Changes in glucosamine and galactosamine levels during conidial germination in Neurospora crassa.

The levels of glucosamine and galactosamine were determined in conidia, germinating conidia, and vegetative mycelia of Neurospora crassa. In the vegetative mycelia about 90% of the amino sugars were shown to be components of the cell wall. The remaining 10% of the amino sugars were tentatively identified as the nucleotide sugars uridine diphospho-2-acetamido-2-deoxy-D-glucose and uridine diphospho-2-acetamido-2-deoxy-D-galactose. Conidia and vegetative mycelia contained about the same levels of glucosamine. During the first 9 h after the initiation of germination, the total glucosamine content had increased 3.1-fold, whereas the residual dry weight of the culture had increased 7.7-fold. This led to a drop in the glucosamine concentration from 100 mumol/g of residual dry weight to 42 mumol/g. During this time, all of the conidia had germinated and the surface area of the new germ tubes had increased to 10 times that of the conidia. Either germ tubes were initially produced without glucosamine-containing polymers, or these polymers (probably chitin) were deposited only at low densities in the germ tube cell walls. The chitin precursor uridine diphospho-2-acetamido-2-deoxy-D-glucose was present at all times during conidial germination. Conida contained very low levels of galactosamine. During germination, galactosamine could not be detected until the culture had reached a cell density of about 0.6 mg of residual dry weight per ml of growth medium. This was observed regardless of the time required to reach this cell density or the fold increase in dry weight. The accumulation of galactosamine-containing polymers does not appear to be necessary for germ tube formation. The levels of soluble galactosamine (uridine diphospho-2-actamido-2-deoxy-D-galatose) were very low in conidia and increased during germination at the same time that galactosamine appeared in the cellular polymers. In addition, under certain culture conditions, the appearance of galactosamine and the increase in the glucosamine concentration occurred simultaneously.     

Loss of NAD(P)-reducing power and glutathione disulfide excretion at the start of induction of aerial growth in Neurospora crassa.

When exponentially growing hyphae of Neurospora crassa in aerated liquid cultures are filtered and the resulting mycelial mat is exposed to air, aerial hyphae develop and synchronous conidiation is obtained. The hyphae in direct contact with air adhere to each other within minutes and form aerial hyphae during the following 12 h; the hyphae which are not in direct contact with air do not adhere to each other and do not form aerial hyphae. Previous data indicated that oxidative stress was generated in the adhering hyphae; proteins and specific enzymes were found to be oxidatively modified and degraded. In this work, we report a dramatic fall in the reduced-to-oxidized ratio of NAD and NADP coenzymes during the first 6 min of exposure to air. This drop did not occur in a mycelial mat exposed to a N2-enriched atmosphere. Adding a carbon source to the mycelial mat did not abolish the loss of NAD(P)-reducing power. After the initial fall, the reducing levels of the coenzymes returned to the starting value in about 30 min. A peak of extracellular glutathione disulfide occurred simultaneously with the loss of NAD(P)-reducing power. The reducing power loss and the excretion of glutathione disulfide are thought to be consequences of a hyperoxidant state; the adhesion of hyphae is thought to be a response to the hyperoxidant state.     

Glutamine requirement for aerial mycelium growth in Neurospora crassa

Five amino acids are accumulated during vegetative growth of Neurospora crassa, particularly.during the prestationary growth phase. Alanine, glutamine, glutamate, arginine and ornithine.comprised over 80% of the total amino acid pool in the mycelium. Amino acid pools of different amino acid auxotrophs were followed during the partial transformation of a mycelial mat into an aerial mycelium. The mycelial mat under starvation and in direct contact with air rapidly formed aerial mycelium, which produced thereafter a burst of conidia. During this process,glutamine and alanine in the mycelial mat were consumed more rapidly than other amino acids;in the growing aerial mycelium, glutamate and glutamine were particularly accumulated. Of the amino acids that were initially accumulated in the mycelial mat, only a high glutamine pool was required for aerial mycelium growth induced by starvation. This requirement for glutamine could not be satisfied by a mixture of the amino compounds that are synthesized via glutamine amidotransferase reactions. It is proposed that glutamine serves as a nitrogen carrier from the mycelial mat to the growing aerial mycelium.     

