Heterokaryon Incompatibility Loci Research Articles

Natural Variation of Heterokaryon Incompatibility Gene het-c in Podospora anserina Reveals Diversifying Selection

In filamentous fungi, allorecognition takes the form of heterokaryon incompatibility, a cell death reaction triggered when genetically distinct hyphae fuse. Heterokaryon incompatibility is controlled by specific loci termed het-loci. In this article, we analyzed the natural variation in one such fungal allorecognition determinant, the het-c heterokaryon incompatibility locus of the filamentous ascomycete Podospora anserina. The het-c locus determines an allogenic incompatibility reaction together with two unlinked loci termed het-d and het-e. Each het-c allele is incompatible with a specific subset of the het-d and het-e alleles. We analyzed variability at the het-c locus in a population of 110 individuals, and in additional isolates from various localities. We identified a total of 11 het-c alleles, which define 7 distinct incompatibility specificity classes in combination with the known het-d and het-e alleles. We found that the het-c allorecognition gene of P. anserina is under diversifying selection. We find a highly unequal allele distribution of het-c in the population, which contrasts with the more balanced distribution of functional groups of het-c based on their allorecognition function. One explanation for the observed het-c diversity in the population is its function in allorecognition. However, alleles that are most efficient in allorecognition are rare. An alternative and not exclusive explanation for the observed diversity is that het-c is involved in pathogen recognition. In Arabidopsis thaliana, a homolog of het-c is a pathogen effector target, supporting this hypothesis. We hypothesize that the het-c diversity in P. anserina results from both its functions in pathogen-defense, and allorecognition.

Genesis of a Fungal Non-Self Recognition Repertoire

Conspecific allorecognition, the ability for an organism to discriminate its own cells from those of another individual of the same species, has been developed by many organisms. Allorecognition specificities are determined by highly polymorphic genes. The processes by which this extreme polymorphism is generated remain largely unknown. Fungi are able to form heterokaryons by fusion of somatic cells, and somatic non self-recognition is controlled by heterokaryon incompatibility loci (het loci). Herein, we have analyzed the evolutionary features of the het-d and het-e fungal allorecognition genes. In these het genes, allorecognition specificity is determined by a polymorphic WD-repeat domain. We found that het-d and het-e belong to a large gene family with 10 members that all share the WD-repeat domain and show that repeats of all members of the family undergo concerted evolution. It follows that repeat units are constantly exchanged both within and between members of the gene family. As a consequence, high mutation supply in the repeat domain is ensured due to the high total copy number of repeats. We then show that in each repeat four residues located at the protein/protein interaction surface of the WD-repeat domain are under positive diversifying selection. Diversification of het-d and het-e is thus ensured by high mutation supply, followed by reshuffling of the repeats and positive selection for favourable variants. We also propose that RIP, a fungal specific hypermutation process acting specifically on repeated sequences might further enhance mutation supply. The combination of these evolutionary mechanisms constitutes an original process for generating extensive polymorphism at loci that require rapid diversification.

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.

Autophagy is induced during cell death by incompatibility and is essential for differentiation in the filamentous fungus Podospora anserina.

In filamentous fungi, a cell death reaction occurs when cells of unlike genotype fuse. This cell death reaction, known as incompatibility reaction, is genetically controlled by a set of loci termed het loci (for heterokaryon incompatibility loci). In Podospora anserina, genes induced during this cell death reaction (idi genes) have been identified. The idi-6/pspA gene encodes a serine protease that is the orthologue of the vacuolar protease B of Saccharomyces cerevisiae involved in autophagy. We report here that the PSPA protease participates in the degradative autophagic pathway in Podospora. We have identified the Podospora orthologue of the AUT7 gene of S. cerevisiae involved in the early steps of autophagy in yeast. This gene is induced during the development of the incompatibility reaction and was designated idi-7. We have used a GFP-IDI7 fusion protein as a cytological marker of the induction of autophagy. Relocalization of this fusion protein and detection of autophagic bodies inside the vacuoles during the development of the incompatibility reaction provide cytological evidence of induction of autophagy during this cell death reaction. Therefore, cell death by incompatibility in fungi appears to be related to type II programmed cell death in metazoans. In addition, we found that pspA and idi-7 null mutations confer differentiation defects such as the absence of female reproductive structures, indicating that autophagy is required for differentiation in Podospora.

