Importance of MAP Kinases during Protoperithecial Morphogenesis in Neurospora crassa

In order to produce multicellular structures filamentous fungi combine various morphogenetic programs that are fundamentally different from those used by plants and animals. The perithecium, the female sexual fruitbody of Neurospora crassa, differentiates from the vegetative mycelium in distinct morphological stages, and represents one of the more complex multicellular structures produced by fungi. In this study we defined the stages of protoperithecial morphogenesis in the N. crassa wild type in greater detail than has previously been described; compared protoperithecial morphogenesis in gene-deletion mutants of all nine mitogen-activated protein (MAP) kinases conserved in N. crassa; confirmed that all three MAP kinase cascades are required for sexual development; and showed that the three different cascades each have distinctly different functions during this process. However, only MAP kinases equivalent to the budding yeast pheromone response and cell wall integrity pathways, but not the osmoregulatory pathway, were essential for vegetative cell fusion. Evidence was obtained for MAP kinase signaling cascades performing roles in extracellular matrix deposition, hyphal adhesion, and envelopment during the construction of fertilizable protoperithecia.     

Probing the growth dynamics of Neurospora crassa with microfluidic structures

Despite occupying physically and chemically heterogeneous natural environments, the growth dynamics of filamentous fungi is typically studied on the surface of homogeneous laboratory media. Fungal exploration and exploitation of complex natural environments requires optimal survival and growth strategies at the colony, hyphal, and intra hyphal level, with hyphal space-searching strategies playing a central role. We describe a new methodology for the characterisation and analysis of hyphal space-searching strategies, which uses purposefully designed three-dimensional microfluidics structures mimicking some of the characteristics of natural environments of the fungi. We also demonstrate this new methodology by running a comparative examination of two Neurospora crassa strains, i.e., the wild type of N. crassa -- a commonly used model organism for the study of filamentous fungi -- and the N. crassa ro-1 mutant strain -- which is deficient in hyphal and mycelial growth. Continuous live imaging showed that both strains responded actively to the geometrically confined microstructured environments without any detectable temporal delay or spatial adjustment. While both strains navigated the test structures exhibiting similar geometry-induced space-searching mechanisms, they presented fundamentally different growth patterns that could not be observed on geometrically unconfined, flat agar surfaces.     

A polypeptide of 59 kDa is associated with bundles of cytoplasmic filaments in Neurospora crassa.

Complex arrangements of filamentous structures have been isolated from vegetative cells of the fungus Neurospora crassa. They were enriched by differential centrifugation and purified by permeation chromatography. The filamentous structures are made up of units of 8-10 nm diameter and were isolated in bundles of up to six to nine units. The main constituent of these structures is a polypeptide with an apparent molecular mass of 59 kDa (P59Nc), which represents 4-5% of the total N. crassa proteins. The filamentous structures are cold-stable and are not affected by high-ionic-strength solutions or by the presence of 10 mM-EDTA or 1% (w/v) Triton X-100; they were disassembled by raising the pH of the solution or by using Tris-based buffers. The disassembled form assembled into structures sedimentable at 105,000 g after dialysis against the isolation buffer. The sedimentable structures were organized in the form of regular aggregates of 42-45 nm polypeptides and reacted weakly with anti-IFA, a monoclonal antibody which recognizes an epitope common to many of the higher-eukaryote intermediate-filament polypeptides. Immunofluorescence examination of wall-digested hyphae of N. crassa using affinity-purified antibodies prepared against P59Nc showed immunostaining of abundant filamentous and dot-shaped structures distributed in the cytoplasm.     

The ham-2 locus, encoding a putative transmembrane protein, is required for hyphal fusion in Neurospora crassa.

Somatic cell fusion is common during organogenesis in multicellular eukaryotes, although the molecular mechanism of cell fusion is poorly understood. In filamentous fungi, somatic cell fusion occurs during vegetative growth. Filamentous fungi grow as multinucleate hyphal tubes that undergo frequent hyphal fusion (anastomosis) during colony expansion, resulting in the formation of a hyphal network. The molecular mechanism of the hyphal fusion process and the role of networked hyphae in the growth and development of these organisms are unexplored questions. We use the filamentous fungus Neurospora crassa as a model to study the molecular mechanism of hyphal fusion. In this study, we identified a deletion mutant that was restricted in its ability to undergo both self-hyphal fusion and fusion with a different individual to form a heterokaryon. This deletion mutant displayed pleiotropic defects, including shortened aerial hyphae, altered conidiation pattern, female sterility, slow growth rate, lack of hyphal fusion, and suppression of vegetative incompatibility. Complementation with a single open reading frame (ORF) within the deletion region in this mutant restored near wild-type growth rates, female fertility, aerial hyphae formation, and hyphal fusion, but not vegetative incompatibility and wild-type conidiation pattern. This ORF, which we named ham-2 (for hyphal anastomosis), encodes a putative transmembrane protein that is highly conserved, but of unknown function among eukaryotes.     

