André Fleißner

Architecture and development of the Neurospora crassa hypha -- a model cell for polarized growth.

Neurospora crassa has been at the forefront of biological research from the early days of biochemical genetics to current progress being made in understanding gene and genetic network function. Here, we discuss recent developments in analysis of the fundamental form of fungal growth, development and proliferation -- the hypha. Understanding the establishment and maintenance of polarity, hyphal elongation, septation, branching and differentiation are at the core of current research. The advances in the identification and functional dissection of regulatory as well as structural components of the hypha provide an expanding basis for elucidation of fundamental attributes of the fungal cell. The availability and continuous development of various molecular and microscopic tools, as utilized by an active and co-supportive research community, promises to yield additional important new discoveries on the biology of fungi.

The Saccharomyces cerevisiae PRM1 Homolog in Neurospora crassa Is Involved in Vegetative and Sexual Cell Fusion Events but Also Has Postfertilization Functions

Cell–cell fusion is essential for a variety of developmental steps in many eukaryotic organisms, during both fertilization and vegetative cell growth. Although the molecular mechanisms associated with intracellular membrane fusion are well characterized, the molecular mechanisms of plasma membrane merger between cells are poorly understood. In the filamentous fungus Neurospora crassa, cell fusion events occur during both vegetative and sexual stages of its life cycle, thus making it an attractive model for studying the molecular basis of cell fusion during vegetative growth vs. sexual reproduction. In the unicellular yeast Saccharomyces cerevisiae, one of the few proteins implicated in plasma membrane merger during mating is Prm1p; prm1Δ mutants show an ∼50% reduction in mating cell fusion. Here we report on the role of the PRM1 homolog in N. crassa. N. crassa strains with deletions of a Prm1-like gene (Prm1) showed an ∼50% reduction in both vegetative and sexual cell fusion events, suggesting that PRM1 is part of the general cell fusion machinery. However, unlike S. cerevisiae, N. crassa strains carrying a Prm1 deletion exhibited complete sterility as either a male or female mating partner, a phenotype that was not complemented in a heterokaryon with wild type (WT). Crosses with ΔPrm1 strains were blocked early in sexual development, well before development of ascogenous hyphae. The ΔPrm1 sexual defect in N. crassa was not suppressed by mutations in Sad-1, which is required for meiotic silencing of unpaired DNA (MSUD). However, mutations in Sad-1 increased the number of progeny obtained in crosses with a ΔPrm1 (Prm1-gfp) complemented strain. These data indicate multiple roles for PRM1 during sexual development.

HAM‐2 and HAM‐3 are central for the assembly of the Neurospora STRIPAK complex at the nuclear envelope and regulate nuclear accumulation of the MAP kinase MAK‐1 in a MAK‐2‐dependent manner

Intercellular communication and somatic cell fusion are important for fungal colony establishment, multicellular differentiation and have been associated with host colonization and virulence of pathogenic species. By a combination of genetic, biochemical and live cell imaging techniques, we characterized the Neurospora crassa STRIPAK complex that is essential for self‐signalling and consists of the six proteins HAM‐2/STRIP, HAM‐3/striatin, HAM‐4/SLMAP, MOB‐3/phocein, PPG‐1/PP2A‐C and PP2A‐A. We describe that the core STRIPAK components HAM‐2 and HAM‐3 are central for the assembly of the complex at the nuclear envelope, while the phosphatase PPG‐1 only transiently associates with this central subcomplex. Our data connect the STRIPAK complex with two MAP kinase pathways: (i) nuclear accumulation of the cell wall integrity MAP kinase MAK‐1 depends on the functional integrity of the STRIPAK complex at the nuclear envelope, and (ii) phosphorylation of MOB‐3 by the MAP kinase MAK‐2 impacts the nuclear accumulation of MAK‐1. In summary, these data support a model, in which MAK‐2‐dependent phosphorylation of MOB‐3 is part of a MAK‐1 import mechanism. Although self‐communication remained intact in the absence of nuclear MAK‐1 accumulation, supporting the presence of multiple mechanisms that co‐ordinate robust intercellular communication, proper fruiting body morphology was dependent on the MAK‐2‐phosphorylated N‐terminus of MOB‐3.

A Sensing Role of the Glutamine Synthetase in the Nitrogen Regulation Network in Fusarium fujikuroi.

