HETEROTHALLISM AND SEXUALITY IN ASCOBOLUS STERCORARIUS

Bistis, G. N., and J. R. Raper. (Harvard U., Cambridge, Mass.) Heterothallism and sexuality in Ascobolus stercorarius. Amer. Jour. Bot. 50(9): 880–891. Illus. 1963.—The steps in the sexual development of the heterothallic ascomycete, Ascobolus stercorarius, are: (1) induction of antheridial hyphae and antheridia; (2) induction of ascogonial hyphae and ascogonia; (3) directed growth of the trichogyne; and (4) plasmogamy. Although this sequence occurs in each of the 2 reciprocal combinations (A –antheridial/a-ascogonial and a-antheridial/A-ascogonial), several differences between the 2 combinations have been found. The differences are especially apparent with regard to antheridial induction and the pattern of proliferation of ascogonial hyphae. A study of the specificity of the agents regulating the sexual reactions between the 2 mating-types has confirmed previously described class-specificity at antheridial induction (sexual activation). Experiments utilizing substituted oidia have demonstrated an absence of mating-class specificity in trichogyne attraction and even at plasmogamy. The incipient fruiting bodies which result from illegitimate fusions (a X a and A X A), however, stop growing after 24 hr. This cessation of development suggests the presence of a second block to self-fertility in the sexual process of this species.     

Parasexual analysis of common-A crosses in the basidiomycete Agrocybe aegerita

Summary Crosses were performed between homokaryons of Agrocybe aegerita having the same allele at the A incompatibility gene but different B alleles. Heterokaryotic mycelia originating from crosses between two complementary auxotrophs were characterized by their instability on complete medium and extensive anastomosis between hyphae. Diploid mycelia were selected by plating oidia recovered from these heterokaryons onto minimal medium. These mycelia were characterized by the production of larger oidia than those of homokaryons, the release of a brown pigment when growing on complete medium and extensive hyphal anastomoses. Diploids retained the two B incompatibility functions of their homokaryotic parents and gave rise to a diploid/haploid dikaryon when crossed with a compatible homokaryon. Nearly 1% of the oidia recovered from heterokaryons were diploid. These nuclear fusion frequencies as well as the production of brown pigments enabled the identification of diploid strains on complete medium. In this way, crosses between wild prototrophic strains were successfully performed. Somatic recombination was induced following the treatment of diploid mycelia with haploidizing compounds. Selection based on the inability of mycelia to produce the brown pigments on complete medium led to selection of strains homoallelic at the B locus.     

The A mating type and blue light regulate all known differentiation processes in the basidiomycete Coprinus cinereus

Monokaryons of Coprinus cinereus constitutively form small spores (oidia) in the aerial mycelium. Some strains also produce large, inflated single cells (chlamydospores) at the agar/air interface, and hyphal aggregates (hyphal knots) that can develop into sclerotia. Monokaryons show various reactions upon transformation with heterologous A mating type genes. Production of oidia in such A-activated transformants is repressed in the dark and induced by blue light. Five of six monokaryons tested following transformation with A genes showed induced production of hyphal knots and sclerotia in the dark, and at least three strains showed enhanced chlamydospore production in the dark. Continuous incubation under blue light inhibited formation of hyphal knots, sclerotia and chlamydospores in both competent monokaryons and in A-activated transformants. On artificial medium and on a 12 h light/12 h dark regime, A-activated transformants of one distinct monokaryon (218) formed fruit-body primordia that were arrested in development before karyogamy. Our studies show that A mating type genes control all major differentiation processes in Coprinus, but whether developmental processes can proceed depends on the genetic background of the strain. Received: 11 May 1998 / Accepted: 15 July 1998     

Isolation and characterization of compatible diploids of Schizophyllum commune.

