Regulation and glutamic acid decarboxylase during Neurospora crassa conidial germination.

Glutamic acid decarboxylase (GAD) from Neurospora crassa was assayed in dormant and germinating conidia that had been permeabilized by toluene and methanol. N. crassa conidia contained 10 times the GAD activity found in vegetativemycelia. During conidial germination, GAD activity rapidly decreased to low levels before germ tubes appeared. GAD activity in germinating conidia closely followed the decreasing rate of glutamic acid metabolism. Inhibiting protein synthesis partially blocked the decrease in GAD activity, but eliminating exogenous carbon sources did not alter the initial rate of decrease in this enzyme. However, when conidia were incubated for more than 3 h in distilled water, GAD activity began to increase and eventually reached levels comparable to those in dormant conidia. Either GAD was reversibly inactivated or this enzyme could be synthesized from endogenous storage compounds when conidia were incubated in distilled water. These results are consistent with the hypothesis that GAD is a developmentally regulated enzyme that is responsible for catalyzing the first step in the metabolism of the large pool of free glutamic acid during conidial germination.     

Biogenesis of mitochondrial membranes in Neurospora crassa. Mitochondrial protein synthesis during conidial germination.

The conidia of Neurospora crassa entered logarithmic growth after a 1-h lag period at 30 degrees C. Although [14C]leucine is incorporated quickly early in growth, cellular protein data indicated that no net protein synthesis occurred until after 2 h of growth. Neurospora is known to produce ethanol during germination even though respiratory enzymes are present. Also, Neurospora mitochondria isolated from cells less than 3-h old are uncoupled. Since oxygen uptake increased during germination, was largely cyanide-sensitive, and reached a maximum at 3 h, it is hypothesized that during early germination the uncoupled electron transport chain merely functions to dispose of reducing equivalents generated by substrate level ATP production. The rate of protein synthesis in vitro by mitochondria isolated from 0-8-h-old cells increased as did cell age. Mitochondrial protein synthesis in vivo, assayed in the presence of 100 mug cycloheximide/ml, increased from low levels in the cinidia to peak levels at 3-4 h of age and then slowly decreased. The rate of mitochondrial protein synthesis in vivo was linear for at least 90 min in 0-4-h-old cells, but declined after 15 min of incorporation in 6 and 8-h-old cells. The products of mitochondrial protein synthesis in vivo were analyzed with dodecylsulfate gel electrophoresis and autoradiography. Early in germination 80% of the synthesis was of two small proteins (molecular weights 7200 and 9000). At 8 h 85% of the radioactivity was in 10 larger proteins (12 200 to 80 000). Within the high-molecular-weight class, proteins of between 12 000 and 21 500 molecular weight were preferentially lavelled early in germination, whereas after 8 h of growth proteins of 27 500 to 80 000 molecular weight were preferentially labelled. It is hypothesized that the 7200 and 9000-molecular-weight products of mitochondrial protein synthesis combine with other proteins to form the larger proteins found later in growth. The availability of these other proteins in cells of different ages could affect the rate of mitochondrial protein synthesis in vivo.     

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.     

Some required events in conidial germination of Neurospora crassa.

A temperature-sensitive mutant of Neurospora crassa, with reduced levels of protein synthesis at 37°C, was used to identify some essential events in conidial germination. Conidia of mutant strain psi-1 were incubated for 2 hr at 37°C and then shifted to 20°C. Germination was inhibited at 37°C, but commenced after 1.5 hr at 20°C. Increases in aspartate transcarbamylase activity, cell wall synthesis, and nuclear number preceded germination. However, increases in glutamate dehydrogenase activity, amino acid uptake, and DNA synthesis were inhibited prior to germination. Although all of these events were correlated with germination in control cultures of the mutant at 20°C and of its parent strain at 20 and 37°C, some events were apparently not essential for germination. The requirement for aspartate transcarbamylase activity was demonstrated independently by the failure of strain pyr-3d (lacking the activity) to germinate in the absence of uridine. The dispensability of glutamate dehydrogenase activity and DNA synthesis for the germination of some conidia was verified by the germination of strain am-1 (lacking glutamate dehydrogenase activity) in the absence of glutamate and by the germination of the parent strain in the presence of hydroxyurea (an inhibitor of DNA synthesis). These findings identify some landmarks in germination which may be useful in further studies of the regulation of a developmental program. They also provide preliminary evidence that the resting conidia may contain nuclei arrested at different stages of their division cycle.     

Live-cell imaging of vegetative hyphal fusion in Neurospora crassa

The process of hyphal fusion (anastomosis) in growing colonies of Neurospora crassa, stained with the membrane-selective dyes FM1-43 and FM4-64, was visualized by confocal microscopy. Time-lapse, live-cell imaging illustrated the dynamics of hyphal growth and anastomosis during its pre-contact, contact and post-contact, and post-fusion stages. Fusion-competent hyphae were morphologically distinct and exhibited remote sensing, resulting in branch initiation and/or re-direction of growth to facilitate contact between participating hyphae. A stained Spitzenk?rper was often observed where fusion-competent hyphae met. It is suggested that this structure contains secretory vesicles responsible for the delivery of cell adhesion molecules at the point of contact, cell wall synthesizing enzymes for the swelling growth of fused hyphal tips, and digestive enzymes required for fusion pore formation. Dramatic changes in cytoplasmic flow frequently occurred between the participating hyphae following fusion. After anastomosis has taken place, septa commonly formed close to the fusion site. The live-cell imaging reported here has clearly shown the complexity of the hyphal homing and fusion process. The control and consequences of repeated anastomoses within a mycelium must be as complex as the process itself.     

