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.     

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.     

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.     

Escape from Het-6 Incompatibility in Neurospora Crassa Partial Diploids Involves Preferential Deletion within the Ectopic Segment

Self-incompatible het-6(OR)/het-6(PA) partial diploids of Neurospora crassa were selected from a cross involving the translocation strain, T(IIL -> IIIR)AR18, and a normal sequence strain. About 25% of the partial diploids exhibited a marked increase in growth rate after 2 weeks, indicating that ``escape' from het-6 incompatibility had occurred. Near isogenic tester strains with different alleles (het-6(OR) and het-6(PA)) were constructed and used to determine that 80 of 96 escape strains tested were het-6(PA), retaining the het-6 allele found in the normal-sequence LGII position; 16 were het-6(OR), retaining the allele in the translocated position. Restriction fragment length polymorphisms in 45 escape strains were examined with probes made from cosmids that spanned the translocated region. Along with electrophoretic analysis of chromosomes from three escape strains, RFLPs showed that escape is associated with deletion of part of one or the other of the duplicated DNA segments. Deletions ranged in size from ~70 kbp up to putatively the entire 270-kbp translocated region but always included a 35-kbp region wherein we hypothesize het-6 is located. The deletion spectrum at het-6 thus resembles other cases where mitotic deletions occur such as of tumor suppressor genes and of the hprt gene (coding for hypoxanthine-guanine phosphoribosyl-transferase) in humans.     

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.     

Nonallelic interactions between het-c and a polymorphic locus, pin-c, are essential for nonself recognition and programmed cell death in Neurospora crassa

Nonself recognition in filamentous fungi is conferred by genetic differences at het (heterokaryon incompatibility) loci. When individuals that differ in het specificity undergo hyphal fusion, the heterokaryon undergoes a programmed cell death reaction or is highly unstable. In Neurospora crassa, three allelic specificities at the het-c locus are conferred by a highly polymorphic domain. This domain shows trans-species polymorphisms indicative of balancing selection, consistent with the role of het loci in nonself recognition. We determined that a locus closely linked to het-c, called pin-c (partner for incompatibility with het-c) was required for het-c nonself recognition and heterokaryon incompatibility (HI). The pin-c alleles in isolates that differ in het-c specificity were extremely polymorphic. Heterokaryon and transformation tests showed that nonself recognition was mediated by synergistic nonallelic interactions between het-c and pin-c, while allelic interactions at het-c increased the severity of the HI phenotype. The pin-c locus encodes a protein containing a HET domain; predicted proteins containing HET domains are frequent in filamentous ascomycete genomes. These data suggest that nonallelic interactions may be important in nonself recognition in filamentous fungi and that proteins containing a HET domain may be a key factor in these interactions.     

Nonself Recognition Through Intermolecular Disulfide Bond Formation of Ribonucleotide Reductase in Neurospora

Type I ribonucleotide reductases (RNRs) are conserved across diverse taxa and are essential for the conversion of RNA into DNA precursors. In Neurospora crassa, the large subunit of RNR (UN-24) is unusual in that it also has a nonself recognition function, whereby coexpression of Oak Ridge (OR) and Panama (PA) alleles of un-24 in the same cell leads to growth inhibition and cell death. We show that coexpressing these incompatible alleles of un-24 in N. crassa results in a high molecular weight UN-24 protein complex. A 63-amino-acid portion of the C terminus was sufficient for un-24^PA incompatibility activity. Redox active cysteines that are conserved in type I RNRs and essential for their catalytic function were found to be required for incompatibility activity of both UN-24^OR and UN-24^PA. Our results suggest a plausible model of un-24 incompatibility activity in which the formation of a complex between the incompatible RNR proteins is potentiated by intermolecular disulfide bond formation.     

A constitutive mutation in a posttranscriptional regulator gene for a phenoloxidase and proteases in Podospora anserina

A recessive mutation in the gene mod-2 results in the synthesis at low temperatures of a phenoloxidase and an arrest of growth, reversible by beta-phenyl-pyruvic acid, a protease inhibitor. Phenoloxidase synthesis is 5-fluorouracil resistant and cycloheximade sensitive. Suppression of both cold sensitivity and phenoloxidase synthesis by common factors (higher NH4+ concentrations or mutations) suggests that the protease, suspected to be responsible for cold sensitivity, also arises from preexisting mRNA molecules. Instead of being recessive and constitutive, the mod-2 mutations is suppressive and dominant when cold sensitivity and phenoloxidases synthesis are induced as the consequence of the nonallelic gene interactions C/D, C/E, or R/V responsible for protoplasmic incompatibility. Combinations of nonallelic incompatibility systems and several mod-2 mutations lead us to hypothesize that the translational control of the above proteins depends on conformational relationships between incompatibility gene products and mod-2.     