Summary Common-AB diploids with several heterozygous biochemical markers were mated with appropriately marked haploid strains of S. commune in an effort to obtain compatible, common-A, and common-B diploid progeny with biochemical markers identical to those of the common-AB parent. The spores from these crosses were germinated on minimal medium. Five compatible diploids, but no common-A or common-B diploids, marked as desired, were isolated by this method. Two possessed some dikaryotic cells and two had many dikaryotic cells. One of the latter was shown to have peculiar behaviour associated with one of its B mating-type factors.     

SEXUALITY AND CULTURAL CHARACTERISTICS OF ASPERGILLUS HETEROTHALLICUS

Aspergillus heterothallicus K., F. and R., isolated from Costa Rican soils, represents the first species in this genus that is truly heterothallic. Each of the eleven strains investigated is functionally hermaphroditic but self-sterile and falls into one of two cross-mating classes, A or a. The mating-type factors A and a appear to be an allelic pair segregating independently of the locus for pigmentation of mycelium which varies from yellow to pinkish orange in different isolates. The striking variation in the development of hülle cell masses that occurred among the progeny from the cross WB 5096(A) × WB 5097(a) was found to be genetically controlled. The gene responsible for a delay in the formation and maturation of cleistothecia appeared to be loosely linked to the a mating-type locus and could be recombined into the A mating type. The mechanism of fertilization has not been completely elucidated. Coiled ascogonia were found within young hülle cell masses developed in cultures where two isolates of opposite mating types were crossed; such coils have not been observed, thus far, within hülle cell masses in unmated cultures. Although no recognizable male structure has been found, the fertilizing element appears to be mycelial in form rather than conidial. Interspecific mating did not occur when strains of Aspergillus heterothallicus were paired with other members of the A. nidulans group.     

Structure and function of a mating-type gene from the homothallic species Neurospora africana

The homothallic Neurospora species, N. africana, contains sequences that hybridize to the A but not to a mating-type sequences of the heterothallic species N. crassa. In this study, the N. africana mating-type gene, mt A-1, was cloned, sequenced and its function analyzed in N. crassa. Although N. africana does not mate in a heterothallic manner, its mt A-1 gene functions as a mating activator in N. crassa. In addition, the N. africana mt A-1 gene confers mating type-associated vegetative incompatibility in N. crassa. DNA sequence analysis shows that the N. africana mt A-1 open reading frame (ORF) is 93% identical to that of N. crassa mt A-1. The mt A-1 ORF of N. africana contains no stop codons and was detected as a cDNA which is processed in a similar manner to mt A-1 of N. crassa. By DNA blot and orthogonal field agarose gel electrophoretic analysis, it is shown that the composition and location of the mating-type locus and the organization of the mating-type chromosome of N. africana are similar to that of N. crassa.     

The A mating type and blue light regulate all known differentiation processes in the basidiomycete Coprinus cinereus

Monokaryons of Coprinus cinereus constitutively form small spores (oidia) in the aerial mycelium. Some strains also produce large, inflated single cells (chlamydospores) at the agar/air interface, and hyphal aggregates (hyphal knots) that can develop into sclerotia. Monokaryons show various reactions upon transformation with heterologous A mating type genes. Production of oidia in such A-activated transformants is repressed in the dark and induced by blue light. Five of six monokaryons tested following transformation with A genes showed induced production of hyphal knots and sclerotia in the dark, and at least three strains showed enhanced chlamydospore production in the dark. Continuous incubation under blue light inhibited formation of hyphal knots, sclerotia and chlamydospores in both competent monokaryons and in A-activated transformants. On artificial medium and on a 12?h light/12?h dark regime, A-activated transformants of one distinct monokaryon (218) formed fruit-body primordia that were arrested in development before karyogamy. Our studies show that A mating type genes control all major differentiation processes in Coprinus, but whether developmental processes can proceed depends on the genetic background of the strain.     

