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.     

Suppressor Mutants of Neurospora Crassa That Tolerate Allelic Differences at Single or at Multiple Heterokaryon Incompatibility Loci

Allelic differences at any one of at least 11 heterokaryon incompatibility (het) loci in Neurospora crassa trigger an incompatibility response: localized cell death at sites of hyphal anastomosis. We have isolated spontaneous and insertional suppressor mutants that are heterokaryon-compatible in spite of allelic differences at one or at several het loci. Some intra- and extragenic mutants tolerated allelic differences only at single het loci. Multi-tolerant spontaneous mutants were isolated by selecting simultaneously for tolerance of differences at het-c, -d and -e, or at each of these plus mating-type. Some suppressor mutants were specific for only one allele at the affected het locus; others suppressed both alleles. Insertional mutations were isolated from banks of transformants, each having a plasmid integrated into a random position in the chromosome. One mutant tolerated allelic differences at het-d. A homologous cosmid from a Neurospora genomic bank complemented the mutant phenotype. A second insertional inactivation mutant was tolerant of het-c differences. Inactivation of the wild-type locus corresponding to the integration site was accomplished by repeat-induced point mutation (RIP). The RIP progeny, like the original mutant, were tolerant of differences at het-c. It may be possible to use such suppressor mutants as universal donors of hypovirulence in pathogenic fungi.     

Characterization of mat A-2, mat A-3 and deltamatA mating-type mutants of Neurospora crassa.

The mating-type locus of Neurospora crassa regulates mating identity and entry into the sexual cycle. The mat A idiomorph encodes three genes, mat A-1, mat A-2, and mat A-3. Mutations in mat A-1 result in strains that have lost mating identity and vegetative incompatibility with mat a strains. A strain containing mutations in both mat A-2 and mat A-3 is able to mate, but forms few ascospores. In this study, we describe the isolation and characterization of a mutant deleted for mat (deltamatA), as well as mutants in either mat A-2 or mat A-3. The deltamatA strain is morphologically wild type during vegetative growth, but it is sterile and heterokaryon compatible with both mat A and mat a strains. The mat A-2 and mat A-3 mutants are also normal during vegetative growth, mate as a mat A strain, and produce abundant biparental asci in crosses with mat a, and are thus indistinguishable from a wild-type mat A strain. These data and the fact that the mat A-2 mat A-3 double mutant makes few asci with ascospores indicate that MAT A-2 and MAT A-3 are redundant and may function in the same pathway. Analysis of the expression of two genes (sdv-1 and sdv-4) in the various mat mutants suggests that the mat A polypeptides function in concert to regulate the expression of some sexual development genes.     

Mating type and mating strategies in Neurospora

In the heterothallic species Neurospora crassa, strains of opposite mating type, A and a, must interact to give the series of events resulting in fruiting body formation, meiosis, and the generation of dormant ascospores. The mating type of a strain is specified by the DNA sequence it carries in the mating type region; strains that are otherwise isogenic can mate and produce ascospores. The DNA of the A and a regions have completely dissimilar sequences. Probing DNA from strains of each mating type with labelled sequences from the A and the a regions has shown that, unlike in Saccharomyces cerevisiae, only a single copy of a mating type sequence is present in a haploid genome. The failure to switch is explainable by the physical absence of DNA sequences characteristic of the opposite mating type. While the mating type sequences must be of the opposite kind for mating to occur in the sexual cycle, two strains of opposite mating type cannot form a stable heterokaryon during vegetative growth; instead, they fuse abortively to give a heterokaryon incompatibility reaction, which results in death of the cells along the fusion line. The DNA sequences responsible for this reaction are coextensive with those sequences in the A and a regions which are necessary to initiate fruiting body formation. The genus Neurospora also includes homothallic species--ones in which a single haploid nucleus carries all the information necessary to form fruiting bodies, undergo meiosis, and produce new haploid spores. One such species, N. terricola, contains one copy each of the A and the a sequences within each haploid genome.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Germline Transformation Using a Prune Cdna Rescues Prune/Killer of Prune Lethality and the Prune Eye Color Phenotype in Drosophila

