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.     

A novel gene, phcA from Pseudomonas syringae induces programmed cell death in the filamentous fungus Neurospora crassa

The phytopathogen Pseudomonas syringae competes with other epiphytic organisms, such as filamentous fungi, for resources. Here we characterize a gene in P. syringae pv. syringae B728a and P. syringae pv. tomato DC3000, termed phcA , that has homology to a filamentous fungal gene called het-c . phcA is conserved in many P. syringae strains, but is absent in one of the major clades, which includes the P. syringae pathovar phaseolicola. In the filamentous fungus Neurospora crassa , HET-C regulates a conserved programmed cell death pathway called heterokaryon incompatibility (HI). Ectopic expression of phcA in N. crassa induced HI and cell death that was dependent on the presence of a functional het-c pin-c haplotype. Further, by co-immunoprecipitation experiments, a heterocomplex between N. crassa HET-C1 and PhcA was associated with phcA- induced HI . P. syringae was able to attach and extensively colonize N. crassa hyphae, while an Escherichia coli control showed no association with the fungus. We further show that the P. syringae is able to use N. crassa as a sole nutrient source. Our results suggest that P. syringae has the potential to utilize phcA to acquire nutrients from fungi in nutrient-limited environments like the phyllosphere by the novel mechanism of HI induction.     

A eukaryotic gene encoding an endonuclease that specifically repairs DNA damaged by ultraviolet light.

Many eukaryotic organisms, including humans, remove ultraviolet (UV) damage from their genomes by the nucleotide excision repair pathway, which requires more than 10 separate protein factors. However, no nucleotide excision repair pathway has been found in the filamentous fungus Neurospora crassa. We have isolated a new eukaryotic DNA repair gene from N.crassa by its ability to complement UV-sensitive Escherichia coli cells. The gene is altered in a N.crassa mus-18 mutant and responsible for the exclusive sensitivity to UV of the mutant. Introduction of the wild-type mus-18 gene complements not only the mus-18 DNA repair defect of N.crassa, but also confers UV-resistance on various DNA repair-deficient mutants of Saccharomyces cerevisiae and a human xeroderma pigmentosum cell line. The cDNA encodes a protein of 74 kDa with no sequence similarity to other known repair enzymes. Recombinant mus-18 protein was purified from E.coli and found to be an endonuclease for UV-irradiated DNA. Both cyclobutane pyrimidine dimers and (6-4)photoproducts are cleaved at the sites immediately 5' to the damaged dipyrimidines in a magnesium-dependent, ATP-independent reaction. This mechanism, requiring a single polypeptide designated UV-induced dimer endonuclease for incision, is a substitute for the role of nucleotide excision repair of UV damage in N.crassa.     

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.     

A two-component histidine kinase of the rice blast fungus is involved in osmotic stress response and fungicide action

We isolated and characterized a histidine kinase gene (HIK1) from the rice blast fungus, Pyricularia oryzae (Magnaporthe grisea). The deduced amino acid sequence of HIK1 showed highest similarity (85.7%) to a hybrid-type histidine kinase, Os-1/Nik-1 of Neurospora crassa. Disruption of HIK1 caused no defect in cell growth on normal media and in pathogenicity to rice plants. The Deltahik1 strain acquired resistance to three groups of fungicides (phenylpyrroles, dicarboximides, and aromatic hydrocarbons) similar to os-1 mutants of N. crassa. The Deltahik1 strain showed increased sensitivity to high concentrations of sugars although its salt sensitivity was not elevated, suggesting that the rice blast fungus can distinguish osmostresses caused by high sugar concentrations and high salt concentrations. In contrast, os-1 mutants of N. crassa are sensitive to high concentrations of both salts and sugars. These findings suggest that P. oryzae and N. crassa partially differ in their os (osmosensitive) signal transduction pathway.     

Ultrastructural changes in Neurospora cells undergoing senescence induced by kalilo plasmids

In N. crassa and N. intermedia, the kalilo plasmid triggers senescence by insertion into mitochondrial DNA. To investigate the cell death pathway induced by this plasmid, juvenile and senescent subcultures of several senescent strains were examined by light and transmission electron microscopy, and at the DNA level. There were no signs of apoptotic events, such as shrinkage of the cytoplasm away from the cell wall, apoptotic bodies, internucleosomal DNA fragmentation or condensation of the cytoplasm while retaining mitochondria and endomembrane structure. Instead, swollen mitochondria lacking cristae and containing amorphous inclusions, and disruption of nuclear and mitochondrial membranes indicated a necrotic mode of cell death.     

