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.     

Molecular characterization of mutations of nlt-4, the pathway-specific regulatory gene which controls nitrate assimilation in Neurospora crassa

The nit-4 genes of three conventional Neurospora crassa mutations and of the closely related species, Neurospora intermedia, have been isolated by amplifying the genomic DNA with the polymerase chain reaction. Nucleotide sequencing has revealed that the three nit-4 mutants, alleles 15, 1214, and 2994, are the result of a missense mutation, a nonsense mutation and a frameshift mutation, respectively. The nucleotide sequence of the NIT4 protein coding region of a nit-4 mutant (allele 2994) and of N. intermedia have been determined and compared with that of wild-type N. crassa. The molecular characteristics confirm that the mutated gene of 2994 originated from N. intermedia and was introgressed into N. crassa. The polyglutamine domains of the N. crassa wild type, the 2994 mutant, or N. intermedia cannot replace an upstream glutamine-rich domain which is essential for nit-4 function.     

Circadian and light-induced expression of luciferase in Neurospora crassa

We have constructed a plasmid vector for expressing firefly luciferase in Neurospora crassa under control of the light- and clock-regulated ccg-2 (eas) promoter. The sequence of the luciferase gene in the vector has been modified to reflect the N. crassa codon bias. Both light-induced activity and circadian activity are demonstrated. Expression of luciferase in strains carrying mutant frequency alleles shows appropriate period length alterations. These data demonstrate that luciferase is a sensitive reporter of gene expression in N. crassa. Our results also show that the modified luciferase is expressed in Aspergillus nidulans.     

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.     

Genetic architecture of a reinforced, postmating, reproductive isolation barrier between Neurospora species indicates evolution via natural selection

A role for natural selection in reinforcing premating barriers is recognized, but selection for reinforcement of postmating barriers remains controversial. Organisms lacking evolvable premating barriers can theoretically reinforce postmating isolation, but only under restrictive conditions: parental investment in hybrid progeny must inhibit subsequent reproduction, and selected postmating barriers must restore parents' capacity to reproduce successfully. We show that reinforced postmating isolation markedly increases maternal fitness in the fungus Neurospora crassa, and we detect the evolutionary genetic signature of natural selection by quantitative trait locus (QTL) analysis of the reinforced barrier. Hybrid progeny of N. crassa and N. intermedia are highly inviable. Fertilization by local N. intermedia results in early abortion of hybrid fruitbodies, and we show that abortion is adaptive because only aborted maternal colonies remain fully receptive to future reproduction. In the first QTL analysis of postmating reinforcement in microbial eukaryotes, we identify 11 loci for abortive hybrid fruitbody development, including three major QTLs that together explain 30% of trait variance. One of the major QTLs and six QTLs of lesser effect are found on the mating-type determining chromosome of Neurospora. Several reinforcement QTLs are flanked by genetic markers showing either segregation distortion or non-random associations with alleles at other loci in a cross between N. crassa of different clades, suggesting that the loci also are associated with local effects on same-species reproduction. Statistical analysis of the allelic effects distribution for abortive hybrid fruitbody development indicates its evolution occurred under positive selection. Our results strongly support a role for natural selection in the evolution of reinforced postmating isolation in N. crassa.     

Allelic Specificity at the Het-C Heterokaryon Incompatibility Locus of Neurospora Crassa Is Determined by a Highly Variable Domain

In filamentous fungi, the ability to form a productive heterokaryon with a genetically dissimilar individual is controlled by specific loci termed het loci. Only strains homozygous for all het loci can establish a heterokaryon. In Neurospora crassa, 11 loci, including the mating-type locus, regulate the capacity to form heterokaryons. An allele of the het-c locus (het-c(OR)) of N. crassa has been previously characterized and encodes a nonessential 966 amino acid glycine-rich protein. Herein, we describe the genetic and molecular characterization of two het-c alleles, het-c(PA) and het-c(GR), that have a different specificity from that of het-c(OR), showing that vegetative incompatibility is mediated by multiple alleles at het-c. By constructing chimeric alleles, we show that het-c specificity is determined by a highly variable domain of 34-48 amino acids in length. In this regard, het-c is similar to loci that regulate recognition in other species, such as the (S) self-incompatibility locus in plants, the sexual compatibility locus in basidiomycetes and the major histocompatibility complex (MHC) genes in vertebrates.     