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.

Identification of specificity determinants and generation of alleles with novel specificity at the het-c heterokaryon incompatibility locus of Neurospora crassa.

The capacity for nonself recognition is a ubiquitous and essential aspect of biology. In filamentous fungi, nonself recognition during vegetative growth is believed to be mediated by genetic differences at heterokaryon incompatibility (het) loci. Filamentous fungi are capable of undergoing hyphal fusion to form mycelial networks and with other individuals to form vegetative heterokaryons, in which genetically distinct nuclei occupy a common cytoplasm. In Neurospora crassa, 11 het loci have been identified that affect the viability of such vegetative heterokaryons. The het-c locus has at least three mutually incompatible alleles, termed het-c(OR), het-c(PA), and het-c(GR). Hyphal fusion between strains that are of alternative het-c specificity results in vegetative heterokaryons that are aconidial and which show growth inhibition and hyphal compartmentation and death. A 34- to 48-amino-acid variable domain, which is dissimilar in HET-C(OR), HET-C(PA), and HET-C(GR), confers allelic specificity. To assess requirements for allelic specificity, we constructed chimeras between the het-c variable domain from 24 different isolates that displayed amino acid and insertion or deletion variations and determined their het-c specificity by introduction into N. crassa. We also constructed a number of artificial alleles that contained novel het-c specificity domains. By this method, we identified four additional and novel het-c specificities. Our results indicate that amino acid and length variations within the insertion or deletion motif are the primary determinants for conferring het-c allelic specificity. These results provide a molecular model for nonself recognition in multicellular eucaryotes.

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.

The protein product of the het-s heterokaryon incompatibility gene of the fungus Podospora anserina behaves as a prion analog.

The het-s locus of Podospora anserina is a heterokaryon incompatibility locus. The coexpression of the antagonistic het-s and het-S alleles triggers a lethal reaction that prevents the formation of viable heterokaryons. Strains that contain the het-s allele can display two different phenotypes, [Het-s] or [Het-s*], according to their reactivity in incompatibility. The detection in these phenotypically distinct strains of a protein expressed from the het-s gene indicates that the difference in reactivity depends on a posttranslational difference between two forms of the polypeptide encoded by the het-s gene. This posttranslational modification does not affect the electrophoretic mobility of the protein in SDS/PAGE. Several results suggest a similarity of behavior between the protein encoded by the het-s gene and prions. The [Het-s] character can propagate in [Het-s*] strains as an infectious agent, producing a [Het-s*] --> [Het-s] transition, independently of protein synthesis. Expression of the [Het-s] character requires a functional het-s gene. The protein present in [Het-s] strains is more resistant to proteinase K than that present in [Het-s*] mycelium. Furthermore, overexpression of the het-s gene increases the frequency of the transition from [Het-s*] to [Het-s]. We propose that this transition is the consequence of a self-propagating conformational modification of the protein mediated by the formation of complexes between the two different forms of the polypeptide.

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.

New round spore mutations in Neurospora crassa accompanying changes in a duplication closely linked to the R locus

Various genotypes can result in round ascospores in N. crassa (Mitchell 1966 Neurospora Newsl. 10:6; Barry et al. 1972 Neurospora Newsl. 19:17). I have observed that round ascospores can also originate in crosses where one parent is the apparent breakdown of a partial diploid heterozygous at the heterokaryon incompatibility locus het-5. The breakdown may remove one of the duplicated chromosome segments carrying a het-5 allele and possibly extend past the duplication and affect the nearby Round spore locus (R).