Effects of auxin and gibberellin on conidial germination in Neurospora crassa

Auxins, IAA, 2,4-D, and NAA, and gibberellin, GA, significantlyenhanced the conidial germination rate in the wild-type Neurosporacrassa. The inhibitory effect of an antiauxin, 2,4,6-T on conidialgermination was overcome by IAA. The present results suggestthat auxin and gibberellin may act as regulators of conidialgermination in Neurospora. (Received November 21, 1977; )     

SO, a protein involved in hyphal fusion in Neurospora crassa, localizes to septal plugs

The colony of a filamentous ascomycete fungus typically grows as a multinucleate syncytium. While this syncytial organization has developmental advantages, it bears the risk of extensive damage caused by local injury of hyphae. Loss of cytoplasm in injured hyphae is restricted by the fast and efficient sealing of the central pores of hyphal crosswalls, or septa, by a peroxisome-derived organelle called the Woronin body. The formation of septal plugs is also associated with development and leads to separation of certain parts of the colony. Septal plugs associated with developmental processes or aging hyphae typically occur by the accumulation of sealing material. Here we report that in Neurospora crassa, a protein necessary for hyphal fusion and proper colony development called SO (SOFT) localizes to septal plugs. In response to injury, SO accumulates at the septal plug in a Woronin body-independent manner. However, the presence of the Woronin body affects the speed of accumulation of SO at the septal pore. We determined that SO contributes to, but is not essential for, septal plugging. SO accumulation was also observed at septal plugs formed during hyphal aging and during programmed cell death mediated by genetic differences at heterokaryon incompatibility (het) loci.     

Isolation and identification of the conidial germination factor of Neurospora crassa.

The germination-essential substance (germination factor [GF]) that is lost from conidia of Neurospora crassa on exposure to solutions of low water activity has been isolated and identified as a group of iron-transport compounds, or siderochromes. The principal siderochrome of conidia is ferricrocin, a cyclic hexapeptide. A closely related substance, ferrichrome C, is tentatively identified as a minor constituent. The same substances are also present in extracts of mycelium along with small amounts of a third siderochrome, which has not been identified. The GF activity of culture filtrates is due to coprogen, the only siderochrome previously identified with N. crassa.     

Neurospora crassa conidial germination: role of endogenous amino acid pools.

The levels of the endogenous amino acid pools in conidia, germinating conidia, and mycelia of wild-type Neurospora crassa were measured. Three different chromatographic procedures employing the amino acid analyzer were used to identify and quantitatively measure 28 different ninhydrin-positive compounds. All of the common amino acids were detected in conidial extracts except proline, methionine, and cystine. The levels of these three amino acid pools were also very low in mycelia. During the first hour of germination in minimal medium, the levels of most of the free amino acid pools decreased. The pool of glutamic acid, the predominant free amino acid in conidia, decreased 70% during the first hour. Very little glutamic acid or any other amino acid was excreted into the medium. During the first 20 min of germination, the decrease in the glutamic acid pool was nearly equivalent to the increase in the aspartic acid pool. The aspartic acid and lambda-aminobutyric acid pools were the only amino acid pools that increased to maximum levels within the first 20 min of germination and then decreased. It is proposed that an important metabolic event that occurs during the early stages of conidial germination is the production of reduced pyridine nucleotides. The degradation of the large glutamic acid pool existing in the conidia (2.5% of the conidial dry weight) could produce these reduced coenzymes.     

A mitogen-activated protein kinase that senses nitrogen regulates conidial germination and growth in Aspergillus fumigatus

We show that the mitogen-activated protein (MAP) kinase pathway that responds to osmotic stress in Aspergillus fumigatus is also involved in nutritional sensing. This MAP kinase regulates conidial germination in response to the nitrogen source and is activated upon starvation for either carbon or nitrogen during vegetative growth.     

cot-1, a gene required for hyphal elongation in Neurospora crassa, encodes a protein kinase.

Neurospora crassa is a filamentous fungus that grows on semisolid media by forming spreading colonies. Mutations at several loci prevent this spreading growth. cot-1 is a temperature sensitive mutant of N.crassa that exhibits restricted colonial growth. At temperatures above 32 degrees C colonies are compact while at lower temperatures growth is indistinguishable from that of the wild type. Restricted colonial growth is due to a defect in hyphal tip elongation and a concomitant increase in hyphal branching. We have isolated a genomic cosmid clone containing the wild type allele of cot-1 by complementation. Sequence analyses suggested that cot-1 encodes a member of the cAMP-dependent protein kinase family. Strains in which we disrupted cot-1 are viable but display restricted colonial growth. Duplication, by ectopic integration of a promoter-containing fragment which includes the first one-third (209 codons) of the structural gene, unexpectedly resulted in restricted colonial growth. Our results suggest that an active COT1 kinase is required for one or more events essential for hyphal elongation.     