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.     

Molecular characterization of vegetative incompatibility genes that restrict hypovirus transmission in the chestnut blight fungus Cryphonectria parasitica

Genetic nonself recognition systems such as vegetative incompatibility operate in many filamentous fungi to regulate hyphal fusion between genetically dissimilar individuals and to restrict the spread of virulence-attenuating mycoviruses that have potential for biological control of pathogenic fungi. We report here the use of a comparative genomics approach to identify seven candidate polymorphic genes associated with four vegetative incompatibility (vic) loci of the chestnut blight fungus Cryphonectria parasitica. Disruption of candidate alleles in one of two strains that were heteroallelic at vic2, vic6, or vic7 resulted in enhanced virus transmission, but did not prevent barrage formation associated with mycelial incompatibility. Detailed characterization of the vic6 locus revealed the involvement of nonallelic interactions between two tightly linked genes in barrage formation, heterokaryon formation, and asymmetric, gene-specific influences on virus transmission. The combined results establish molecular identities of genes associated with four C. parasitica vic loci and provide insights into how these recognition factors interact to trigger incompatibility and restrict virus transmission.     

A new gene for resistance to green leafhopperNephotettix virescens (Distant) in rice

The mode of inheritance of resistance to green leafhopper in 12 cultivars of riceOryza saliva L. was studied. Seedlings of parent and hybrid populations were artificially infested with second- and third-instar virus-free green leafhopper nymphs. Seedling reaction was scored when TNI, the susceptible check, was completely killed. The results revealed that single dominant genes confer resistance in six varieties, two independent dominant genes in four varieties, and single recessive genes in two varieties. The single dominant genes in Sri Gaya, ARC 7320, and T23 and one of the two genes in Aswina and Bhura Rata 2 are allelic toGlh-1; while Bhawalia hasGlh-5 gene. The second gene of Bhura Rata 2 is allelic to IR28 gene. Resistance in Chamar is controlled by two independent genes one of which is allelic toGlh-5 and the other allelic to IR28 gene. Bazal hasGlh-2 andGlh-5. The single recessive gene in ARC 7012 is allelic toglh-4 but the single recessive gene in DV85 is nonallelic to and independent ofglh-4. This new recessive gene is designated asglh-8. The single dominant genes of Dumai, Gadur, and the second gene of Aswina are nonallelic to all the known genes for resistance.     

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.     

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.     

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.     

Self/nonself recognition in Tuber melanosporum is not mediated by a heterokaryon incompatibility system

Vegetative incompatibility is a widespread phenomenon in filamentous ascomycetes, which limits formation of viable heterokaryons. Whether this phenomenon plays a role in maintaining the homokaryotic state of the hyphae during the vegetative growth of Tuber spp. Gene expression, polymorphism analysis as well as targeted in vitro experiments allowed us to test whether a heterokaryon incompatibility (HI) system operates in Tuber melanosporum. HI is controlled by different genetic systems, often involving HET domain genes and their partners whose interaction can trigger a cell death reaction. Putative homologues to HI-related genes previously characterized in Neurospora crassa and Podospora anserina were identified in the T. melanosporum genome. However, only two HET domain genes were found. In many other ascomycetes HET domains have been found within different genes including some members of the NWD (NACHT and WD-repeat associated domains) gene family of P. anserina. More than 50 NWD homologues were found in T. melanosporum but none of these contain a HET domain. All these T. melanosporum paralogs showed a conserved gene organization similar to the microexon genes only recently characterized in Schistosoma mansoni. Expression data of the annotated HI-like genes along with low allelic polymorphism suggest that they have cellular functions unrelated to HI. Moreover, morphological analyses did not provide evidence for HI reactions between pairs of genetically different T. melanosporum strains. Thus, the maintenance of the genetic integrity during the vegetative growth of this species likely depends on mechanisms that act before hyphal fusion.