Adaptive Significance of Vegetative Incompatibility in NEUROSPORA CRASSA

Certain features reminiscent of sexuality occur in the vegetative life cycle of some filamentous fungi such as Neurospora crassa. Hyphal fusions can occur between genetically different individuals, thereby endowing the new composite mycelium, a heterokaryon, with some of the advantages of heterozygosity usually associated with diploid organisms. In N. crassa, however, there are a number of incompatibility loci which prevent formation of heterokaryons unless the alleles at the incompatibility loci are identical in the two mycelia. The selection pressures that maintain incompatibility polymorphisms are not known. We suggest here that they are maintained because they prevent a kind of exploitation of heterokaryons by nuclei that are nonadaptive in homokaryons but that enjoy a proliferative advantage over other nuclei in heterokaryons. A mathematical model that abstracts the major features of the vegetative life cycle of Neurosopra crassa has been developed, and the action of selection in this model and various extensions of it is such as to maintain polymorphisms of vegetative incompatibility factors.     

Isolation and characterization of Neurospora crassa mutants resistant to antifungal plant defensins

Twenty-five Neurospora crassa mutants obtained by chemical mutagenesis were screened for increased resistance to various antifungal plant defensins. Plant defensin-resistant N. crassa mutants were further tested for their cross-resistance towards other families of structurally different antimicrobial peptides. Two N. crassa mutants, termed MUT16 and MUT24, displaying resistance towards all plant defensins tested but not to structurally different antimicrobial peptides were selected for further characterization. MUT16 and MUT24 were more resistant towards plant defensin-induced membrane permeabilization as compared to the N. crassa wild-type. Based on the previously demonstrated key role of fungal sphingolipids in the mechanism of growth inhibition by plant defensins, membrane sphingolipids of MUT16 and MUT24 were analysed. Membranes of these mutants contained structurally different glucosylceramides, novel glycosylinositolphosphorylceramides, and an altered level of steryl glucosides. Evidence is provided to link these clear differences in sphingolipid profiles of N. crassa mutants with their resistance towards different plant defensins.     

A eukaryote without catalase-containing microbodies: Neurospora crassa exhibits a unique cellular distribution of its four catalases

Microbodies usually house catalase to decompose hydrogen peroxide generated within the organelle by the action of various oxidases. Here we have analyzed whether peroxisomes (i.e., catalase-containing microbodies) exist in Neurospora crassa. Three distinct catalase isoforms were identified by native catalase activity gels under various peroxisome-inducing conditions. Subcellular fractionation by density gradient centrifugation revealed that most of the spectrophotometrically measured activity was present in the light upper fractions, with an additional small peak coinciding with the peak fractions of HEX-1, the marker protein for Woronin bodies, a compartment related to the microbody family. However, neither in-gel assays nor monospecific antibodies generated against the three purified catalases detected the enzymes in any dense organellar fraction. Furthermore, staining of an N. crassa wild-type strain with 3,3'-diaminobenzidine and H(2)O(2) did not lead to catalase-dependent reaction products within microbodies. Nonetheless, N. crassa does possess a gene (cat-4) whose product is most similar to the peroxisomal type of monofunctional catalases. This novel protein indeed exhibited catalase activity, but was not localized to microbodies either. We conclude that N. crassa lacks catalase-containing peroxisomes, a characteristic that is probably restricted to a few filamentous fungi that produce little hydrogen peroxide within microbodies.     

Molecular analysis of mutants of the <Emphasis Type="Italic">Neurospora</Emphasis> adenylosuccinate synthetase locus

The ad-8 gene of Neurospora crassa, in addition to being used for the study of purine biology, has been extensively studied as a model for gene structure, mutagenesis and intralocus recombination. Because of this there is an extensive collection of well-characterized N. crassa ad-8 mutants in the Fungal Genetics Stock Center collection. Among these are spontaneous mutants and mutants induced with X-ray, UV or chemical mutagens. The specific lesions in these mutants have been genetically mapped at high resolution. We have sequenced the ad-8 locus from 13 of these mutants and identified the molecular nature of the mutation in each strain. We compare the historical fine-structure map to the DNA and amino acid sequence of each allele. The placement of the individual lesions in the fine-structure map was more accurate at the 5' end of the gene and no mutants were identified in the 3' untranslated region of this gene. We additionally analysed ad-8(+) alleles in 18 N. crassa strains subjected to whole-genome sequence analysis and describe the variability among Neurospora strains and among fungi and other organisms.     

Analysis of two additional loci in Neurospora crassa related to Spore killer-2

Two new loci found in one strain of Neurospora crassa (P2604) collected in Malaya are related to the meiotic drive system Spore killer Sk-2. Sk-2 was found in Neurospora intermedia and introgressed into N. crassa. P2604 showed high resistance to killing when crossed to Sk-2. This resistance was found to be linked to, but not allelic to, resistance locus r(Sk-2) on LGIIIL. Analysis showed that the high resistance phenotype of P2604 requires resistance alleles at two different loci on LGIIIR. Strains carrying a resistance allele at only the proximal or the distal locus, respectively, were obtained and intercrossed. Highly resistant strains were obtained by rejoining the two genes. The proximal locus alone confers a low level of resistance. This locus was named pr(Sk-2) for partial resistance to Sk-2. The distal locus was named mod(pr) because its only known phenotype is to modify pr(Sk-2).     