Filamentous fungi are capable of hyphal fusion, but heterokaryon formation between different isolates is controlled by specific loci termed het loci. Heterokaryotic cells formed between strains of different het genotype are rapidly destroyed or strongly inhibited in their growth. In Neurospora crassa, at least 11 loci, including the mating type locus, affect the capacity to form a heterokaryon between different isolates. In this report, we describe the molecular characterization of the vegetative incompatibility locus, het-C. The het-C(OR) allele was cloned by genetically identifying the het-C locus in a chromosome walk, and the activity of clones containing the het-C(OR) allele was tested in a functional transformation assay. The het-C(OR) allele encodes a 966-amino acid polypeptide with a putative signal peptide, a coiled-coil motif and a C-terminal glycine-rich domain, similar to glycine-rich domains detected in various extracellular and structural cell envelope proteins. Both the coiled-coil and one-third of the glycine-rich carboxyl terminal domains were required for full het-C(OR) activity. Mutants of het-C(OR) were obtained by repeat-induced point mutation (RIP); these mutants were indistinguishable from wild type during vegetative growth and sexual reproduction but displayed dual compatibility with both of two mutually incompatible het-C(OR) and het-c(PA) strains.     

The effect of Wolbachia versus genetic incompatibilities on reinforcement and speciation

Wolbachia is a widespread group of intracellular bacteria commonly found in arthropods. In many insect species, Wolbachia induce a cytoplasmic mating incompatibility (CI). If different Wolbachia infections occur in the same host species, bidirectional CI is often induced. Bidirectional CI acts as a postzygotic isolation mechanism if parapatric host populations are infected with different Wolbachia strains. Therefore, it has been suggested that Wolbachia could promote speciation in their hosts. In this article we investigate theoretically whether Wolbachia-induced bidirectional CI selects for premating isolation and therefore reinforces genetic divergence between parapatric host populations. To achieve this we combined models for Wolbachia dynamics with a well-studied reinforcement model. This new model allows us to compare the effect of bidirectional CI on the evolution of female mating preferences with a situation in which postzygotic isolation is caused by nuclear genetic incompatibilities (NI). We distinguish between nuclear incompatibilities caused by two loci with epistatic interactions, and a single locus with incompatibility among heterozygotes in the diploid phase. Our main findings are: (1) bidirectional CI and single locus NI select for premating isolation with a higher speed and for a wider parameter range than epistatic NI; (2) under certain parameter values, runaway sexual selection leads to the increase of an introduced female preference allele and fixation of its preferred male trait allele in both populations, whereas under others it leads to divergence in the two populations in preference and trait alleles; and (3) bidirectional CI and single locus NI can stably persist up to migration rates that are two times higher than seen for epistatic NI. The latter finding is important because the speed with which mutants at the preference locus spread increases exponentially with the migration rate. In summary, our results show that bidirectional CI selects for rapid premating isolation and so generally support the view that Wolbachia can promote speciation in their hosts.     

Directed Replacement of Mt a by Mt a-1 Effects a Mating Type Switch in Neurospora Crassa

To test the functions of a mating type genes, we developed an efficient strategy to select transformants of Neurospora crassa in which resident A mating type DNA was replaced by cloned DNA from the mt a idiomorph. Cloned a idiomorphic DNA could specify all functions, including fertility, of a mating type, but only when it replaced A DNA at the mating type locus. Only the mt a-1 region of the a idiomorph was necessary in order to specify a mating type. Gene replacement events involved the homologous sequences flanking the unique mating type idiomorphic DNA, resulting in apparently isogenic a and A strains. These isogenic strains were fertile when crossed with one another, indicating that no determinants outside the transforming DNA are necessary for fertility as a and that no host sequences of A strains interfere with fertility as a. One a replacement strain bore a duplication of the transforming mt a-1 and hph DNA. The duplication strain had unexpected properties. Although mating type segregated 1:1 in crosses of this strain to A, the duplicated regions were efficiently altered during the sexual process to generate a single copy in the progeny. No progeny were recovered that had undergone RIP (repeat induced point mutation) sufficient to inactivate the mt a-1 gene. We infer that the mt a-1 gene is necessary and sufficient to specify a mating type identity in all vegetative and sexual activities. Mt a-1 may also play an essential role in ascosporogenesis after fertilization.     

