Vegetative incompatibility in the het-6 region of Neurospora crassa is mediated by two linked genes

Non-self-recognition during asexual growth of Neurospora crassa involves restriction of heterokaryon formation via genetic differences at 11 het loci, including mating type. The het-6 locus maps to a 250-kbp region of LGIIL. We used restriction fragment length polymorphisms in progeny with crossovers in the het-6 region and a DNA transformation assay to identify two genes in a 25-kbp region that have vegetative incompatibility activity. The predicted product of one of these genes, which we designate het-6(OR), has three regions of amino acid sequence similarity to the predicted product of the het-e vegetative incompatibility gene in Podospora anserina and to the predicted product of tol, which mediates mating-type vegetative incompatibility in N. crassa. The predicted product of the alternative het-6 allele, HET-6(PA), shares only 68% amino acid identity with HET-6(OR). The second incompatibility gene, un-24(OR), encodes the large subunit of ribonucleotide reductase, which is essential for de novo synthesis of DNA. A region in the carboxyl-terminal portion of UN-24 is associated with incompatibility and is variable between un-24(OR) and the alternative allele un-24(PA). Linkage analysis indicates that the 25-kbp un-24-het-6 region is inherited as a block, suggesting that a nonallelic interaction may occur between un-24 and het-6 and possibly other loci within this region to mediate vegetative incompatibility in the het-6 region of N. crassa.     

Molecular and functional analyses of incompatibility genes at het-6 in a population of Neurospora crassa

Two closely linked genes, un-24 and het-6, associated with the het-6 heterokaryon incompatibility functional haplotype were examined in 40 Neurospora crassa strains from a Louisiana sugarcane field. Partial diploid analyses were used to determine that half of the strains were functionally Oak Ridge (OR) and half were non-OR and indistinguishable from the standard Panama (PA) form. PCR-based markers were developed to identify polymorphisms within both un-24 and het-6. Two common forms of each gene occur based on these molecular markers. Rare forms of both un-24 and het-6 were identified as variants of the non-OR form by a DNA transformation assay. The heterokaryon incompatibility function of haplotypes, based on partial diploid analyses, was perfectly correlated with the PCR-based markers at both loci. This correlation indicates that the two loci are in severe linkage disequilibrium in this population sample and may act as an incompatibility gene complex. Southern hybridizations using OR- and PA-derived cloned probes from the region that spans un-24 and het-6 showed that the apparent absence of recombination in this approximately 25-kbp region is associated with low levels of overall sequence identity between the PA and OR forms.     

Nuclear DNA degradation during heterokaryon incompatibility in Neurospora crassa

Many filamentous fungi are capable of undergoing conspecific hyphal fusion with a genetically different individual to form a heterokaryon. However, the viability of such heterokaryons is dependent upon vegetative (heterokaryon) incompatibility (het) loci. If two individuals undergo hyphal anastomosis, but differ in allelic specificity at one or more het loci, the fusion cell is usually compartmentalized and self-destructs. Many of the microscopic features associated with vegetative incompatibility resemble apoptosis in metazoans and plants. To test the hypothesis whether vegetative incompatibility results in nuclear degradation, a characteristic of apoptosis, the cytology of hyphal fusions between incompatible Neurospora crassa strains that differed at three het loci, mat, het-c and het-6, and the cytology of transformants containing incompatible het-c alleles were examined using fluorescent DNA stains and terminal deoxynucleotidyl transferase-mediated dUTP-X nick end labeling (TUNEL). Hyphal fusion cells between het incompatible strains and hyphal segments in het-c incompatible transformants were compartmentalized by septal plugging and contained heavily degraded nuclear DNA. Hyphal fusion cells in compatible self-pairings and hyphal cells in het-c compatible transformants were not compartmentalized and rarely showed TUNEL-positive nuclei. Cell death events also were observed in senescent, older hyphae. Morphological features of hyphal compartmentation and death during vegetative incompatibility and the extent to which it is genetically controlled can best be described as a form of programmed cell death.     

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.     

Spreading the synaptonemal complex of Neurospora crassa

A protocol was developed to spread the synaptonemal complex (SC) of the fungus Neurospora crassa. It involves direct mechanical breakage of meiotic cells before spreading. This technique makes it possible to examine the SC of the same nucleus with both light and electron microscopy. This protocol is potentially applicable for other Pyrenomycetes. The SCs were examined at zygotene, pachytene and diplotene. The central elements and the recombination nodules (RN) were well revealed by silver staining. Ten to 13 RNs were counted at pachytene. The total genomic SC length varied with the stage. This whole mount electron microscopy of the SC is particularly useful for studying chromosomal rearrangements.     