Analysis of three separate probes suggests the absence of endocytosis in Neurospora crassa hyphae

Reports of the existence of endocytosis in filamentous fungi have been conflicting and inconclusive. For this reason, we have tested three independent markers in Neurospora crassa: the electron opaque marker lanthanum (La) and the fluorescent probes Lucifer yellow (LY) and FM4-64. Both La and LY were endocytosed by Saccharomyces cerevisiae cells, which were used as positive controls for endocytosis, but the probes did not accumulate in N. crassa hyphae. Only FM4-64 became internalized into N. crassa hyphae, but it induced abnormal changes in membrane systems and its internalization could be explained by mechanisms other than endocytosis. Together, our results suggest that endocytosis does not occur in N. crassa hyphae and question whether the styryl dyes do in fact reliably report normal endocytosis in filamentous fungi.     

Neurospora crassa ve-1 affects asexual conidiation

The velvet factor of the homothallic fungus Aspergillus nidulans promotes sexual fruiting body formation. The encoding veA gene is conserved among fungi, including the ascomycete Neurospora crassa. There, the orthologous ve-1 gene encodes a deduced protein with high similarity to A. nidulans VeA. Cross-complementation experiments suggest that both the promoter and the coding sequence of N. crassa ve-1 are functional to complement the phenotype of an A. nidulans deletion mutant. Moreover, ve-1 expression in the heterologous host A. nidulans results in development of reproductive structures in a light-dependent manner, promoting sexual development in the darkness while stimulating asexual sporulation under illumination. Deletion of the N. crassa ve-1 locus by homologous gene replacement causes formation of shortened aerial hyphae accompanied by a significant increase in asexual conidiation, which is not light-dependent. Our data suggest that the conserved velvet proteins of A. nidulans and N. crassa exhibit both similar and different functions to influence development of these two ascomycetes.     

The so locus is required for vegetative cell fusion and postfertilization events in Neurospora crassa

The process of cell fusion is a basic developmental feature found in most eukaryotic organisms. In filamentous fungi, cell fusion events play an important role during both vegetative growth and sexual reproduction. We employ the model organism Neurospora crassa to dissect the mechanisms of cell fusion and cell-cell communication involved in fusion processes. In this study, we characterized a mutant with a mutation in the gene so, which exhibits defects in cell fusion. The so mutant has a pleiotropic phenotype, including shortened aerial hyphae, an altered conidiation pattern, and female sterility. Using light microscopy and heterokaryon tests, the so mutant was shown to possess defects in germling and hyphal fusion. Although so produces conidial anastomosis tubes, so germlings did not home toward wild-type germlings nor were wild-type germlings attracted to so germlings. We employed a trichogyne attraction and fusion assay to determine whether the female sterility of the so mutant is caused by impaired communication or fusion failure between mating partners. so showed no defects in attraction or fusion between mating partners, indicating that so is specific for vegetative hyphal fusion and/or associated communication events. The so gene encodes a protein of unknown function, but which contains a WW domain; WW domains are predicted to be involved in protein-protein interactions. Database searches showed that so was conserved in the genomes of filamentous ascomycete fungi but was absent in ascomycete yeast and basidiomycete species.     

The nuclear Dbf2-related kinase COT1 and the mitogen-activated protein kinases MAK1 and MAK2 genetically interact to regulate filamentous growth, hyphal fusion and sexual development in Neurospora crassa

Ndr kinases, such as Neurospora crassa COT1, are important for cell differentiation and polar morphogenesis, yet their input signals as well as their integration into a cellular signaling context are still elusive. Here, we identify the cot-1 suppressor gul-4 as mak-2 and show that mutants of the gul-4/mak-2 mitogen-activated protein (MAP) kinase pathway suppress cot-1 phenotypes along with a concomitant reduction in protein kinase A (PKA) activity. Furthermore, mak-2 pathway defects are partially overcome in a cot-1 background and are associated with increased MAK1 MAPK signaling. A comparative characterization of N. crassa MAPKs revealed that they act as three distinct modules during vegetative growth and asexual development. In addition, common functions of MAK1 and MAK2 signaling during maintenance of cell-wall integrity distinguished the two ERK-type pathways from the p38-type OS2 osmosensing pathway. In contrast to separate functions during vegetative growth, the concerted activity of the three MAPK pathways is essential for cell fusion and for the subsequent formation of multicellular structures that are required for sexual development. Taken together, our data indicate a functional link between COT1 and MAPK signaling in regulating filamentous growth, hyphal fusion, and sexual development.     

Characterization of mat A-2, mat A-3 and deltamatA mating-type mutants of Neurospora crassa.