Sexual compatibility between serotypes ofFilobasidiella neoformans (Cryptococcus neoformans)

Twenty-five of 37Cryptococcus neoformans strains of known serotype produced the basidiomycetousFilobasidiella state either alone or when paired with a strain of compatible mating-type. Sixteen strains were mating-type, four strains were a mating-type, and five strains were self-fertile.F. neoformans serotypes A and D were interfertile with compatible mating-types ofF. bacillispora serotypes B and C.C. neoformans var.gattii was interfertile with compatible mating-types ofF. neoformans andF. bacillispora. F. bacillispora strains, which utilized creatinine andl-malic acid, were interfertile with compatible mating-types ofF. neoformans, which did not utilize creatinine andl-malic acid. The interfertility between serotypes and biotypes eliminates the need for recognizing the names ofC. neoformans var.gattii, C. bacillisporus, andF. bacillispora. It is proposed thatC. neoformans var.gattii andC. bacillisporus be regarded as later, facultative synonyms ofC. neoformans and thatF. bacillispora be regarded as a later, facultative synonym ofF. neoformans.     

Comparative genomics of the mating-type loci of the mushroom Flammulina velutipes reveals widespread synteny and recent inversions

Background

Mating-type loci of mushroom fungi contain master regulatory genes that control recognition between compatible nuclei, maintenance of compatible nuclei as heterokaryons, and fruiting body development. Regions near mating-type loci in fungi often show adapted recombination, facilitating the generation of novel mating types and reducing the production of self-compatible mating types. Compared to other fungi, mushroom fungi have complex mating-type systems, showing both loci with redundant function (subloci) and subloci with many alleles. The genomic organization of mating-type loci has been solved in very few mushroom species, which complicates proper interpretation of mating-type evolution and use of those genes in breeding programs.

Methodology/Principal Findings

We report a complete genetic structure of the mating-type loci from the tetrapolar, edible mushroom Flammulina velutipes mating type A3B3. Two matB3 subloci, matB3a that contains a unique pheromone and matB3b, were mapped 177 Kb apart on scaffold 1. The matA locus of F. velutipes contains three homeodomain genes distributed over 73 Kb distant matA3a and matA3b subloci. The conserved matA region in Agaricales approaches 350 Kb and contains conserved recombination hotspots showing major rearrangements in F. velutipes and Schizophyllum commune. Important evolutionary differences were indicated; separation of the matA subloci in F. velutipes was diverged from the Coprinopsis cinerea arrangement via two large inversions whereas separation in S. commune emerged through transposition of gene clusters.

Conclusions/Significance

In our study we determined that the Agaricales have very large scale synteny at matA (∼350 Kb) and that this synteny is maintained even when parts of this region are separated through chromosomal rearrangements. Four conserved recombination hotspots allow reshuffling of large fragments of this region. Next to this, it was revealed that large distance subloci can exist in matB as well. Finally, the genes that were linked to specific mating types will serve as molecular markers in breeding.     

Morphological and cytological aspects of oidium formation in a basidiomycete,Pholiota nameko

Pholiota nameko produced abundant oidia on aerial hyphae from monokaryotic and dikaryotic test stocks, but oidia were rare on submerged hyphae. The oidia from the former stocks had a layer of hydrophobic protein between the cell wall and the inner cell membrane which was absent in the oidia from the latter. The only remarkable differences in the morphological features of the oidia from monokaryotic and dikaryotic mycelia was the slightly larger size of the latter. Observation of various test stocks on slide cultures revealed that about 80% of oidia were produced from the secondary branched hypha, and about 20% from the terminal hyphal, cell of the main hypha. In the former, the secondary hyphae were segmented to form several oidium cells; in the latter, a single or several oidia were formed at the terminal end of the main hypha. Most oidia from monokaryons and dikaryons had only one haploid nucleus, while the remainders were multinucleate. Among the stocks tested, most oidia had a DNA content with a haploid amount at the G1 phase of the cell cycle, but a few contained twice that amount corresponding to the G2 phase     