Ultraviolet Light-Induced Heterokaryon Formation and Parasexuality in Cryphonectria parasitica

Rizwana, R., and Powell, W. A. 1995. Ultraviolet light-induced heterokaryon formation and parasexuality in Cryphonectria parasitica. Experimental Mycology 19, 48-60. The effect of ultraviolet-light on heterokaryon formation, vegetative compatibility, and parasexuality in Cryphonectria parasitica was examined. Heterokaryons of complementary auxotrophic strains could not be made by hyphal anastomosis if the strains belonged to different vegetative compatibility groups. Protoplast fusions overcame incompatibility of strains differing in the alleles of a single but not multiple vegetative incompatibility loci. Fusion of protoplasts from ultraviolet light-treated complementary auxotrophs increased heterokaryon formation by 10⁴ to 10⁵ using the strains differing in alleles of a single vegetative incompatibility gene but had no detectable effect on strains differing in multiple vegetative incompatibility genes. Vegetative compatibility tests of single conidial isolates resolved from these heterokaryons suggest that diploids had formed followed by the loss of one of the VIC alleles. Presence of both auxotrophic markers in some of these single conidial isolates confirms the occurrence of a parasexual cycle. These experiments demonstrate that ultraviolet-light can enhance heterokaryon formation and parasexuality in C. parasitica .     

Mutations of the a Mating-Type Gene in NEUROSPORA CRASSA

In Neurospora, the mating-type locus controls both mating ( A + a is fertile) and heterokaryosis (A + a is incompatible). The two alleles appear stable: no novel fertility reactions have ever been reported, and attempts to separate fertility and heterokaryon incompatibility functions by recombination have been unsuccessful. In the present approach the locus was studied through a mutational analysis of heterokaryon incompatibility function. A selection system was used that detects vigorous (A + a) heterokaryotic colonies against a background of inhibited growth. Twenty-five mutants of an a strain were produced following mutagenic treatment with UV and NG: 15 were viable as homokaryons and 10 were not. All but one were infertile, but most showed an abortive mating reaction involving the production of barren, well-developed perithecia with A and (surprisingly) a testers. None of the mutants complement each other to restore fertility. Seven mutants have been mapped to the mating-type locus region of chromosome 1. Restoration of fertility was used to detect revertants, and these were found in five out of the eight mutants tested. (A dose response was observed). In four cases incompatibility was fully restored and in one case it was not.—The results suggest two positive actions of the locus when in heterozygous (A/a) combination (the stimulation of some stage of ascus production and the inhibition of vegetative heterokaryosis), and one positive action in homozygous combination (the production of a perithecial inhibitor).     

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.     

Mating Type Linked Mutations Which Disrupt the Uniparental Transmission of Chloroplast Genes in Chlamydomonas

In Chlamydomonas reinhardtii, chloroplast genomes are normally transmitted by the mating type plus (mt+) parent and mitochondrial genomes by the mating type minus (mt-) parent. In this paper we describe three new nuclear mutations, designated mat-3-1 to -3, which are tightly linked to the mt+ allele and permit high transmission of chloroplast genomes from the mt- parent, but have no effect on transmission of mitochondrial genomes. We also show that mat-1, reported by others to be a nuclear mutation linked to mt- which promotes transmission of chloroplast genomes by the mt- parent, is probably a vegetative diploid since it contains both mt+ and mt- alleles. Vegetative diploids behave as if they are mt- with respect to mating, but possess a level of chloroplast gene transmission intermediate between that of haploid mt- and mt+ stocks.     

Recurrent locus-specific mutation resulting from a cryptic ectopic insertion in Neurospora

New mutations are found among approximately 20% of progeny when one or both parents carry eas allele UCLA191 (eas(UCLA), easily wettable, hydrophobin-deficient, linkage group II). The mutations inactivate the wild-type allele of cya-8 (cytochrome aa3 deficient, linkage group VII), resulting in thin, "transparent" mycelial growth. Other eas alleles fail to produce cya-8 mutant progeny. The recurrent cya-8 mutations are attributed to repeat-induced point mutation (RIP) resulting from a duplicated copy of cya-8+ that was inserted ectopically at eas when the UCLA191 mutation occurred. As expected for RIP, eas(UCLA)-induced cya-8 mutations occur during nuclear proliferation prior to karyogamy. When only one parent is eas(UCLA), the new mutations arise exclusively in eas(UCLA) nuclei. Mutation of cya-8 is suppressed when a long unlinked duplication is present. Stable cya-8 mutations are effectively eliminated in crosses homozygous for rid, a recessive suppressor of RIP. The eas(UCLA) allele is associated with a long paracentric inversion. A discontinuity is present in eas(UCLA) DNA. The eas promoter is methylated in cya-8 progeny of eas(UCLA), presumably by the spreading of methylation beyond the adjoining RIP-inactivated duplication. These findings support a model in which an ectopic insertion that created a mutation at the target site acts as a locus-specific mutator via RIP.     

