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.     

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.     

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.     

The het-c heterokaryon incompatibility gene in Aspergillus niger

Heterokaryon incompatibility among Aspergillus niger strains is a widespread phenomenon that is observed as the inability to form stable heterokaryons. The genetic basis of heterokaryon incompatibility reactions is well established in some sexual filamentous fungi but largely unknown in presumed asexual species, such as A. niger. To test whether the genes that determine heterokaryon incompatibility in Neurospora crassa, such as het-c, vib-1 and pin-c, have a similar function in A. niger, we performed a short in silico search for homologues of these genes in the A. niger and several related genomes. For het-c, pin-c and vib-1 we did indeed identify putative orthologues. We then screened a genetically diverse worldwide collection of incompatible black Aspergilli for polymorphisms in the het-c orthologue. No size variation was observed in the variable het-c indel region that determines the specificity in N. crassa. Sequence comparison showed only minor variation in the number of glutamine coding triplets. However, introduction of one of the three N. crassa alleles (het-c2) in A. niger by transformation resulted in an abortive phenotype, reminiscent of the heterokaryon incompatibility in N. crassa. We conclude that although the genes required are present and the het-c homologue could potentially function as a heterokaryon incompatibility gene, het-c has no direct function in heterokaryon incompatibility in A. niger because the necessary allelic variation is absent.     

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.     

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.     

HET-E and HET-D belong to a new subfamily of WD40 proteins involved in vegetative incompatibility specificity in the fungus Podospora anserina

Vegetative incompatibility, which is very common in filamentous fungi, prevents a viable heterokaryotic cell from being formed by the fusion of filaments from two different wild-type strains. Such incompatibility is always the consequence of at least one genetic difference in specific genes (het genes). In Podospora anserina, alleles of the het-e and het-d loci control heterokaryon viability through genetic interactions with alleles of the unlinked het-c locus. The het-d2(Y) gene was isolated and shown to have strong similarity with the previously described het-e1(A) gene. Like the HET-E protein, the HET-D putative protein displayed a GTP-binding domain and seemed to require a minimal number of 11 WD40 repeats to be active in incompatibility. Apart from incompatibility specificity, no other function could be identified by disrupting the het-d gene. Sequence comparison of different het-e alleles suggested that het-e specificity is determined by the sequence of the WD40 repeat domain. In particular, the amino acids present on the upper face of the predicted beta-propeller structure defined by this domain may confer the incompatible interaction specificity.     

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.     

Molecular genetics of heterokaryon incompatibility in filamentous ascomycetes.

Filamentous fungi spontaneously undergo vegetative cell fusion events within but also between individuals. These cell fusions (anastomoses) lead to cytoplasmic mixing and to the formation of vegetative heterokaryons (i.e., cells containing different nuclear types). The viability of these heterokaryons is genetically controlled by specific loci termed het loci (for heterokaryon incompatibility). Heterokaryotic cells formed between individuals of unlike het genotypes undergo a characteristic cell death reaction or else are severely inhibited in their growth. The biological significance of this phenomenon remains a puzzle. Heterokaryon incompatibility genes have been proposed to represent a vegetative self/nonself recognition system preventing heterokaryon formation between unlike individuals to limit horizontal transfer of cytoplasmic infectious elements. Molecular characterization of het genes and of genes participating in the incompatibility reaction has been achieved for two ascomycetes, Neurospora crassa and Podospora anserina. These analyses have shown that het genes are diverse in sequence and do not belong to a gene family and that at least some of them perform cellular functions in addition to their role in incompatibility. Divergence between the different allelic forms of a het gene is generally extensive, but single-amino-acid differences can be sufficient to trigger incompatibility. In some instances het gene evolution appears to be driven by positive selection, which suggests that the het genes indeed represent recognition systems. However, work on nonallelic incompatibility systems in P. anserina suggests that incompatibility might represent an accidental activation of a cellular system controlling adaptation to starvation.     

Escape from mating-type incompatibility in bisexual (A + a) Neurospora heterokaryons.

In Neurospora crassa, strains of opposite mating type generally do not form stable heterokaryons because the mating type locus acts as a heterokaryon incompatibility locus. However, when one A and one a strain, having complementing auxotrophic mutants, are placed together on minimal medium, growth may occur, although the growth is generally slow. In this study, escape from such slow growth to that at a wild type or near-wild type rate was observed. The escape cultures are stable heterokaryons, mostly having lost the mating type allele function from one component nucleus, so that the nuclear types are heterokaryon compatible. Either A or a mating type can be lost. This loss of function has been attributed to deletion since only one nuclear type could be recovered in all heterokaryons except one, but deletion spanning adjacent loci has been directly demonstrated in a minority of cases. Alternatively when one component strain is tol and the other tol+ (tol being a recessive mutant suppressing the heterokaryon incompatibility associated with mating type), escape may occur by the deletion or mutation of tol+, also resulting in heterokaryon compatibility. An induction mechanism for escape is speculated upon.     