Induction of multiple germ tubes in Neurospora crassa by antitubulin agents

The antitubulin fungicide benomyl suppressed the linear growth of Neurospora crassa wild type strain St. Lawrence 74 at micromolar concentrations. The rate of germination of macroconidia was not affected. Macroconidia exposed to 1.7 microM benomyl for 5 h formed multiple germ tubes. When germlings incubated for 4 h were exposed to 1.7 microM benomyl for 3 h, their germ tube stopped growing, swelled and emitted several branches. Normal linear growth was restored after removal of the fungicide. Linear growth of N. crassa was resistant up to 16 microM nocodazole. This drug induced multipolar germination at 8 microM, and griseofulvin only at 140 microM. The microtubule (MT) cytoskeleton of N. crassa could be revealed by indirect immunofluorescence with the monoclonal antibody YOL 1/34 directed against yeast alpha-tubulin. We detected no striking effects of the benomyl treatments on MT organization. The MT-stabilizing agents deuterium oxide (D2O) and cAMP have no antagonistic effects on the benomyl-induced multipolar germination. The positioning of nuclei and mitochondria was determined from the DAPI and Rhodamine 123 fluorescence patterns, respectively. Benomyl inhibited nuclear migration into multiple germ tubes. Quantitative scanning cytophotometry revealed a peak in the intensity of the mitochondria-associated Rhodamine 123 fluorescence near the apex of untreated germlings. This peak disappeared in multiple germ tubes. Benomyl-resistant mutant bml 511 (r), mutated in its beta-tubulin gene, germinated normally in the presence of the fungicide. This strongly suggests that multiple germ tube formation was due to the effect of benomyl on beta-tubulin. Benomyl-resistant strain 74-3, constructed by reintroducing the cloned mutant N. crassa beta-tubulin gene into the cells by transformation, displayed a partial resistance to benomyl with respect to multipolar germination. Its rate of germination was slow (50% germination reached after 4 h at 37 degrees C as compared to 2.5 h for the wild type). In contrast to N. crassa, the other ascomycete Aspergillus nidulans is nocodazole-sensitive (linear growth suppressed at 1.6 microM). It did not respond to the MT inhibitors benomyl and nocodazole with respect to the pattern of germ tube emergence. Our results suggest that microtubule or membrane beta-tubulin is involved in the maintenance of developmental polarity during germ tube emergence and growth of N. crassa.     

Intergeneric complementation of a circadian rhythmicity defect: phylogenetic conservation of structure and function of the clock gene frequency.

The Neurospora crassa frequency locus encodes a 989 amino acid protein that is a central component, a state variable, of the circadian biological clock. We have determined the sequence of all or part of this protein and surrounding regulatory regions from additional fungi representing three genera and report that there is distinct, preferential conservation of the frequency open reading frame (ORF) as compared with non-coding sequences. Within the coding region, many of the domain hallmarks of the N. crassa protein are highly conserved, especially an internal region bearing the causative mutations in frq1 and frq7, the most extreme alleles in the frequency allelic series. Despite considerable diversity among the strains analyzed in terms of morphology, growth, circadian clock output and frq sequence, the ORF from the most distantly related fungus included in this study (Sordaria fimicola) rescues rhythmicity in a N.crassa frequency null strain. Both sequence conservation, and the ability of frequency from a genus displaying one developmental program to complement circadian defects in a separate genus with a distinct, clock-regulated developmental program, are consistent with a central role of the frequency gene product in a general circadian oscillator capable of controlling diverse outputs in a variety of systems.     

Dominance Modifiers in NEUROSPORA CRASSA: Phenocopy Selection and Influence on Certain Ascus Mutants