Evidence for tryptophan being a signal molecule that inhibits conidial anastomosis tube fusion during colony initiation in Neurospora crassa

Mycelial interconnectedness achieved by hyphal fusion has been hypothesized to facilitate the distribution and sharing of nutrients between different parts of a mycelium, especially when nutrients are heterogeneously distributed in the environment. However, the link between environmental nutrient availability and hyphal fusion is little understood. Here, we report that amino acids and extracellular pH regulate conidial anastomosis tube (CAT) fusion during colony initiation in Neurospora crassa. Quantitative analyses revealed that low extracellular pH and certain amino acids, particularly tryptophan, inhibit CAT fusion. Conidial germination was also inhibited by tryptophan but this inhibition was mitigated by the presence of other amino acids. This provides evidence for tryptophan having a role as a signal molecule that regulates CAT fusion. Tryptophan acts intracellularly because two amino acid permease mutants (Δmtr and Δaap-20) exhibited resistance against tryptophan-mediated inhibition of CAT fusion. Tryptophan and low pH did not significantly affect vegetative hyphal fusion in mature colonies, indicating that the latter is regulated in a different manner to CAT fusion.     

Microtubule dynamics and organization during hyphal growth and branching in Neurospora crassa

By confocal microscopy, we analyzed microtubule (Mt) behavior during hyphal growth and branching in a Neurospora crassa strain whose Mts had been tagged with GFP. Images were assembled spatially and temporally to better understand the 3-D organization of the microtubular cytoskeleton and a clearer view of its dynamics. Cytoplasmic Mts were mainly arranged longitudinally along the hyphal tube. Straight segments were rare; most Mts showed a distinct helical curvature with a long pitch and a tendency to intertwine with one another to form a loosely braided network throughout the cytoplasm. This study revealed that the microtubular cytoskeleton of a hypha advances as a unit, i.e., as the cell elongates, it moves forward by bulk flow. Nuclei appeared trapped in the microtubular network and were carried forward in unison as the hypha elongated. During branching, one or more cortical Mts became associated with the incipient branch and were pulled into the emergence of the branch. As extension of the branch and distortion of the Mts continued, Mts soon were severed with both new Mt ends (+ and -) present in the new branch. Although the exact mechanisms for addition Mt recruitment into the branch remains an open question, the recorded evidence indicates both bulk insertion of established cortical parent-hypha Mts as well as in situ polymerization were involved. The latter conclusion was supported by FRAP studies showing evidence of Mt nucleation and polymerization assembly in the growing tip of the developing branch. Nuclei entered the branch entrapped in the advancing network of Mts.     

Cyclic AMP-dependent protein kinase of Neurospora crassa

$\text{Neurospora}\text{crassa}$ was surveyed for cyclic AMP-dependent protein kinase activity. Two peaks (I and II) of protein kinase activity were demonstrated by DEAE-cellulose chromatography of wild type $\text{Neurospora}$ extracts. Peak I was stimulated by cyclic AMP, eluted below 60 mM NaCl and had high activity using histone H2B as substrate. Peak II eluted at 200–250 mM NaCl; its activity was not cyclic AMP stimulated and was highest with dephosphorylated casein as a substrate. Cyclic AMP binding to a protein associated with the protein kinase is specifically inhibited by certain cyclic AMP analogs.     

Cell biology of conidial anastomosis tubes in Neurospora crassa

Although hyphal fusion has been well documented in mature colonies of filamentous fungi, it has been little studied during colony establishment. Here we show that specialized hyphae, called conidial anastomosis tubes (CATs), are produced by all types of conidia and by conidial germ tubes of Neurospora crassa. The CAT is shown to be a cellular element that is morphologically and physiologically distinct from a germ tube and under separate genetic control. In contrast to germ tubes, CATs are thinner, shorter, lack branches, exhibit determinate growth, and home toward each other. Evidence for an extracellular CAT inducer derived from conidia was obtained because CAT formation was reduced at low conidial concentrations. A cr-1 mutant lacking cyclic AMP (cAMP) produced CATs, indicating that the inducer is not cAMP. Evidence that the transduction of the CAT inducer signal involves a putative transmembrane protein (HAM-2) and the MAK-2 and NRC-1 proteins of a mitogen-activated protein kinase signaling pathway was obtained because ham-2, mak-2, and nrc-1 mutants lacked CATs. Optical tweezers were used in a novel experimental assay to micromanipulate whole conidia and germlings to analyze chemoattraction between CATs during homing. Strains of the same and opposite mating type were shown to home toward each other. The cr-1 mutant also underwent normal homing, indicating that cAMP is not the chemoattractant. ham-2, mak-2, and nrc-1 macroconidia did not attract CATs of the wild type. Fusion between CATs of opposite mating types was partially inhibited, providing evidence of non-self-recognition prior to fusion. Microtubules and nuclei passed through fused CATs.     

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.