Plasma Membrane Fusion Is Specifically Impacted by the Molecular Structure of Membrane Sterols During Vegetative Development of Neurospora crassa.

Cell-to-cell fusion is crucial for the development and propagation of most eukaryotic organisms. Despite this importance, the molecular mechanisms mediating this process are only poorly understood in biological systems. In particular, the step of plasma membrane merger and the contributing proteins and physicochemical factors remain mostly unknown. Earlier studies provided the first evidence of a role of membrane sterols in cell-to-cell fusion. By characterizing different ergosterol biosynthesis mutants of the fungus Neurospora crassa, which accumulate different ergosterol precursors, we show that the structure of the sterol ring system specifically affects plasma membrane merger during the fusion of vegetative spore germlings. Genetic analyses pinpoint this defect to an event prior to engagement of the fusion machinery. Strikingly, this effect is not observed during sexual fusion, suggesting that the specific sterol precursors do not generally block membrane merger, but rather impair subcellular processes exclusively mediating fusion of vegetative cells. At a colony-wide level, the altered structure of the sterol ring system affects a subset of differentiation processes, including vegetative sporulation and steps before and after fertilization during sexual propagation. Together, these observations corroborate the notion that the accumulation of particular sterol precursors has very specific effects on defined cellular processes rather than nonspecifically disturbing membrane functioning. Given the phenotypic similarities of the ergosterol biosynthesis mutants of N. crassa during vegetative fusion and of Saccharomyces cerevisiae cells undergoing mating, our data support the idea that yeast mating is evolutionarily and mechanistically more closely related to vegetative than sexual fusion of filamentous fungi.

Evidence of repeated horizontal transfer of sterol C-5 desaturase encoding genes among dikarya fungi.

Gene duplication and horizontal gene transfer (HGT) are two important but different forces for adaptive genome evolution. In eukaryotic organisms, gene duplication is considered to play a more important evolutionary role than HGT. However, certain fungal lineages have developed highly efficient mechanisms that avoid the occurrence of duplicated gene sequences within their genomes. While these mechanisms likely originated as a defense against harmful mobile genetic elements, they come with an evolutionary cost. A prominent example for a genome defense system is the RIP mechanism of the ascomycete fungus Neurospora crassa, which efficiently prevents sequence duplication within the genome and functional redundancy of the subsequent paralogs. Despite this tight control, the fungus possesses two functionally redundant sterol C-5 desaturase enzymes, ERG-10a and ERG-10b, that catalyze the same step during ergosterol biosynthesis. In this study, we addressed this conundrum by phylogenetic analysis of the two proteins and supporting topology tests. We obtained evidence that a primary HGT of a sterol C-5 desaturase gene from Tremellales (an order of Basidiomycota) into a representative of the Pezizomycotina (a subphylum of Ascomycota) is the origin of the ERG-10b sequence. The reconstructed phylogenies suggest that this HGT event was followed by multiple HGT events among other members of the Pezizomycotina, thereby generating a diverse group with members in the four classes Sordariomycetes, Xylonomycetes, Eurotiomycetes and Dothideomycetes, which all harbor the second sterol C-5 desaturase or maintained in some cases only the ERG-10b version of this enzyme. These results furnish an example for a gene present in numerous ascomycetous fungi but primarily acquired by an ancestral HGT event from another fungal phylum. Furthermore, these data indicate that HGT represents one mechanism to generate functional redundancy in organisms with a strict avoidance of gene duplications.

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.

The dynamics of signal complex formation mediating germling fusion in Neurospora crassa.

Colony initiation of filamentous fungi commonly involves fusion of germinating vegetative spores. Studies in Neurospora crassa revealed an unusual cell-cell communication mechanism mediating this process, in which the fusion partners coordinately alternate between two physiological stages, probably related to signal sending and receiving. This "cell dialog" involves the alternating, oscillatory recruitment of the SO protein and the MAK-2 MAP kinase module to the apical plasma membrane of growing fusion tips. In this review video article, we show the dynamics of the fluorescent labeled proteins SO and MAK-2 and provide an animated graphical model of the "cell dialog" process.

Signal exchange and integration during self-fusion in filamentous fungi.

Growth and propagation of filamentous ascomycete fungi commonly involves vegetative cell fusion. In the red bread mold Neurospora crassa and many other ascomycete species, fusion occurs between germinating spores during colony formation and between hyphal branches in established mycelia. Both fusion processes promote the development and behavior of the fungal colony as a supra-cellular network. Germling and hyphal fusion in N. crassa rely on an unusual mode of cellular communication, in which the two fusion partners likely alternate between signal emission and reception, thereby establishing a kind of "cell dialog". In recent years, numerous molecular factors mediating this unique cellular behavior have been identified, including several conserved signal transmission pathways, as well as proteins specific for ascomycete fungi. Analysis of their molecular interactions revealed the presence of an intricate signaling network, whose sophisticated interconnections are still unfolding. Despite this complexity, germling and hyphal fusion provide experimentally easily amenable model systems and might therefore advance as paradigms for signal transmission and cell fusion. In this article, we strive to highlight some of the recent advances in this field of research and to discuss the current working model of the "cell dialog".

Cell fusion in Neurospora crassa.

In recent years, the filamentous fungus Neurospora crassa has advanced as a model organism for studying eukaryotic cell-cell communication and fusion. Cell merger in this fungus employs an unusual mode of communication, in which the fusion partners appear to switch between signal sending and receiving. Many molecular factors mediating this intriguing mechanism and the subsequent membrane merger have been identified. It has become apparent that conserved factors, such as MAP kinases, NADPH oxidases and the STRIPAK complex, together with fungal specific proteins are wired into an intricate signaling network. Here, we will present an overview of recent findings on the molecular mechanism mediating fusion in N. crassa and will discuss the current working model.

Biochemical characterization of molybdenum cofactor-free nitrate reductase from Neurospora crassa.

Nitrate reductase (NR) is a complex molybdenum cofactor (Moco)-dependent homodimeric metalloenzyme that is vitally important for autotrophic organism as it catalyzes the first and rate-limiting step of nitrate assimilation. Beside Moco, eukaryotic NR also binds FAD and heme as additional redox active cofactors, and these are involved in electron transfer from NAD(P)H to the enzyme molybdenum center where reduction of nitrate to nitrite takes place. We report the first biochemical characterization of a Moco-free eukaryotic NR from the fungus Neurospora crassa, documenting that Moco is necessary and sufficient to induce dimer formation. The molybdenum center of NR reconstituted in vitro from apo-NR and Moco showed an EPR spectrum identical to holo-NR. Analysis of mutants unable to bind heme or FAD revealed that insertion of Moco into NR occurs independent from the insertion of any other NR redox cofactor. Furthermore, we showed that at least in vitro the active site formation of NR is an autonomous process.