ANN3, is less stable in nutrient-poor environment or when V. dahliae encounters antagonists. Ann1 promotes resting structure formation but suppresses conidiation and antibacterial activity. Possible products of the ANN cluster were revealed by comparing metabolites extracted from ANN3 regulator mutants and from the bacterial-fungal interaction zone. Our findings revealed that V. dahliae perceives different nutrient environments and changes its survival strategy by differential expression of the ANN secondary metabolite gene cluster. Author summaryVerticillium dahliae is an economically significant phytopathogen that is widely distributed. The fungus adjusts and adapts its survival strategy according to the surrounding environment. Transcriptome data revealed that the core biosynthetic gene ANN3 of the adapt-to-nutrient NRPS-like (ANN) cluster is most expressed in nutrient-poor environments. The expression of ANN3 is governed by in-cluster repressor Ann1 and global transcriptional regulators that regulate other metabolic processes. Transcription factors Som1 and Vta2 are involved in the early plant-root infection process, whereas Mtf1 regulates late stage of disease development. ANN3 is activated by Som1 and Vta2, but repressed by Mtf1. The repressor Ann1 is less stable in nutrient-poor environments or when bacterial competitors are present. Ann1 promotes dormancy and represses spreading by conidiation. Vegetative growth is reduced but antibacterial activity is promoted when ANN1 is deleted. Possible chemical products of the ANN cluster were identified by comparing the metabolites extracted from the regulator mutant strains and the bacterial-fungal interaction zone. In summary, our findings show how V. dahliae react to environmental signals to balance growth, survival, and competition through the ANN cluster.

genetics↗

bioRxiv2025

The homeobox transcription factor HbxB coordinates distinct gene regulatory networks for asexual development and secondary metabolism in Aspergillus nidulans

Formation of conidia as asexual spores and sometimes worldwide distribution through the air is a very important feature of the fungal life style. This process is controlled by several regulatory proteins, including homeobox domain transcription factors. HbxB is one such regulator with implications in the control of development, secondary metabolism and various stress responses in the filamentous fungus Aspergillus nidulans. However, the molecular mechanism of the regulatory role of HbxB during asexual development is still elusive. Here we show that HbxB is a nuclear localized protein with great impact on asexual sporogenesis. Employment of high throughput assays like chromatin immunoprecipitation (ChIP-seq) and transcriptomics (RNA-seq), elucidated the in vivo binding landscape of HbxB in a genome-wide scale. A set of 238 genes as direct targets of HbxB were identified. A nine bases DNA motif where HbxB prefers to bind in vivo was discovered as HbxB response element (HRE). HbxB is influencing the expression of genes encoding master regulators of the asexual development such as SclB, PpoC, FlbA and FlbC. Moreover, the direct transcriptional control of the secondary metabolites sterigmatocystin and emericellamides biosynthesis by HbxB was discovered. Lastly also a previously elusive mutual regulatory control circuit between HbxB and two major regulators of the asexual development SclB and MsnA was found. Both of these regulators can directly induce the expression of hbxB. This study provides a detailed molecular mechanism on how HbxB controls A. nidulans asexual sporulation. ImportanceFungal distribution mainly relies on the formation of spores that are subsequently dispersed in different media to ensure colonization of substrates and the survival of the fungus. The asexual developmental program is a widely used strategy in the fungal kingdom for production of spores (conidia). The HbxB transcription factor is a nuclear localized, homeobox domain protein, with a strong impact on asexual sporulation of presumably numerous fungal species. This study enhances our understanding of the mechanism with which HbxB exerts its regulatory actions. HbxB is binding in vivo to specific DNA regulatory elements of genes encoding proteins with key roles in asexual development (like SclB, MsnA and PpoC), secondary metabolism (such as genes from the sterigmatocystin and emericellamide clusters) and stress response/tolerance. Overall, these findings open a window into how Hbx regulators orchestrate and coordinate fungal asexual developmental programs genome-wide at the molecular level.

molecular biology↗

bioRxiv2025

The Aspergillus nidulans transcription factor SclB governs the transition from vegetative to asexual development