Loci independent of mating type that condition secondary sexual incompatibility in the fungus Cochliobolus heterostrophus

A full sib mating design was used to select for increased perithecial number in matings between sexually compatible pairs of ascospore progeny of the fungus Cochliobolus heterostrophus. In the third generation of selection approximately one-half of the isolate pairs produced abnormally low numbers of perithecia, indicating segregation at a single locus controlling fertility, which was independent of the mating type locus. We termed the phenomenon secondary sexual incompatibility, although it may also be related to vegetative compatibility. Large numbers of perithecia formed when the paired sexually compatible isolates had different alleles at the locus; few formed when they had identical alleles. Additional studies of two full sib families from another population showed the presence of at least four loci that condition secondary sexual incompatibility among these isolates. High fertility occurred only between isolates with different alleles at least one of the four loci. This phenomenon appears similar to the enhancement of fertility in Ceratocystis ulmi by genes for heterokaryon incompatibility, but the relationship of secondary sexual incompatibility loci to heterokaryon incompatibility loci in C. heterostrophys has not been determined.

HETEROMORPHISM FOR HETEROKARYON INCOMPATIBILITY GENES IN NATURAL POPULATIONS OF NEUROSPORA CRASSA

Five Neurospora crassa isolates from each of three sites in Louisiana were compared for genotype at five heterokaryon incompatibility (het) loci. The comparisons were made using duplications (partial diploids), based on the fact that duplications heterozygous for het loci have strikingly abnormal phenotypes which greatly facilitate the study of such genes. Duplications were synthesized in crosses between the wild strains (normal chromosome sequence) and testers of defined het genotype and having duplication-producing chromosome rearrangements. Crosses segregating for phenotypes characteristic of duplications heterozygous for het loci indicated allelic differences between testers and wild strains for specific het genes. Whenever a wild strain differed from a tester for a specific het locus, but another wild strain did not, the two wild strains could be inferred to differ from each other.-No two isolates from any site were heterokaryon compatible (of identical het genotype), despite the fact that all isolates from each of two sites occurred within several meters of each other. Heteromorphism was found for all five genes studied at one site, four genes at another site, and three at another. Intra- and interpopulation differences between strains were approximately the same.-Confirmation is also provided that two het genes originally detected in duplications are in fact heterokaryon incompatibility loci.

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.

A search for complexity at the mating-type locus of Neurospora crassa.

Evidence for complexity at the mating-type locus of Neurospora crassa was sought by selecting recombinants between closely linked markers on either side. All recombinants were tested for crossing ability, to test the hypothesis that the two mating-type alleles are actually closely linked self-sterile mutants; such tests should also detect subunits analogous to the α and β subunits of the A factor of Schizophyllum or Coprinus. No change in crossing ability was found among the 5,019 recombinants tested, representing 235,000 viable ascospores. The results indicate that if subunits exist, they are not more than 0.002 units apart. Twelve hundred and forty of the recombinants were tested in a way that should also have detected subunits analogous to the A and B factors of Schizophyllum and Coprinus, except that A and B would be closely linked. No such subunits were detected.N. crassa strains of opposite mating type are heterokaryon-incompatible during vegetative growth, and observations of various investigators have suggested that the heterokaryon incompatibility might be controlled by a separate closely-linked gene rather than by mating type itself. A sample of the recombinants was therefore tested for separation of the heterokaryon-incompatibility and crossing-compatibility functions. (Heterokaryon-incompatibility was scored by the presence of an incompatibility reaction in duplications heterozygous for mating type; this technique is simple and eliminates complications due to unlinked heterokaryon-incompatibility loci, several of which are known in N. crassa.) No separation was found. The results indicate that if an adjacent gene is responsible for the heterokaryon-incompatibility, it is not more than 0.0078 units from mating type, if on the left, and not more than 0.018 units from mating type, if on the right.