In the plant pathogenic ascomycete Fusarium fujikuroi the synthesis of several economically important secondary metabolites (SM) depends on the nitrogen status of the cells. Of these SMs, gibberellin and bikaverin synthesis is subject to nitrogen catabolite repression (NCR) and is therefore only executed under nitrogen starvation conditions. How the signal of available nitrogen quantity and quality is sensed and transmitted to transcription factors is largely unknown. Earlier work revealed an essential regulatory role of the glutamine synthetase (GS) in the nitrogen regulation network and secondary metabolism as its deletion resulted in total loss of SM gene expression. Here we present extensive gene regulation studies of the wild type, the Δgln1 mutant and complementation strains of the gln1 deletion mutant expressing heterologous GS-encoding genes of prokaryotic and eukaryotic origin or 14 different F. fujikuroi gln1 copies with site-directed mutations. All strains were grown under different nitrogen conditions and characterized regarding growth, expression of NCR-responsive genes and biosynthesis of SM. We provide evidence for distinct roles of the GS in sensing and transducing the signals to NCR-responsive genes. Three site directed mutations partially restored secondary metabolism and GS-dependent gene expression, but not glutamine formation, demonstrating for the first time that the catalytic and regulatory roles of GS can be separated. The distinct mutant phenotypes show that the GS (1) participates in NH4 +-sensing and transducing the signal towards NCR-responsive transcription factors and their subsequent target genes; (2) affects carbon catabolism and (3) activates the expression of a distinct set of non-NCR GS-dependent genes. These novel insights into the regulatory role of the GS provide fascinating perspectives for elucidating regulatory roles of GS proteins of different organism in general.

The AngFus3 Mitogen-Activated Protein Kinase Controls Hyphal Differentiation and Secondary Metabolism in Aspergillus niger

ABSTRACT Adaptation to a changing environment is essential for the survival and propagation of sessile organisms, such as plants or fungi. Filamentous fungi commonly respond to a worsening of their growth conditions by differentiation of asexually or sexually produced spores. The formation of these specialized cell types is, however, also triggered as part of the general life cycle by hyphal age or density. Spores typically serve for dispersal and, therefore, translocation but can also act as resting states to endure times of scarcity. Eukaryotic differentiation in response to environmental and self-derived signals is commonly mediated by three-tiered mitogen-activated protein (MAP) kinase signaling cascades. Here, we report that the MAP kinase Fus3 of the black mold Aspergillus niger (AngFus3) and its upstream kinase AngSte7 control vegetative spore formation and secondary metabolism. Mutants lacking these kinases are defective in conidium induction in response to hyphal density but are fully competent in starvation-induced sporulation, indicating that conidiation in A. niger is triggered by various independent signals. In addition, the mutants exhibit an altered profile of volatile metabolites and secrete dark pigments into the growth medium, suggesting a dysregulation of the secondary metabolism. By assigning the AngFus3 MAP kinase pathway to the transduction of a potentially self-derived trigger, this work contributes to the unraveling of the intricate signaling networks controlling fungal differentiation. Moreover, our data further support earlier observations that differentiation and secondary metabolism are tightly linked in filamentous fungi.

Molecular Mechanisms Regulating Cell Fusion and Heterokaryon Formation in Filamentous Fungi

For the majority of fungal species, the somatic body of an individual is a network of interconnected cells sharing a common cytoplasm and organelles. This syncytial organization contributes to an efficient distribution of resources, energy, and biochemical signals. Cell fusion is a fundamental process for fungal development, colony establishment, and habitat exploitation and can occur between hyphal cells of an individual colony or between colonies of genetically distinct individuals. One outcome of cell fusion is the establishment of a stable heterokaryon, culminating in benefits for each individual via shared resources or being of critical importance for the sexual or parasexual cycle of many fungal species. However, a second outcome of cell fusion between genetically distinct strains is formation of unstable heterokaryons and the induction of a programmed cell death reaction in the heterokaryotic cells. This reaction of nonself rejection, which is termed heterokaryon (or vegetative) incompatibility, is widespread in the fungal kingdom and acts as a defense mechanism against genome exploitation and mycoparasitism. Here, we review the currently identified molecular players involved in the process of somatic cell fusion and its regulation in filamentous fungi. Thereafter, we summarize the knowledge of the molecular determinants and mechanism of heterokaryon incompatibility and place this phenomenon in the broader context of biotropic interactions and immunity.