The location and analysis of two heterokaryon incompatibility (het) loci in strains of Aspergillus nidulans

The heterokaryon incompatibility system in Aspergillus nidulans has been investigated by parasexual methods. The use of complementary auxotrophs with a repeated serial transfer method or with a protoplast fusion technique has enabled heterokaryons and diploid strains to be recovered from heterokaryon incompatible combinations of strains. The effects of allelic interaction at heterokaryon incompatibility (het) loci on the morphologies of the heterokaryon and diploid colonies isolated are described. Parasexual analyses conducted among strains belonging to the heterokaryon compatibility groups, h-cGl and h-cB, and the two recombinant compatibility classes, have located the hetA and hetB genes to linkage groups V and VI respectively.     

Heterokaryon incompatibility function of barrage-associated vegetative incompatibility genes (vic) in Cryphonectria parasitica

Six vegetative incompatibility (vic) loci have been identified in Cryphonectria parasitica based on barrage formation during mycelial interactions. We used hygromycin B- and benomyl-resistance as forcing markers in C. parasitica strains to test whether heteroallelism at each vic locus prevents heterokaryon formation following mycelial interactions. Paired strains that had allelic differences at any of vic1, 2, 3, 6 or 7 but not vic4 displayed heterokaryon incompatibility function, as recognized by slow growth or aberrant morphology. While clearly forming barrages in mycelial interactions, paired strains with different alleles at vic4 formed stable heterokaryons. With examples from other fungi, this inconsistency at vic4 suggests that barrage formation and heterokaryon incompatibility are not different manifestations of the same process. Rather, the evidence indicates that heterokaryon incompatibility represents a component of a vegetative incompatibility system that may also use cell-surface or extracellular factors to trigger programmed cell death to modulate nonself recognition in fungi.     

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.     

The Use of Duplication-Generating Rearrangements for Studying Heterokaryon Incompatibility Genes in Neurospora

Heterokaryon (vegetative) incompatibility, governing the fusion of somatic hyphal filaments to form stable heterokaryons, is of interest because of its widespread occurrence in fungi and its bearing on cellular recognition. Conventional investigations of the genetic basis of heterokaryon incompatibility in N. crassa are difficult because in commonly used stocks differences are present at several het loci, all with similar incompatibility phenotypes. This difficulty is overcome by using duplications (partial diploids) that are unlikely to contain more than one het locus. A phenotypically expressed incompatibility reaction occurs when unlike het alleles are present within the same somatic nucleus, and this parallels the heterokaryon incompatibility reaction that occurs when unlike alleles in different haploid nuclei are introduced into the same somatic hypha by mycelial fusion.—Nontandem duplications were used to confirm that the incompatibility reactions in heterokaryons and in duplications are alternate expressions of the same genes. This was demonstrated for three loci which had previously been established by conventional heterokaryon tests—het-e, het-c and mt. These were each obtained in duplications as recombinant meiotic segregants from crosses heterozygous for duplication-generating chromosome rearrangements. The particular method of producing the duplications is irrelevant so long as the incompatibility alleles are heterozygous.—The duplication technique has made it possible to determine easily the het-e and het-c genotypes of numerous laboratory and wild strains of unknown constitution. In laboratory strains both loci are represented simply by two alleles. Analysis of het-c is more complicated in some wild strains, where differences have been demonstrated at one or more additional het loci within the duplication used and multiple allelism is also possible.—The results show that the duplication method can be used to identify and map additional vegetative incompatibility loci, without the necessity of heterokaryon tests.     

A chromosome assay method for the detection of heterokaryon incompatibility (het) genes operating between members of different heterokaryon compatibility (h-c) groups in Aspergillus nidulans

Protoplast fusion has made possible the isolation of a diploid strain from haploid parents belonging to heterokaryon compatibility (h-c) groups Q and Gl of Aspergillus nidulans. This diploid was not fully heterokaryon compatibility tests conducted between selected pairs of parasexually derived progeny strains facilitated a chromosome assay method for the detection of heterokaryon incompatibility (het) genes. Despite the lack of segregation for the linkage group VI marker, it proved possible to locate het genes on linkage groups III, V, VI and VII. Backcross data detected five het gene differences operating between the h-cQ and h-cGl parental strains. Two het loci were located on linkage group III.