Use of 1H nuclear magnetic resonance to measure intracellular metabolite levels during growth and asexual sporulation in Neurospora crassa

Conidiation is an asexual sporulation pathway that is a response to adverse conditions and is the main mode of dispersal utilized by filamentous fungal pathogens for reestablishment in a more favorable environment. Heterotrimeric G proteins (consisting of α, β, and γ subunits) have been shown to regulate conidiation in diverse fungi. Previous work has demonstrated that all three of the Gα subunits in the filamentous fungus Neurospora crassa affect the accumulation of mass on poor carbon sources and that loss of gna-3 leads to the most dramatic effects on conidiation. In this study, we used (1)H nuclear magnetic resonance (NMR) to profile the metabolome of N. crassa in extracts isolated from vegetative hyphae and conidia from cultures grown under conditions of high or low sucrose. We compared wild-type and Δgna-3 strains to determine whether lack of gna-3 causes a significant difference in the global metabolite profile. The results demonstrate that the global metabolome of wild-type hyphae is influenced by carbon availability. The metabolome of the Δgna-3 strain cultured on both high and low sucrose is similar to that of the wild type grown on high sucrose, suggesting an overall defect in nutrient sensing in the mutant. However, analysis of individual metabolites revealed differences in wild-type and Δgna-3 strains cultured under conditions of low and high sucrose.     

Rhythms of Enzyme Activity Associated with Circadian Conidiation in Neurospora crassa

The mycelial growth front of the band strain of Neurospora grown on a solid surface exhibits a circadian rhythm of conidiation. Enzyme assays on extracts from that mycelium have shown that the activities of 6 of 13 enzymes (nicotinamide adenine dinucleotide nucleosidase, isocitrate lyase, citrate synthase, glyceraldehydephosphate dehydrogenase, phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase) and soluble-protein content oscillate with the visible morphological change. The rhythmic enzymes associated with the Krebs and glyoxylate cycles are more active during conidiogenesis, whereas the activities of the rhythmic enzymes of glycolysis and the hexose monophosphate shunt are reduced during that phase. The absence of enzyme oscillations in wild-type and fluffy strains which do not form conidia under the conditions employed suggests that the enzyme fluctuations are associated with conidiogenesis itself. Oscillations of enzyme activity as a function of time are restricted to the growth front. A permanent record of rhythmicity associated with conidial and nonconidial regions does, however, exist in the mycelial mat behind the growth front. The activities of three enzymes (nicotinamide adenine dinucleotide nucleosidase, glucose-6-phosphate dehydrogenase, and phosphogluconate dehydrogenase) are not directly influenced by CO(2) concentration, but are correlated with the prescence or absence of conidiation which is controlled by CO(2) concentration. In contrast, citrate synthase and malate dehydrogenase activities are correlated with changes in CO(2) concentration.     

Asexual development is increased in Neurospora crassa cat-3-null mutant strains

We use asexual development of Neurospora crassa as a model system with which to determine the causes of cell differentiation. Air exposure of a mycelial mat induces hyphal adhesion, and adherent hyphae grow aerial hyphae that, in turn, form conidia. Previous work indicated the development of a hyperoxidant state at the start of these morphogenetic transitions and a large increase in catalase activity during conidiation. Catalase 3 (CAT-3) increases at the end of exponential growth and is induced by different stress conditions. Here we analyzed the effects of cat-3-null strains on growth and asexual development. The lack of CAT-3 was not compensated by other catalases, even under oxidative stress conditions, and cat-3^RIP colonies were sensitive to H₂O₂, indicating that wild-type (Wt) resistance to external H₂O₂ was due to CAT-3. cat-3^RIP colonies grown in the dark produced high levels of carotenes as a consequence of oxidative stress. Light exacerbated oxidative stress and further increased carotene synthesis. In the cat-3^RIP mutant strain, increased aeration in liquid cultures led to increased hyphal adhesion and protein oxidation. Compared to the Wt, the cat-3^RIP mutant strain produced six times more aerial hyphae and conidia in air-exposed mycelial mats, as a result of longer and more densely packed aerial hyphae. Protein oxidation in colonies was threefold higher and showed more aerial hyphae and conidia in mutant strains than did the Wt. Results indicate that oxidative stress due to lack of CAT-3 induces carotene synthesis, hyphal adhesion, and more aerial hyphae and conidia.