Mutational analysis of the glycosylphosphatidylinositol (GPI) anchor pathway demonstrates that GPI-anchored proteins are required for cell wall biogenesis and normal hyphal growth in Neurospora crassa

Using mutational and proteomic approaches, we have demonstrated the importance of the glycosylphosphatidylinositol (GPI) anchor pathway for cell wall synthesis and integrity and for the overall morphology of the filamentous fungus Neurospora crassa. Mutants affected in the gpig-1, gpip-1, gpip-2, gpip-3, and gpit-1 genes, which encode components of the N. crassa GPI anchor biosynthetic pathway, have been characterized. GPI anchor mutants exhibit colonial morphologies, significantly reduced rates of growth, altered hyphal growth patterns, considerable cellular lysis, and an abnormal "cell-within-a-cell" phenotype. The mutants are deficient in the production of GPI-anchored proteins, verifying the requirement of each altered gene for the process of GPI-anchoring. The mutant cell walls are abnormally weak, contain reduced amounts of protein, and have an altered carbohydrate composition. The mutant cell walls lack a number of GPI-anchored proteins, putatively involved in cell wall biogenesis and remodeling. From these studies, we conclude that the GPI anchor pathway is critical for proper cell wall structure and function in N. crassa.     

WSC-1 and HAM-7 Are MAK-1 MAP Kinase Pathway Sensors Required for Cell Wall Integrity and Hyphal Fusion in Neurospora crassa

A large number of cell wall proteins are encoded in the Neurospora crassa genome. Strains carrying gene deletions of 65 predicted cell wall proteins were characterized. Deletion mutations in two of these genes (wsc-1 and ham-7) have easily identified morphological and inhibitor-based defects. Their phenotypic characterization indicates that HAM-7 and WSC-1 function during cell-to-cell hyphal fusion and in cell wall integrity maintenance, respectively. wsc-1 encodes a transmembrane protein with extensive homology to the yeast Wsc family of sensor proteins. In N. crassa, WSC-1 (and its homolog WSC-2) activates the cell wall integrity MAK-1 MAP kinase pathway. The GPI-anchored cell wall protein HAM-7 is required for cell-to-cell fusion and the sexual stages of the N. crassa life cycle. Like WSC-1, HAM-7 is required for activating MAK-1. A Δwsc-1;Δham-7 double mutant fully phenocopies mutants lacking components of the MAK-1 MAP kinase cascade. The data identify WSC-1 and HAM-7 as the major cell wall sensors that regulate two distinct MAK-1-dependent cellular activities, cell wall integrity and hyphal anastomosis, respectively.     

Isolation and characterization of non-neuronal enolase (NNE) from Neurospora crassa and comparison with neuron specific enolase isolated from neuroblastoma cell line NG108

Enolase is a vital enzyme of the glycolytic pathway. It exists mainly in two forms, non-neuronal enolase (NNE) and neuron specific enolase (NSE). Neurospora crassa, a filamentous fungus, was used as the source of pure NNE, and by using DEAE-cellulose and a Sephadex G-150 column chromatography highly purified enzyme (20.4 fold purification with 54.7 percent recovery) was obtained. The development profile of the enzyme shows a peak value after 90 hours of mycelial growth from conidia of N. crassa. In this respect, it differs from neuroblastoma NSE where the peak value of the enzyme activity appears 7 1/2 hours after the splitting of the cells. N. crassa enolase (NNE) is more thermolabile than NG108 NSE and N. crassa enolase is more sensitive to urea, chloride, and fluorophosphate. The Km values for 2-phosphoglycerate and Mg++ were 0.34 mM and 0.47 mM, respectively, for N. crassa enolase, whereas these values were 1.1 mM and 3.1 mM, respectively, in the case of neuroblastoma NSE. N. crassa enolase is a dimer molecule of molecular weight 85,000 daltons. N. crassa enolase is not neutralized by NSE antisera and neutralized by NNE antisera as opposed to neuroblastoma NSE.     