Asexual reproduction in filamentous fungi is a common, efficient and fast differentiation process, for producing large numbers of asexual spores (conidia), which can be distributed through the air to colonize new environments. The whole process is tightly controlled by specific regulatory proteins. Among those major regulators is the zinc-finger domain protein of SclB (Sclerotia like B), known to influence various aspects of asexual growth and secondary metabolism in Aspergillus nidulans as well as other filamentous fungi. Two different growth conditions of A. nidulans were compared to obtain a mechanistic overview for the role of SclB, mainly during the transition of the fungus from vegetative to asexual growth. Chromatin immunoprecipitation was coupled with next generation sequencing (ChIP-seq) and combined with transcriptomic analyses (RNA-seq). SclB coordinates this developmental shift mainly by controlling the expression of genes encoding for few, however, prominent regulators of conidiation. They include the transcription factors BrlA, VelB and SclB and the pheromone oxygenase PpoC. Association of SclB to promoter regions requires the newly identified SclB response element (SRE) with a nine base-pair DNA motif. Scl2 is the corresponding protein in the fungal plant pathogen Verticillium dahliae and partially complements the {Delta}sclB A. nidulans asexual deficiency. This supports a conserved function of this regulator among different fungal species. In summary, SclB coordinates transition from vegetative growth to asexual reproduction in A. nidulans through in vivo transcriptional control over genes coding for established players of conidia formation. ImportanceFungi constantly adapt to environmental changes in their various habitats. Asexual spore formation allows to quickly leave an unfriendly habitat through dispersal into the air. The asexual developmental program of fungi ensures large number of spores, in a short period of time and in energetically efficient manner. SclB transcription factor is a key regulator of asexual growth and secondary metabolism in numerous fungal species. The mechanism through which SclB orchestrates the transition of the Aspergillus nidulans filamentous fungus from the vegetative to the asexual growth was revealed. This regulator directly controls in vivo itself as well as expression of master genes for the asexual program such as brlA for transcriptional control or ppoC for pheromone production. This study enhances the molecular understanding, how fungal asexual differentiation is initiated and coordinated, which supports the development of better strategies to control fungal pathogens, improving human health, safety and crop management.

molecular biology↗

bioRxiv2025

The VelB intrinsically disordered domain promotes selective heterodimer formation of velvet domain regulatory proteins for fungal development

Fungi possess several transcription factors with a characteristic velvet domain for DNA-binding and homo- or heterodimerization, which is structurally similar to the mammalian NF-B Rel homology domain. Velvet dimers control fungal development, virulence and mycotoxin formation. VelB is the only regulator, which carries an intrinsically disordered domain (IDD) within the velvet domain. The IDD as well as the positioning within VelB is conserved in the fungal kingdom. Intrinsically disordered regions contribute to transcription activation and DNA binding and frequently appear in eukaryotic transcription factors. The VelB IDD provides selective heterodimerization as well as protein stability control. The IDD is not required for the formation of the VelB-VeA heterodimer of Aspergillus nidulans or Verticillium dahliae, but promotes the formation of the VelB-VosA heterodimer. The IDD destabilizes VelB single molecules and also balances its distribution and ratio between both velvet heterodimers. These balances contribute to control appropriate mycotoxin production and sexual development. Herewith, the VelB IDD represents a novel control mechanism of velvet protein stability and heterodimer formation for precise priming of fungal development.

molecular biology↗

bioRxiv2025

The Aspergillus nidulans velvet domain containing transcription factor VeA is shuttled from cytoplasm into nucleus during vegetative growth and stays there for sexual development, but has to return into cytoplasm for asexual development

Survival of multicellular organisms requires the coordinated interplay between networks regulating gene expression and controlled intracellular transport of respective regulators. Velvet domain proteins are fungal transcription factors, which form various heterodimers and play key roles in controlling early developmental decisions towards more either asexual or sexual differentiation. VeA is the central subunit of the trimeric velvet complex VelB-VeA-LaeA, which links transcriptional to epigenetic control for the coordination of fungal developmental programs to specific secondary metabolite synthesis. Nuclear localization of the VeA bridging factor is carefully controlled in fungi. VeA carries three nuclear localization signals NLS1, NLS2 and NLS3, which all contribute to nuclear import. An additional VeA nuclear export sequence (NES) provides a shuttle function, which allows the cell to relocate VeA to the cytoplasm. VeA is nuclear during vegetative growth, but has to be exported from the nucleus to allow and promote asexual development. In contrast, progression of the sexual pathway requires continuous nuclear VeA localization. Accurate nuclear import and export control of velvet proteins is further connected to specific stability control mechanism as prerequisites for fungal development and secondary metabolism. These results illustrate the various complex mutual dependencies of velvet regulatory proteins for coordinating fungal development and secondary metabolism.

molecular biology↗

bioRxiv2025

Molecular circuit between Aspergillus nidulans transcription factors MsnA and VelB to coordinate fungal stress and developmental responses

Development and secondary metabolism of the filamentous fungus Aspergillus nidulans are tightly controlled by concerted actions of several master regulator transcription factors. The connection between fungal development and cellular stress response programs is often elusive. Here we show that the MsnA zinc finger transcription factor, which controls salt-stress response, is a novel major player in fungal development. A molecular circuit among MsnA and the velvet domain regulator VelB was discovered, which mutually fosters the actions of both regulatory proteins during development. MsnA controls the expression of several genes encoding master transcriptional regulators of asexual as well as sexual development. In addition, MsnA affects directly and indirectly the synthesis of specific secondary metabolites relevant for fungal defense against other organisms and growth, in addition to salt-stress responses. Moreover, the expression of genes encoding the epigenetic regulators VapA and VipC are also directly controlled by MsnA. These subunits of the VapA-VipC-VapB methyltransferase signal transduction complex promote asexual differentiation. MsnA is therefore placed at a novel prominent position of the central regulatory network, which coordinates stress responses with the developmental and metabolic fate of the fungus.

microbiology↗

bioRxiv2021

Hülle-cell-mediated protection of fungal reproductive and overwintering structures against fungivorous animals