The mating-type locus of Neurospora crassa regulates mating identity and entry into the sexual cycle. The mat A idiomorph encodes three genes, mat A-1, mat A-2, and mat A-3. Mutations in mat A-1 result in strains that have lost mating identity and vegetative incompatibility with mat a strains. A strain containing mutations in both mat A-2 and mat A-3 is able to mate, but forms few ascospores. In this study, we describe the isolation and characterization of a mutant deleted for mat (deltamatA), as well as mutants in either mat A-2 or mat A-3. The deltamatA strain is morphologically wild type during vegetative growth, but it is sterile and heterokaryon compatible with both mat A and mat a strains. The mat A-2 and mat A-3 mutants are also normal during vegetative growth, mate as a mat A strain, and produce abundant biparental asci in crosses with mat a, and are thus indistinguishable from a wild-type mat A strain. These data and the fact that the mat A-2 mat A-3 double mutant makes few asci with ascospores indicate that MAT A-2 and MAT A-3 are redundant and may function in the same pathway. Analysis of the expression of two genes (sdv-1 and sdv-4) in the various mat mutants suggests that the mat A polypeptides function in concert to regulate the expression of some sexual development genes.     

A Putative Rhamnogalacturonase Required for Sexual Development of Neurospora Crassa

In previous work, the asd-1 (ascus development) gene of the filamentous fungus Neurospora crassa was identified as a gene expressed preferentially during the sexual cycle and shown to be essential for normal sexual development. The asd-1 gene has been sequenced and further characterized. It contains two introns, the first of which is in-frame and inefficiently or differentially spliced. The predicted ASD-1 protein has extensive homology with rhamnogalacturonase B of Aspergillus aculeatus, which cleaves the backbone within the ramified hairy regions of pectin. In homozygous asd-1 crosses, sexual development is initiated and large numbers of normal-sized asci are formed. Ascospore delineation does not occur, however, and no sexual progeny are produced. As most asd-1 asci contain eight nuclei, the two meiotic divisions and subsequent mitotic division typical of normal crosses seem to occur, but the haploid nuclei are not partitioned into ascospores. In wild-type crosses, the ASD-1 protein is present in large amounts in croziers and young asci, but it is only faintly detectable in more mature asci containing developing ascospores. Models to explain the possible role of a rhamnogalacturonase in sexual development are presented.     

Control of mating type heterokaryon incompatibility by the tol gene in Neurospora crassa and N. tetrasperma.

The mating-type of Neurospora crassa (A and a) have a dual function: A and a individuals are required for sexual reproduction, but only strains of the same mating type will form a stable vegetative heterokaryon. Neurospora tetrasperma, in contrast, is a naturally occurring A+a heterokaryon. It was shown previously that the mating-type genes of both species are functionally the same and are not responsible for this difference in heterokaryon incompatibility. This suggests that a separate genetic system determines the heterokaryon incompatibility function of mating type. The mutant tolerant (tol) in N. crassa, unlinked to mating type, acts as a specific suppressor of A+a heterokaryon incompatibility. In the present study, the wild-type alleles at the tol locus were introgressed reciprocally, from N. crassa into N. tetrasperma and from N. tetrasperma into N. crassa, to investigate the action of these alleles in the A+a heterokaryon incompatibility systems of these species. The wild-type allele from N. tetrasperma (tolT) acts as a recessive suppressor of A+a heterokaryon incompatibility in N. crassa. Furthermore, the wild-type allele from N. crassa (tolC) causes A and a to become heterokaryon incompatible in N. tetrasperma, while having no effect on the sexual reproduction. Therefore, the tol gene plays a major role in determining the heterokaryon compatibility of mating type in these species: tolC is an active allele that causes incompatibility and tolT an inactive allele that suppresses incompatibility by its inactivity.     

Localization of RHO-4 indicates differential regulation of conidial versus vegetative septation in the filamentous fungus Neurospora crassa

Rho-4 mutants of the filamentous fungus Neurospora crassa lack septa and asexual spores (conidia) and grow slowly. In this report, localization of green fluorescent protein-tagged RHO-4 is used to elucidate the differences in factors controlling RHO-4 localization during vegetative growth versus asexual development. RHO-4 forms a ring at incipient vegetative septation sites that constricts with the formation of the septum toward the septal pore; RHO-4 persists around the septal pore after septum completion. During the formation of conidia, RHO-4 localizes to the primary septum but subsequently is relocalized to the cytoplasm after the placement of the secondary septum. Cytoplasmic localization and inactivation of RHO-4 are mediated by a direct physical interaction with RDI-1, a RHO guanosine nucleotide dissociation inhibitor. Inappropriate activation of the cyclic AMP-dependent protein kinase A pathway during vegetative growth causes mislocalization of RHO-4 away from septa to the cytoplasm, a process which was dependent upon RDI-1. An adenylate cyclase cr-1 mutant partially suppresses the aconidial defect of rho-4 mutants but only rarely suppresses the vegetative septation defect, indicating that conidial septation is negatively regulated by CR-1. These data highlight the differences in the regulation of septation during conidiation versus vegetative septation in filamentous fungi.