Neurospora Mutants Affecting Polyamine-Dependent Processes and Basic Amino Acid Transport Mutants Resistant to the Polyamine Inhibitor, α-Difluoromethylornithine

Polyamines (spermidine and spermine) are required by living cells, but their functions are poorly understood. Mutants of Neurospora crassa with enhanced or diminished sensitivity to interference with polyamine synthesis, originally selected to study the regulation of the pathway, were found to have unexpected defects. A group of four non-allelic mutations, causing no interference with polyamine synthesis, each imparted spermidine auxotrophy to a genotype already partially impaired in spermidine synthesis. Strains carrying only the new mutations displayed unconditional delay or weakness at the onset of growth, but grew well thereafter and had a normal or overly active polyamine pathway. These mutants may have defects in vital macromolecular activities that are especially dependent upon the polyamines-activities that have not been identified with certainty in studies to date. Another group of mutants, selected as resistant to the polyamine inhibitor difluoromethylornithine (DFMO), had normal activity and regulation of ornithine decarboxylase, the target of the drug. All but one of thirty mutants were allelic, and were specifically deficient in the basic amino acid permease. This mechanism of DFMO resistance is unprecedented among the many DFMO-resistant cell types of other organisms and demonstrates that DFMO can be used for efficient genetic studies of this transport locus in N. crassa.     

Genetic evidence for a regulatory pathway controlling alternative oxidase production in Neurospora crassa

When the cytochrome-mediated mitochondrial electron transport chain of Neurospora crassa is disrupted, an alternative oxidase encoded by the nuclear aod-1 gene is induced. The alternative oxidase donates electrons directly to oxygen from the ubiquininol pool and is insensitive to chemicals such as antimycin A and KCN that affect the standard electron transport chain. To facilitate isolation of mutants affecting regulation of aod-1, a reporter system containing the region upstream of the aod-1 coding sequence fused to the coding sequence of the N. crassa tyrosinase gene (T) was transformed into a strain carrying a null allele of the endogenous T gene. In the resulting reporter strain, growth in the presence of chloramphenicol, an inhibitor of mitochondrial translation whose action decreases the level of mitochondrial translation products resulting in impaired cytochrome-mediated respiration, caused induction of both alternative oxidase and tyrosinase. Conidia from the reporter strain were mutagenized, plated on medium containing chloramphenicol, and colonies that did not express tyrosinase were identified as potential regulatory mutants. After further characterization, 15 strains were found that were unable to induce both the reporter and the alternative oxidase. Complementation analysis revealed that four novel loci involved in aod-1 regulation had been isolated. The discovery that several genes are required for regulation of aod-1 suggests the existence of a complex pathway for signaling from the mitochondria to the nucleus and/or for expression of the gene.     

Functional analysis of the two zinc fingers of SRE, a GATA-type factor that negatively regulates siderophore synthesis in Neurospora crassa

Multiple GATA factors, zinc finger DNA binding proteins that recognize consensus GATA elements, exist in Neurospora crassa. One of them, SRE, is involved in controlling the iron metabolic pathway of N. crassa. In N. crassa, iron transport is mediated by a number of small cyclic peptides, known as siderophores. The siderophore synthesis pathway is negatively regulated by SRE; a loss-of-function sre mutant strain showed partial constitutive synthesis of siderophore. In the research presented here, the negative function of SRE was further confirmed by a heterokaryon test and by gene complementation. SRE was expressed as a GST fusion protein. In vitro EMSA revealed that SRE binds specifically to DNA molecules containing GATA sequence elements. Autoregulation of sre gene expression appears possible because the sre gene promoter itself contains GATA sequences. Mutations were introduced into sre that lead to amino acid substitutions in each of the zinc fingers that will disrupt their function. In vitro EMSA revealed that both N-terminal and C-terminal zinc fingers of SRE are involved in DNA binding. This feature is different from that found with the vertebrate two zinc finger GATA factors. Invivo tests, accomplished by transforming the mutant sre genes into sre rip mutant, showed that SRE with mutations in either or both zinc fingers still maintained its function under low-iron conditions. In contrast, these mutant SRE proteins fail to function under high-iron conditions. Our results predict the presence of other positive or negative regulators of the siderophore synthetic pathway.     