Accumulation of specific sterol precursors targets a MAP kinase cascade mediating cell-cell recognition and fusion

Significance Deficiencies in sterol biosynthesis resulting in the accumulation of precursor sterol molecules are commonly associated with cellular malfunctioning and disease, including neurodegenerative and inflammatory disorders. However, the molecular and cellular consequences of the aberrant accumulation of sterol precursors are not understood. In particular, it is unclear whether specific biochemical or signaling pathways are targeted by the precursors and to what extent their specific structures contribute to their disruptive effects. Here we show that the accumulation of ergosterol precursors specifically targets a conserved ERK MAP kinase pathway that mediates fungal cell–cell communication and fusion. This effect is only caused by precursors with a conjugated double bond in their aliphatic side chain, indicating specific structure–function relationships in the mechanism of action. Sterols are vital components of eukaryotic cell membranes. Defects in sterol biosynthesis, which result in the accumulation of precursor molecules, are commonly associated with cellular disorders and disease. However, the effects of these sterol precursors on the metabolism, signaling, and behavior of cells are only poorly understood. In this study, we show that the accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain specifically disrupts cell–cell communication and fusion in the fungus Neurospora crassa. Genetically identical germinating spores of this fungus undergo cell–cell fusion, thereby forming a highly interconnected supracellular network during colony initiation. Before fusion, the cells use an unusual signaling mechanism that involves the coordinated and alternating switching between signal sending and receiving states of the two fusion partners. Accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain disrupts this coordinated cell–cell communication and suppresses cell fusion. These specific sterol precursors target a single ERK-like mitogen-activated protein (MAP) kinase (MAK-1)-signaling cascade, whereas a second MAP kinase pathway (MAK-2), which is also involved in cell fusion, is unaffected. These observations indicate that a minor specific change in sterol structure can exert a strong detrimental effect on a key signaling pathway of the cell, resulting in the absence of cell fusion.

Anastomosis and Heterokaryon Formation

Colonies of filamentous fungi comprise of a network of syncytic, multinucleate hyphae. Establishment and growth of the mycelial colony commonly involve fusion of specialized hyphal structures, a process termed anastomosis formation. This unusual biological phenomenon has long attracted the attention of basic and applied researchers. It provides basic experimental research with an easily amenable model for various cell biological questions, including cell-cell communication, directed growth, or plasma membrane merger. In addition, anastomosis formation and subsequent heterokaryon formation have long been used as research tools for genetically manipulating fungi. Examples include genetic mapping in asexual fungi or the use of heterokaryotic mycelia in biotechnological applications. Recent years have seen a great revival of interest in the process of hyphal fusion, cummulating in the conclusion that we are currently still far from fully understanding the biology or appreciating the rich applicable potential of this fascinating process.

7 The Art of Networking: Vegetative Hyphal Fusion in Filamentous Ascomycete Fungi

Hyphal fusion is a common feature of the growth and development of filamentous ascomycete fungi. It occurs at various developmental stages, most prominently during colony establishment by germinating spores and during the formation of cross connections within mature mycelial colonies. Recent years have seen great advances in understanding the biological roles and the molecular mechanisms of this fascinating biological process. It has become apparent that hyphal fusion promotes the formation of the mycelial network, thereby increasing fitness and competitiveness of the fungal colony. On the molecular level, an intricate signaling network controlling communication, attraction, and merger of fusing hyphae has been identified. This network comprises many well-conserved factors, including MAP kinases, reactive oxygen-generating systems, Ca2+-binding regulators, the STRIPAK complex, and cell polarity factors, which are partially adopted in novel and surprising ways. Studying the role and function of hyphal fusion therefore holds much potential to further our understanding not only of fungal growth and development but also of general eukaryotic cell biology.

Spatio-temporal MAPK dynamics mediate cell behavior coordination during fungal somatic cell fusion

ABSTRACT Mitogen-activated protein kinases (MAPKs) are conserved regulators of proliferation, differentiation and adaptation in eukaryotic cells. Their activity often involves changes in their subcellular localization, indicating an important role for these spatio-temporal dynamics in signal transmission. A striking model illustrating these dynamics is somatic cell fusion in Neurospora crassa. Germinating spores of this fungus rapidly alternate between signal sending and receiving, thereby establishing a cell-cell dialog, which involves the alternating membrane recruitment of the MAPK MAK-2 in both fusion partners. Here, we show that the dynamic translocation of MAK-2 is essential for coordinating the behavior of the fusion partners before physical contact. The activation and function of the kinase strongly correlate with its subcellular localization, indicating a crucial contribution of the MAPK dynamics in establishing regulatory feedback loops, which establish the oscillatory signaling mode. In addition, we provide evidence that MAK-2 not only contributes to cell-cell communication, but also mediates cell-cell fusion. The MAK-2 dynamics significantly differ between these two processes, suggesting a role for the MAPK in switching of the cellular program between communication and fusion. Summary: Spatio-temporal dynamics of MAPK mediate the interaction of fusing Neurospora crassa cells, which communicate by taking turns in signal sending and receiving.