Fungal Hulle cells with nuclear storage and developmental backup functions are reminiscent of multipotent stem cells. In the soil, Hulle cells nurse the overwintering fruiting bodies of Aspergillus nidulans. The genome of A. nidulans harbors genes for the biosynthesis of xanthones. We show that enzymes and metabolites of this biosynthetic pathway accumulate in Hulle cells under the control of the regulatory velvet complex, which coordinates development and secondary metabolism. Deletion strains blocked in the conversion of anthraquinones to xanthones are delayed in maturation and growth of fruiting bodies. Xanthones are not required for sexual development but exert antifeedant effects on fungivorous animals such as springtails and woodlice. These findings reveal a novel role of Hulle cells in establishing secure niches for A. nidulans by accumulating metabolites with antifeedant activity that protect reproductive structures from animal predators.

biochemistry↗

bioRxiv2020

V. longisporum elicits media-dependent secretome responses with a further capacity to distinguish between plant-related environments

Verticillia cause a vascular wilt disease affecting a broad range of economically valuable crops. The fungus enters its host plants through the roots and colonizes the vascular system. It requires extracellular proteins for a successful plant colonization. The exoproteome of the allodiploid Verticillium longisporum was analyzed upon cultivation in different media. Secreted fungal proteins were identified by label free LC-MS/MS screening. V. longisporum induced two main secretion patterns. One response pattern was elicited in various non-plant related environments. The second pattern includes the exoprotein responses to the plant-related media, pectin-rich simulated xylem medium and pure xylem sap, which exhibited similar but additional distinct features. These exoproteomes include a shared core set of 223 secreted and similarly enriched fungal proteins. The pectin-rich medium significantly induced the secretion of 144 proteins including a number of pectin degrading enzymes, whereas xylem sap triggered a smaller but unique fungal exoproteome pattern with 32 enriched proteins. The latter pattern included proteins with domains of known effectors, metallopeptidases and carbohydrate-active enzymes. The most abundant and uniquely enriched proteins of these different groups are the necrosis and ethylene inducing-like proteins Nlp2 and Nlp3, the cerato-platanin proteins Cp1 and Cp2, the metallopeptidases Mep1 and Mep2 and the CAZys Gla1, Amy1 and Cbd1. Deletion of the majority of the corresponding genes caused no phenotypic changes during ex planta growth or invasion and colonization of tomato plants. However, we discovered that the NLP2 and NLP3 deletion strains were compromised in plant infections. Overall, our exoproteome approach revealed that the fungus induces specific secretion responses in different environments. The fungus has a general response to non-plant related media whereas it is able to fine-tune its exoproteome in the presence of plant material. Importantly, the xylem sap-specific exoproteome pinpointed Nlp2 and Nlp3 as single effectors required for successful V. dahliae colonization. Author SummaryVerticillium spp. infect hundreds of different plants world-wide leading to enormous economic losses. Verticillium wilt is a disease of the vasculature. The fungus colonizes the xylem of its host plant where it exploits the vascular system to colonize the whole plant. Therefore, the fungus spends part of its lifetime in this nutrient-low and imbalanced environment where it is inaccessible for disease control treatments. This lifestyle as well requires the fungus to react to plant defense responses by secreting specific effector molecules to establish a successful infection. We addressed the differences in media-dependent secretion responses of Verticillium longisporum. We identified a broad response pattern induced by several media, and a similar response (but with some distinct differences) for the plant-related environments: the pectin-rich medium SXM and xylem sap from the host rapeseed. Importantly, we show that the necrosis and ethylene inducing-like proteins Nlp2 and Nlp3 are xylem sap-specific proteins that are required for full V. dahliae pathogenicity on tomato. These factors play a role during the colonization phase and represent potential targets for new control strategies for Verticillium wilt.

genetics↗

bioRxiv2020

Unfolded protein response and scaffold independent pheromone MAP kinase signalling control Verticillium dahliae growth, development and plant pathogenesis

O_LIDevelopment and virulence of the vascular plant pathogen Verticillium dahliae are connected and depend on a complex interplay between the unfolded protein response, a Ham5 independent pheromone MAP kinase module and formation of precursors for oxylipin signal molecules. C_LIO_LIGenes coding for the unfolded protein response regulator Hac1, the Ham5 MAPK scaffold protein, and the oleate {Delta}12-fatty acid desaturase Ode1 were deleted and their functions in growth, differentiation, and virulence on plants were studied using genetic, cell biology, and plant infection experiments. C_LIO_LIThe unfolded protein response transcription factor Hac1 is required for initial root colonization, fungal conidiation and propagation inside the host and is essential for resting structure formation. Microsclerotia development, growth and virulence require the pheromone response MAPK pathway, but without the Ham5 scaffold function. Single ER-associated enzymes for linoleic acid production make important contributions to fungal growth but have only a minor impact on the pathogenicity of V. dahliae. C_LIO_LIFungal growth, sporulation, dormant structure formation and plant infection require a network of the Hac1-regulated unfolded protein response, a scaffold-independent pheromone response MAPK pathway and formation of precursors for signalling. This network includes interesting targets for disease management of the vascular pathogen V. dahliae. C_LI

genetics↗