     

Circadian Rhythms of Nucleic Acid Metabolism in Neurospora crassa

Wild-type, band, and fluffy strains of Neurospora crassa exhibit circadian rhythms of ribonucleic acid and deoxyribonucleic acid content in the growth-front hyphae of cultures grown on a solid medium. There is also a rhythm of (3)H-uridine incorporation into the nucleic acids of the band strain. Maximum incorporation precedes the peaks of nucleic acid content which occur during conidiation. As cultures age, ribonucleic acid content decreases rapidly and deoxyribonucleic acid content decreases gradually in standing, shake, and bubble cultures. A reduction of ribonuclease activity with age is also noted in standing and shake cultures. The nucleic acid content, nuclease activity, and changes associated with age vary with the culture conditions.     

Lipid and cell wall changes in an inositol-requiring mutant of Neurospora crassa.

An inositol deficiency in the inositol-requiring (inl) mutant of Neurospora crassa led to changes in the composition of the inositol-containing lipids and the cell wall. On deficient levels of inositol, phosphatidyl inositol decreased by 23-fold, di(inositolphosphoryl) ceramide decreased by 4-fold, and monoinositolphosphoryl ceramide increased slightly. The inositol deficiency also led to an aberrant hyphal morphology and changes in both the amount of cell wall and the amino sugar content of the cell wall. The glucosamine content of the cell wall decreased by 50%, the galactosamine increased by 50%, but no significant changes were found in the content of the cell wall amino sugar precursors, or in the amino acid, glucose, or total hexose content of the cell wall. Inositol-containing compounds were found associated with purified cell wall material. These compounds were bound tightly to the cell wall but could be removed by treatment with alkali, a treatment which disrupts the cell wall integrity. Possible mechanisms of how changes in lipid composition can affect cell wall biosynthesis are discussed.     

Mutants with Altered Sensitivity to a Calmodulin Antagonist Affect the Circadian Clock in Neurospora Crassa

Two newly isolated mutant strains of Neurospora crassa, cpz-1 and cpz-2, were hypersensitive to chlorpromazine with respect to mycelial growth but responded differently to the drug with respect to the circadian conidiation rhythm. In the wild type, chlorpromazine caused shortening of the period length of the conidiation rhythm. Pulse treatment with the drug shifted the phase and inhibited light-induced phase shifting in Neurospora. By contrast to the wild type, the cpz-2 strain was resistant to these inhibitory effects of chlorpromazine. Inhibition of cpz-2 function by chlorpromazine affected three different parameters of circadian conidiation rhythm, namely, period length, phase and light-induced phase shifting. These results indicate that the cpz-2 gene must be involved in or related closely to the clock mechanism of Neurospora. By contrast, the cpz-1 strain was hypersensitive to chlorpromazine with respect to the circadian conidiation rhythm.     

Regulation of conidiation and adenylyl cyclase levels by the Galpha protein GNA-3 in Neurospora crassa