Insulin-induced stimulation of protein phosphorylation in Neurospora crassa cells.

1) Insulin stimulated the phosphorylation of at least 14 discrete proteins in Neurospora crassa cells. Specific proteins were phosphorylated at serine, threonine, and tyrosine residues, as determined by phosphoamino acid analysis of discrete spots on two-dimensional gels. 2) Insulin stimulated the phosphorylation by [gamma-32P]ATP of at least six discrete proteins in solubilized N. crassa membrane preparations at serine and tyrosine residues. 3) A phosphotyrosine-containing protein of 38 kDa, pI 7.0-7.2, reacted by both immunoblotting and immunoprecipitation with antiserum to P2, a peptide from the human insulin receptor that contains an autophosphorylated tyrosine residue. In N. crassa cells, therefore, as in mammalian cells, insulin induces a variety of protein phosphorylations, some of which may be part of an evolutionarily conserved signal transduction pathway.     

Inducible beta-oxidation pathway in Neurospora crassa.

An inducible beta-oxidation system was demonstrated in a particulate fraction from Neurospora crassa. The activities of all individual beta-oxidation enzymes were enhanced in cells after a shift from a sucrose to an acetate medium. The induction was even more pronounced in transfer to a medium containing oleate as sole carbon and energy source. Since an acyl-coenzyme A (CoA) dehydrogenase was detected instead of acyl-CoA oxidase, the former enzyme seems to catalyze the first step of the beta-oxidation sequence in N. crassa. After isopycnic centrifugation in a linear sucrose gradient, the intracellular organelles housing the fatty acid degradation pathway cosedimented (1.21 g/cm3) with the glyoxylate bypass enzymes isocitrate lyase and malate synthase and were clearly resolved from both mitochondrial marker enzymes (1.19 g/cm3) and catalase (1.26 g/cm3). On the basis of biochemical as well as morphological properties, these particles from N. crassa have recently been designated as glyoxysome-like particles (G. Wanner and T. Theimer, Ann. N.Y. Acad. Sci. 386:269-284, 1982). The failure to detect catalase, urate oxidase, and acyl-CoA oxidase indicate that these glyoxysome-like microbodies in N. crassa lack peroxisomal function and thus are clearly different from the various microbodies reported so far to contain a beta-oxidation pathway.     

Metabolism of mandelic acid by Neurospora crassa.

Preliminary studies on the metabolism of manelic acid by Neurospora crassa reveal the operation of a pathway for its degradation which involves benzoyl formic acid, benzaldehyde, benzoic acid, 4-hydroxybenzoic acid, and protocatechuic acid as the intermediates. This pathway is different from the followed by bacterial systems and is the same as that observed in Aspergillus niger.     

Ribosomal RNA genes of Neurospora crassa: multiple copies and specificities

Ribosomal RNA genes were isolated from the germinated conidial and mycelial cells of N. crassa by repeated cycles of 3H-DNA:rRNA reactions followed by hydroxyapatite chromatography. Specificity of multiple copies of those rDNAs with respect to N. crassa cell types was studied. The fraction of N. crassa germinated conidial in vitro labelled 3H-DNA recovered in the presence of rRNA isolated from the same cell type was about 2.2%, when compared with approximately 1.2% rDNAs obtained in mycelial cells. These isolated rDNAs reacted specifically to 26S and 17S rRNAs of eukaryotic (N. crassa) organisms and did not react with 4S tRNAs. rRNA:rDNA reassociation kinetics studies indicate that 90% of the rRNA genes were homogeneous and not identical with the other 10% rRNA genes isolated from N. crassa mycelia. These studies suggest that the possible heterogeneity of rDNA sequences of N. crassa cannot be attributed to inclusion of any tDNA sequences as has been shown in the heterogeneity of rDNA sequences of the bacterium Escherichia coli. The heterogeneity of multiple copies of N. crassa rDNAs could be due to differences in internal or external spacer regions of N. crassa rRNA genes.     