Ancestral polymorphism and linkage disequilibrium at the het-6 region in pseudohomothallic Neurospora tetrasperma

In species of Neurospora, non-self recognition is mediated by at least 11 heterokaryon (het) incompatibility loci. Previously, we identified ancient allelic variation at het-c in pseudohomothallic N. tetrasperma, which confirmed outcrossing in this species. Here, we report distinct ancestral alleles at het-6 and un-24, two closely linked genes with het incompatibility function in N. crassa. The pattern of variation at het-6 and un-24 in N. tetrasperma is similar to that observed for N. crassa, where two ancestral allele specificities exist for each locus, Oak Ridge (het-6(OR), un-24(OR)) and Panama (het-6(PA), un-24(PA)). Only het-6(OR)/un-24(OR) and het-6(PA)/un-24(PA) allele combinations have been observed. The absence of recombinant haplotypes (e.g., het-6(OR)/un-24(PA)) appears to derive from an ancestral chromosomal rearrangement that limits recombination. Allelic variation at het-6 and un-24 in N. tetrasperma provides further evidence of outcrossing in this predominantly selfing species and indicates that selection maintains ancient allelic diversity at het loci.     

A nonself recognition gene complex in Neurospora crassa

Nonself recognition is exemplified in the fungal kingdom by the regulation of cell fusion events between genetically different individuals (heterokaryosis). The het-6 locus is one of approximately 10 loci that control heterokaryon incompatibility during vegetative growth of N. crassa. Previously, it was found that het-6-associated incompatibility in Oak Ridge (OR) strains involves two contiguous genes, het-6 and un-24. The OR allele of either gene causes "strong" incompatibility (cell death) when transformed into Panama (PA)-background strains. Several remarkable features of the locus include the nature of these incompatibility genes (het-6 is a member of a repetitive gene family and un-24 also encodes the large subunit of ribonucleotide reductase) and the observation that un-24 and het-6 are in severe linkage disequilibrium. Here, we identify "weak" (slow, aberrant growth) incompatibility activities by un-24PA and het-6PA when transformed separately into OR strains, whereas together they exhibit an additive, strong effect. We synthesized strains with the new allelic combinations un-24PA het-6OR and un-24OR het-6PA, which are not found in nature. These strains grow normally and have distinct nonself recognition capabilities but may have reduced fitness. Comparing the Oak Ridge and Panama het-6 regions revealed a paracentric inversion, the architecture of which provides insights into the evolution of the un-24-het-6 gene complex.     

Identification of vib-1, a locus involved in vegetative incompatibility mediated by het-c in Neurospora crassa

A non-self-recognition system called vegetative incompatibility is ubiquitous in filamentous fungi and is genetically regulated by het loci. Different fungal individuals are unable to form viable heterokaryons if they differ in allelic specificity at a het locus. To identify components of vegetative incompatibility mediated by allelic differences at the het-c locus of Neurospora crassa, we isolated mutants that suppressed phenotypic aspects of het-c vegetative incompatibility. Three deletion mutants were identified; the deletions overlapped each other in an ORF named vib-1 (vegetative incompatibility blocked). Mutations in vib-1 fully relieved growth inhibition and repression of conidiation conferred by het-c vegetative incompatibility and significantly reduced hyphal compartmentation and death rates. The vib-1 mutants displayed a profuse conidiation pattern, suggesting that VIB-1 is a regulator of conidiation. VIB-1 shares a region of similarity to PHOG, a possible phosphate nonrepressible acid phosphatase in Aspergillus nidulans. Native gel analysis of wild-type strains and vib-1 mutants indicated that vib-1 is not the structural gene for nonrepressible acid phosphatase, but rather may regulate nonrepressible acid phosphatase activity.     

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.     

Assembly of Tom6 and Tom7 into the TOM core complex of Neurospora crassa

Translocation of preproteins across the mitochondrial outer membrane is mediated by the translocase of the outer mitochondrial membrane (TOM) complex. We report the molecular identification of Tom6 and Tom7, two small subunits of the TOM core complex in the fungus Neurospora crassa. Cross-linking experiments showed that both proteins were found to be in direct contact with the major component of the pore, Tom40. In addition, Tom6 was observed to interact with Tom22 in a manner that depends on the presence of preproteins in transit. Precursors of both proteins are able to insert into the outer membrane in vitro and are assembled into authentic TOM complexes. The insertion pathway of these proteins shares a common binding site with the general import pathway as the assembly of both Tom6 and Tom7 was competed by a matrix-destined precursor protein. This assembly was dependent on the integrity of receptor components of the TOM machinery and is highly specific as in vitro-synthesized yeast Tom6 was not assembled into N. crassa TOM complex. The targeting and assembly information within the Tom6 sequence was found to be located in the transmembrane segment and a flanking segment toward the N-terminal, cytosolic side. A hybrid protein composed of the C-terminal domain of yeast Tom6 and the cytosolic domain of N. crassa Tom6 was targeted to the mitochondria but was not taken up into TOM complexes. Thus, both segments are required for assembly into the TOM complex. A model for the topogenesis of the small Tom subunits is discussed.     