We have identified a new gene encoding the G protein alpha subunit, gna-3, from the filamentous fungus Neurospora crassa. The predicted amino acid sequence of GNA-3 is most similar to the Galpha proteins MOD-D, MAGA, and CPG-2 from the saprophytic fungus Podospora anserina and the pathogenic fungi Magnaporthe grisea and Cryphonectria parasitica, respectively. Deletion of gna-3 leads to shorter aerial hyphae and premature, dense conidiation during growth on solid medium or in standing liquid cultures and to inappropriate conidiation in submerged culture. The conidiation and aerial hypha defects of the Deltagna-3 strain are similar to those of a previously characterized adenylyl cyclase mutant, cr-1. Supplementation with cyclic AMP (cAMP) restores wild-type morphology to Deltagna-3 strains in standing liquid cultures. Solid medium augmented with exogenous cAMP suppresses the premature conidiation defect, but aerial hypha formation is still reduced. Submerged-culture conidiation is refractory to cAMP but is suppressed by peptone. In addition, Deltagna-3 submerged cultures express the glucose-repressible gene, qa-2, to levels greatly exceeding those observed in the wild type under carbon-starved conditions. Deltagna-3 strains exhibit reduced fertility in homozygous crosses during the sexual cycle; exogenous cAMP has no effect on this phenotype. Intracellular steady-state cAMP levels of Deltagna-3 strains are decreased 90% relative to the wild type under a variety of growth conditions. Reduced intracellular cAMP levels in the Deltagna-3 strain correlate with lower adenylyl cyclase activity and protein levels. These results demonstrate that GNA-3 modulates conidiation and adenylyl cyclase levels in N. crassa.     

Microcycle conidiation and its genetic basis in Neurospora crassa.

Some wild isolates of Neurospora show microcycle conidiation in liquid culture under continuous agitation. Macroconidia from agar-grown mycelial cultures germinated in liquid and the germlings spontaneously produced conidia with no intervening mycelial phase. Three types of microcycle conidiation were seen among progeny of N. crassa Vickramam A x N. crassa a wild-type: (1) multinucleate blastoconidia produced by apical budding and septation, (2) multinucleate arthroconidia produced by holothallic septation and disarticulation of cells, and (3) uninucleate microconidia produced directly from conidiogenous cells of the germlings. Two genes were identified which control specific patterns of microcycle conidiogenesis. A single gene mcb in linkage group VR near al-3 (3.2% recombination) controls blastoconidiation. This gene is epistatic to gene mcm located in linkage group IIL, very near ro-7 (1.4%). mcm controls both microconidiation and arthroconidiation depending on temperature. Strains of genotype mcm produce microconidia almost exclusively at 18-22 degrees C, but arthroconidia with few or no microconidia at 30 degrees C. Because they result in rapid and synchronized conidiation in liquid culture, the two genes should be useful for studies of developmental gene regulation. mcm makes it possible to obtain large quantities of pure microconidia rapidly for experimentation.     

Ribosomal genes of Neurospora crassa: constancy of gene number in the conidial and mycelial phases, and homogeneity in length and restriction enzyme cleavage sites within strains

We report the results of experiments which, while not specifically designed to study the possibility of rDNA amplification during different developmental stages in the N. crassa life cycle, clearly indicate a relative constancy in the rDNA content of conidia (asexual spores) and mycelial cells. We also report the results of restriction enzyme studies which indicate that the Neurospora rDNA repeat units are homogeneous in length and restriction site pattern within any given Neurospora strain. These results directly contradict the recent report of Dutta et al. (1983), in which the authors concluded that the rDNA of germinating conidia is amplified, relative to mycelia, and that up to 10% of the rDNA units are heterogeneous.     

Cell Wall Alterations Associated with the Hyperproduction of Extracellular Enzymes in Neurospora crassa

A pleiotropic mutation in Neurospora (exo-1), which confers derepression of alpha-amylase, glucoamylase, beta-fructofuranosidase, and trehalase, appears to also affect the composition of the cell wall. Segregants resulting from the backcross of exo-1 to the wild-type strain from which it derived are altered in the ratio of galactosamine to glucosamine in hydrolysates of isolated cell walls. Conidial cell walls exhibit a marked decrease in the amount of galactosamine in both exo-1 and exo-1(+) strains. Increased levels (approximately sevenfold) of amylase are found in conidia of exo-1, as compared with those of exo-1(+).     

Synchronous Conidiation of Piricularia oryzae by the Replacement Culture

For the purpose of studying the mechanism of conidiogenesis in Piricularia oryzae, methods were developed to separate the phase of mycelial growth from that of conidiation and also to evaluate them quantitatively.