Highlights from this issue

Autophagy is acknowledged as an important cellular defense mechanism against intracellular pathogens. As with other innate immune responses, pathogens have adapted to evade autophagy and in some cases, subvert the pathway to promote their own replication. Poliovirus, a prototypical small positive-strand RNA virus that replicates and assembles in the cytoplasm of the host cell, utilizes membranes derived from the autophagic pathway to aid viral replication and egress from the cell. Recently we made the surprising discovery that GFP-LC3-staining vesicles are physically immobilized during poliovirus infection. Here we discuss our model for the mechanism of vesicle immobilization and the predictions it makes for pathogens that subvert the autophagic pathway to their own ends.     

Lysine catabolism in Rhizoctonia leguminicola and related fungi.

The catabolism of lysine was studied in several yeasts and fungi. Results with cell-free extracts of Rhizoctonia leguminicola support a proposed pathway involving (D- and L-) EPSILON-N-acetyllysine, alpha-keto-epsilon-acetamidohexanoic acid, delta-acetamidovaleric acid, and delta-aminovaleric acid in the conversion of L-lysine to shortchain organic acids. Label from radioactive L-lysine was found to accumulate in D- and L-epsilon-N-acetyllysine, delta-acetamidovaleric acid, delta-aminovaleric acid, and glutaric acid in cultures of R. leguminicola, Neurospora crassa, Saccharomyces cerevisiae, and Hansenula saturnus, suggesting that the proposed omega-acetyl pathway of lysine catabolism is generalized among yeasts and fungi. In N. crassa, as is the case in R. leguminicola, the major precursor of L-pipecolic acid was the L-isomer of lysine; 15N experiments were consistent with delta1-piperideine-2-carboxylic acid as an intermediate in the transformation.     

Establishment of the platform for reverse chemical genetics targeting novel protein-protein interactions

In the "drug discovery" era, protein-protein interaction modules are becoming the most exciting group of targets for study. Although combinatorial libraries and active natural products are rapidly and systemically being equipped by both for-profit and not-for-profit organizations, complete drug-screening systems have not been achieved. There is a growing need for the establishment of drug discovery assays for highly effective utilization of the collected small molecules on a large scale. To generate drug-screening systems, we plan to identify novel protein-protein interactions that may participate in human diseases. The interactions have been identified by MS/MS analysis following immunoprecipitation using antibodies prepared from our cDNA projects. The intracellular pathway involving the identified interaction is computationally constructed, which then clarifies its relationship to the candidate disease. The development of reverse chemical genetics based on such information should help us to realize a significant increment in the number of drug discovery assays available for use. In this article, I describe our strategy for drug discovery and then introduce the applicability of fluorescence intensity distribution analysis (FIDA) and the expression-ready constructs called "ORF trap clones" to reverse chemical genetics.     

Neurospora mrc1 homologue is involved in replication stability and is required for normal cell growth and chromosome integrity in mus-9 and mus-21 mutants

Stalled replication forks easily collapse and such structures can induce DNA strand breaks or toxic recombination products. Therefore, factors involved in stabilization of replication should be important for genome integrity. In our previous study, loss of both ATM and ATR homologues was shown to cause growth defects and chromosome instability in Neurospora crassa. To elucidate the relationships between these defects and replication instability, we focused on one of the viable replication factors, mrc1. We identified an mrc1 homologue from the N. crassa genome database. The mrc1 disruptant was sensitive to the replication inhibitor hydroxyurea (HU) and delayed restart of the cell cycle from HU treatment. Importantly, HU treatment induced histone H2A phosphorylation in the mrc1 mutant but not in the wild type. Furthermore, the HU-induced H2A phosphorylation was completely dependent on the ATM homologue mus-21, and dysfunction of mus-21 increased HU sensitivity of the mrc1 mutant. These results indicate that Neurospora mrc1 is important for stabilization of replication forks and that loss of mrc1 causes activation of the DNA damage checkpoint. Unexpectedly, loss of mrc1 did not affect cell growth, but the deletion of mrc1 reduced hyphal growth speed and conidia viability in the mus-9 and mus-21 mutants. The mrc1 mus-9 and mrc1 mus-21 double mutants also showed accumulation of micronuclei, which is a typical marker of chromosome instability. These results imply that activation of the checkpoint pathway can protect cells from instability of DNA replication caused by loss of mrc1.