When homozygous in zygotes, mutant alleles at the peak locus in linkage group V of Neurospora crassa initiate aberrant asci that are nonlinear, in contrast to the linear asci characteristic of wild type. Most mutant alleles are recessive, inasmuch as crosses of the mutant strains with wild type give linear asci. However, five different mutant alleles, when heterozygous with the wild-type allele, act in varying degrees as zygote dominants, initiating both linear and nonlinear asci, the relative proportions depending on the allele. Five modifiers that act on the dominance relationships of at least one of the five possible heterozygotes of a dominant peak and its wild-type allele have been characterized, four of them having been obtained by selection directed against a phenocopy of these mutants induced by treatment of wild type with l-sorbose. The pattern of modifier specificity observed among the various dominant peak heterozygotes indicates that the phenotypic effects are produced by a complex relationship between the modifiers and the dominant peak alleles in relation to their wild-type allele. In all but two cases the direction of modification, where present, is towards decreasing the dominance of the mutant allele in the heterozygote, evidenced by an increase in the percentage of linear asci when compared with control data. The modifiers exert their maximum modification when they themselves are heterozygous with their wild-type alleles and when the dominant peak allele is heterozygous with its wild-type allele. No modification occurs when heterozygous modifiers are included in zygotes homozygous for a dominant peak allele, reinforcing the notion that the modifiers act on the dominance relationship existent between a dominant peak allele and its wild-type allele, rather than influencing some activity of the mutant allele itself. The modifiers have no detectable effect of their own on ascus morphology, since homozygous modifier zygotes initiate entirely linear asci when only wild-type alleles of peak are present in the zygotes. Their only detectable effect, other than dominance modification, appears to be in conferring sorbose resistance to the mycelium. The modifiers are unlinked to the peak locus, and, except for two of them, they are nonallelic.     

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.     

On the independence of barrage formation and heterokaryon incompatibility in Neurospora crassa

A barrage is a line or zone of demarcation that may develop at the interface where genetically different fungi meet. Barrage formation represents a type of nonself recognition that has often been attributed to the heterokaryon incompatibility system, which limits the co-occurrence of genetically different nuclei in the same cytoplasm during the asexual phase of the life cycle. While the genetic basis of the heterokaryon incompatibility system is well characterized in Neurospora crassa, barrage formation has not been thoroughly investigated. In addition to the previously described Standard Mating Reaction barrage, we identified at least three types of barrage in N. crassa; dark line, clear zone, and raised aggregate of hyphae. Barrage formation in N. crassa was evident only when paired mycelia were genetically different and only when confrontations were carried out on low nutrient growth media. Barrages were observed to occur in some cases between strains that were identical at all major heterokaryon incompatibility (het) loci and the mating-type locus, mat, which acts as a heterokaryon incompatibility locus during the vegetative phase of N. crassa. We also found examples where barrages did not form between strains that had genetic differences at het-6, het-c, and/or mat. Taken together, these results suggest that the genetic control of barrage formation in N. crassa can operate independently from that of heterokaryon incompatibility and mating type. Surprisingly, barrages were not observed to form when wild-collected strains of N. crassa were paired. However, an increase in the frequency of pairings that produced barrages was observed among strains obtained by back-crossing wild strains to laboratory strains, or through successive rounds of inbreeding of wild-derived strains, suggesting the presence in wild strains of genes that suppress barrage.     

Characterization of a cobalt-resistant mutant of Neurospora crassa with transport block

A cobalt-resistant strain of Neurospora crassa (cor) was obtained by repeated subculturing of the wild type on cobalt-containing agar medium. N. crassa cor is twentyfold more resistant to cobalt ions compared with the wild type. Resistance was stable on repeated subculturing of cor on cobalt-free media. N. crassa cor is also cross-resistant to nickel (fourfold), but not to zinc or copper. Higher concentrations of iron and magnesium ions are required to reverse growth inhibition due to cobalt toxicity in N. crassa cor, compared with the wild type. Germinating conidia and mycelia of the cor strain accumulated lower levels of cobalt ions compared with the parent N. crassa. The partial transport block for cobalt uptake is shown to be primarily due to decreased surface binding of cobalt to mycelia and cell walls. Efflux of mycelial cobalt was also observed in wild type and cobalt-resistant N. crassa. The characteristics of cor in comparison with wild type N. crassa are discussed in relation to the mechanisms of cobalt resistance.     

Calmodulin from neurospora crassa. General properties and conformational changes

Calmodulin from Neurospora crassa has been purified to electrophoretic homogeneity. Equilibrium gel filtration experiments suggest that its Ca-binding properties are indistinguishable from those of vertebrate calmodulins. The isoelectric point of 4.04 and electrophoretic behavior under nondenaturing conditions indicate that N. crassa calmodulin is slightly less acidic than its vertebrate counterpart. The amino acid composition is typical of plant calmodulins with the exception that trimethyllysine is absent and that the content of Ser is unusually high. The tryptic peptide map of N. crassa calmodulin reveals an important number of point mutations as compared to vertebrate calmodulin. Differences in primary structure may explain why N. crassa calmodulin is less potent in the activation of myosin light chain kinase than calmodulins from higher organisms. The far UV circular dichroic spectra of the Ca-, Mg-, and metal-free forms of N. crassa calmodulin are similar to those of vertebrate calmodulin; in contrast, the near UV circular dichroic spectra are very different, apparently due to the differences in Tyr content. The single Tyr residue of N. crassa calmodulin, presumably located in position 138, undergoes an inversion of optical chirality upon addition of Ca2+, but not of Mg2+, to the metal-free protein.