Uniparental Inheritance and Replacement of Mitochondrial DNA in Neurospora Tetrasperma

This study tested mechanisms proposed for maternal uniparental mitochondrial inheritance in Neurospora: (1) exclusion of conidial mitochondria by the specialized female reproductive structure, trichogyne, due to mating locus heterokaryon incompatibility and (2) mitochondrial input bias favoring the larger trichogyne over the smaller conidium. These mechanisms were tested by determining the modes of mitochondrial DNA (mtDNA) inheritance and transmission in the absence of mating locus heterokaryon incompatibility following crosses of uninucleate strains of Neurospora tetrasperma with trichogyne (trichogyne inoculated by conidia) and without trichogyne (hyphal fusion). Maternal uniparental mitochondrial inheritance was observed in 136 single ascospore progeny following both mating with and without trichogyne using mtDNA restriction fragment length polymorphisms to distinguish parental types. This suggests that maternal mitochondrial inheritance following hyphal fusions is due to some mechanism other than those that implicate the trichogyne. Following hyphal fusion, mututally exclusive nuclear migration permitted investigation of reciprocal interactions. Regardless of which strain accepted nuclei following seven replicate hyphal fusion matings, acceptor mtDNA was the only type detected in 34 hyphal plug and tip samples taken from the contact and acceptor zones. No intracellular mtDNA mixtures were detected. Surprisingly, 3 days following hyphal fusion, acceptor mtDNA replaced donor mtDNA throughout the entire colony. To our knowledge, this is the first report of complete mitochondrial replacement during mating in a filamentous fungus.     

Heteroallelism at the het-c locus contributes to sexual dysfunction in outcrossed strains of Neurospora tetrasperma.

Neurospora tetrasperma is naturally heterokaryotic, with cells possessing haploid nuclei of both a and A mating types. As a result, isolates are self-fertile (pseudohomothallic). Occasional homokaryotic ascospores and conidia arise, however, and they produce strains that are self-sterile and must outcross to complete sexual reproduction. Invariably, laboratory crosses employing sibling a and A strains from the same parental heterokaryon restore the pseudohomothallic, heterokaryotic state. In contrast, outcrosses employing a and A strains from different wild isolates typically result in sexual dysfunction. Diverse sexual dysfunction types have been observed, ranging from complete sterility to reduced viability. We report that one type of dysfunction, characterized by spontaneous loss of the heterokaryotic state upon ascospore germination, can result from the interaction of incompatible alleles at heterokaryon incompatibility loci. Specifically, we demonstrate that homoallelism at the het-c locus in N. tetrasperma is required for heterokaryon stability. Heterokaryon incompatibility therefore provides an obstacle to outcrossing in this species, an observation with important implications for fungal life-cycle evolution.     

An osmotic-remedial, temperature-sensitive mutation in the allosteric activity site of ribonucleotide reductase in Neurospora crassa

An osmotic-remedial, temperature-sensitive conditional mutant (un-24) was generated by Repeat Induced Point mutation (RIP) from a cross between a wild-type N. crassa strain and a strain carrying a approximately 250-kb duplication of the left arm of linkage group II (LGII). The mutation was mapped to the duplicated segment, within 2.6 map units of the heterokaryon incompatibility locus het-6. DNA transformation identified a 3.75-kb fragment that complemented the temperature-sensitive phenotype. A large ORF within this fragment was found to have a high degree of sequence identity to the large subunit of ribonucleotide reductase (RNR) from diverse organisms. Conserved amino acids at the active site and the allosteric activity sites are also evident. An unusual feature of the Neurospora sequence is a large insertion near the C-terminus relative to otherwise homologous sequences from other organisms. Three transition mutations, indicative of RIP, were identified in the N-terminal region of the temperature-sensitive mutant allele. One of these mutations results in a non-conservative amino acid substitution within the four-helix bundle that is important in the allosteric control of ribonucleotide reductase activity. This substitution appears to disrupt proper folding of the allosteric activity site during synthesis of the protein.