Epistatic and synergistic interactions between circadian clock mutations in Neurospora crassa

We identified a series of epistatic and synergistic interactions among the circadian clock mutations of Neurospora crassa that indicate possible physical interactions among the various clock components encoded by these genes. The period-6 (prd-6) mutation, a short-period temperature-sensitive clock mutation, is epistatic to both the prd-2 and prd-3 mutations. The prd-2 and prd-3 long-period mutations show a synergistic interaction in that the period length of the double mutant strain is considerably longer than predicted. In addition, the prd-2 prd-3 double mutant strain also exhibits overcompensation to changes in ambient temperature, suggesting a role in the temperature compensation machinery of the clock. The prd-2, prd-3, and prd-6 mutations also show significant interactions with the frq(7) long-period mutation. These results suggest that the gene products of prd-2, prd-3, and prd-6 play an important role in both the timing and temperature compensation mechanisms of the circadian clock and may interact with the FRQ protein.     

Photoperiodism in Neurospora crassa

Plants and animals use day or night length for seasonal control of reproduction and other biological functions. Overwhelming evidence suggests that this photoperiodic mechanism relies on a functional circadian system. Recent progress has defined how flowering time in plants is regulated by photoperiodic control of output pathways, but the underlying mechanisms of photoperiodism remain to be described. The authors investigate photoperiodism in a genetic model system for circadian rhythms research, Neurospora crassa. They find that both propagation and reproduction respond systematically to photoperiod. Furthermore, a nonreproductive light-regulated function is also enhanced under certain photoperiodic conditions. All of these photoperiodic responses require a functional circadian clock, in that they are absent in a clock mutant. Night break experiments show that measuring night length is one of the mechanisms used for photoperiod assessment. This represents the first formal report of photoperiodism in the fungi.     

Uptake of Nitrite by Neurospora crassa

Like the nitrate transport system, the nitrite uptake system in Neurospora crassa is induced by either nitrate or nitrite. This induction is prevented by cycloheximide, puromycin, or 6-methyl purine. The K(m) for nitrite of the induced nitrite uptake system is 86 muM, and the V(max) is 100 mumol of nitrite per g (wet weight) per h. Nitrite uptake is inhibited by metabolic poisons such as arsenate, dinitrophenol, cyanide, and antimycin A. No repression or inhibition of the nitrite transport system by ammonia, nitrate, or Casamino Acids was observed.     

Metabolism of ricinoleate by Neurospora crassa

Neurospora crassa is a potential expression system for evaluating fatty-acid-modifying genes from plants producing uncommon fatty acids. One such gene encodes the hydroxylase that converts oleate to ricinoleate, a fatty acid with important industrial uses. To develop this expression system, it is critical to evaluate the metabolism and physiological effects of the expected novel fatty acid(s). We therefore examined effects of ricinoleate on lipid biosynthesis and growth of N. crassa. Ricinoleate inhibited growth and reduced levels of phospholipids and of 2-hydroxy fatty acids in glycolipids, but led to increased lipid accumulation on a mass basis. To evaluate incorporation and metabolism of ricinoleate, we followed the fate of 14 M–3 mM [1-¹⁴C]ricinoleate. The fate of the [¹⁴C]ricinoleate was concentration-dependent. At higher concentrations, ricinoleate was principally incorporated into triacylglycerols. At lower concentrations, ricinoleate was principally metabolized to other compounds. Thus, N. crassa transformants expressing the hydroxylase gene can be detected if the level of hydroxylase expression allows both growth and ricinoleate accumulation.     

Peptide utilization by nitrogen-starved Neurospora crassa

Peptides ranging in size from a mean number of 30 residues down to dipeptides supported growth of a leucine auxotroph when used as both a nitrogen and leucine source. Under nitrogen-limiting conditions, the peptides induced extracellular peptidohydrolytic activity, hydrolyzing peptides to monomer amino acids. Growth of a leu-2 mutant of Neurospora crassa on those peptides transportable by the oligopeptide transport system did not result in induction of hydrolytic activity, whereas growth of a leu-2; gltR mutant on these same peptides resulted in induction of peptidohydrolytic activity. The induced extracellular proteolytic activity was shown to be analogous to that inducible by growth on proteins, e.g., bovine serum albumin.     

Cryobiology of Neurospora crassa. II. Alteration in freeze response of Neurospora crassa conidia by additives

Neurospora crassa conidia in aqueous suspensions were frozen and thawed in the presence of various agents. Colony counts with these treatments were compared with those of the following (a) unfrozen, agent-treated, (b) unfrozen water suspended, and (c) frozen, water suspended. It was found that dimethyl sulfoxide (0.5–20%) resulted in total protection against freeze damage. Glycerol and calcium chloride decreased survival as much as 90% with fast freeze. The latter agents have properties which should decrease the rate of outflow of cellular water during temperature lowering. The results are consistent with the proposal that intracellular ice crystal growth to membrane rupturing dimensions is the damaging freeze mechanism under these conditions.