When P. oryzae was grown on media with low carbon : nitrogen ratio and high nitrogen concentration, conidiation did not take place, in spite of its vegetative growth. Conidiation occurred in a very short period of time when the above mycelia were replaced on nutritionally poor media. Cellophane membrane was overlaid on solid medium and conidia were spread uniformly. Evenly grown mycelial mat which could be easily transferred onto the post-culture medium was obtained. As the preculture medium, MSA medium with carbon: nitrogen ratio of 6.3 and nitrogen concentration of 1.5 g/liter was used. The evenly grown mycelial mat was cut into small square mats of 1.44 cm² each and the small square mycelial mats were transferred onto the post-culture medium together with the cellophane membrane. The conidiation took place in the post-culture and the vegetative growth in the preculture and the conidiation in the post-culture could be observed separately and quantitatively.

Conidiation did not occur at all in the preculture and the degree of conidiation which took place in the post-culture varied according to the precultural conditions. This means that it is a certain state of physiological condition in the preculture which determines the degree of conidiation in the post-culture. The authers designated this state of physiological condition as the “latent activity of conidiation” (LAC). For the purpose of the quantitative estimation of this activity, we expressed LAC in terms of the degree of conidiation in the post-culture under a defined cultural condition. The LAC was subject to change very easily, declined rapidly and disappeared upon prolonged preculture. Only young mycelia showed to have this activity.

The influences of the precultural condition on the development of the LAC and vegetative growth were generally parallel. However, the LAC was generally more sensitive to the environmental condition than the vegetative growth, especially to the temperature change.

The conidia formed were uniform in size and had high rate of germination. Several strains of P. oryzae tested showed very similar behavior.     

The relation between growth, conidiation and trehalase activity in Neurospora crassa

The extent to which trehalose is accumulated in the vegetative mycelium of strains of Neurospora crassa is significantly affected by conidiation. In heavily conidiating strains a rapid decrease in mycelial trehalose occurs following the initiation of conidiation. Meanwhile, trehalase activity in the vegetative mycelium of heavily conidiating strains increases rapidly following the initiation of conidiation, although apparently it is not directly caused by the sporulation process. High levels of both trehalase and trehalose appear concomitantly in the newly formed conidia.     

Aspergillus fumigatus chsE: A Gene Related to CHS3 of Saccharomyces cerevisiae and Important for Hyphal Growth and Conidiophore Development but Not Pathogenicity

The Aspergillus fumigatus chsE (AfchsE) gene was isolated from an A. fumigatus DNA library on the basis of hybridization to a segment of Saccharomyces cerevisiae CHS3 (ScCHS3). The amino acid sequence derived from AfchsE is 28% identical with ScCHS3 and 80% identical with the product of Aspergillus nidulans chsD (AnchsD). A mutant strain constructed by disruption of AfchsE has reduced levels of mycelial chitin, periodic swellings along the length of hyphae, and a block in conidiation that can be partially restored by growth in osmotic stabilizer. This phenotype is different from that reported for an AnchsD mutant, in which germinating conidia and hyphal tips undergo lysis and the colonial growth rate is significantly reduced. Despite the defects associated with the AfchsE- strain, its virulence was not significantly reduced when compared with the wild-type parental strain in a mouse model of pulmonary aspergillosis.     

Genes expressed during conidiation in Neurospora crassa: molecular characterization of con-13

Asexual development in Neurospora crassa proceeds through a series of discrete morphological stages that culminate in the production of dormant spores called conidia. Changes in the pattern of gene expression parallel the morphological transformations associated with conidiation. As a prerequisite to the analysis of developmental gene expression in N. crassa, several genes of unknown function that are preferentially expressed during conidiation were isolated [Berlin and Yanofsky, Mol. Cell. Biol. 5 (1985) 849-855]. The molecular structure and nucleotide sequence of one of these genes, designated con-13, is presented. The con-13 gene specifies a relatively rare 1.35-kb message which is first detected about 8 h following the induction of conidiation. Sequence analysis of both cDNA and genomic clones indicates that the con-13 gene consists of three exons divided by two small introns. It encodes a polypeptide of 340 amino acid residues (37.1 kDa). The Con-13 protein is weakly acidic and hydrophilic. A comparison of the regions upstream from the con-8, con-10, and con-13 genes revealed several short sequence motifs which may be important in developmental gene regulation.