THE KINETICS OF INITIAL NUCLEAR EXCHANGE IN COMPATIBLE AND NONCOMPATIBLE MATINGS OF SCHIZOPHYLLUM COMMUNE

The effect of mating-type factors on exchange of nuclei between mycelial fragments mated in liquid culture was studied. Evidence for exchange of nuclei was based on three criteria for detecting nuclei from both mating partners in mycelial fragments of the developing dikaryon or hetero-karyon. The kinetics of nuclear exchange were shown to be relatively independent of the method used. It was shown that the kinetics of nuclear exchange in the first 96 hr are different in the compatible (A41 B41 × A42 B42), common-A (A42 B42 × A42 B41), common-B (A41 B42 × A42 B42), and common-AB (A41 B41 × A41 B41) matings. In all four types of matings, the percentage of fragments possessing both types of nuclei 12-24 hr after mating is nearly equal. After this time, significant differences appear in the patterns for compatible vs. the three non-compatible matings and the common-A vs. the common-B and common-AB matings. The percentage of mycelial fragments possessing both types of nuclei throughout the 96-hr test period is similar for both the common-B and common-AB matings. The kinetics of nuclear exchange were shown to be independent of the particular mating-type alleles and nutritional markers used. When the efficiency of nuclear exchange in complete and minimal media was compared, it was shown that nuclear exchange occurred more rapidly and synchronously in minimal medium. This difference is not due to growth differences in the two media. These data indicate that the earliest mating interactions, i.e., hyphal anastomosis and nuclear exchange, are independent of the mating-type factors but that subsequent events are determined by these genes.     

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 MAT A locus of the dimorphic yeast Yarrowia lipolytica consists of two divergently oriented genes

The MAT A locus of Yarrowia lipolytica, which was on the basis of its ability to induce sporulation in a diploid B/B strain, represses the mating capacity of this strain. The gene functions required for induction of sporulation and repression of conjugation could be separated by subcloning. Sequence analysis revealed two ORFs in the MAT A locus. One of them (MAT A1) codes for a protein of 119 amino acids which is required to induce sporulation. The other (MAT A2) codes for a protein of 291 amino acids that is able to repress conjugation. Both genes are oriented divergently from a central promoter region, which possesses putative TATA and CAAT boxes for both genes. The product of MAT A1 shows no homology to any known protein and seems to represent a new class of mating-type genes. MAT A2 contains a HMG box with homology to other mating-type genes. Both MAT A1 and MAT A2 are mating-type specific. In cells of both mating types, the regions flanking the MAT A locus contain sequences with homology to either S. cerevisiae SLA2 and ORF YBB9, respectively. From hybridization and subcloning data we estimate that the MAT A region is approximately 2 kb long and is present only once in the genome. Received: 25 January 1999 / Accepted: 16 April 1999     

Mutants of SACCHAROMYCES CEREVISIAE Resistant to the α Mating-Type Factor

Mutants that are resistant to α-factor have been isolated from a mating-type haploid strains of yeast by direct selection on agar medium containing partially purified α-factor. All resistant mutants isolated were found to be sterile. They were characterized and compared with mutants previously isolated as nonmating. Among 93 able to mate at low frequency and to sporulate, none showed linkage to the mating-type locus. The results support the hypothesis that the response to α-factor by cells of mating-type a is essential for mating.     

Cloning and comparison of Aα mating-type alleles of the basidiomycete Schizophyllum commune

Summary An A mating-type allele (A4) was isolated by walking the chromosome from the closely linked PAB1 gene. A cosmid clone containing the A1 allele isolated from the walk was used as a probe to recover the A1 allele from another cosmid library. Cosmids encoding mating-type activity were identified by transforming Schizophyllum cells and screening for activation of A-regulated development. Putative mating-type transformants were confirmed in mating tests and genetic analyses of progeny. The identity of the specific alleles isolated was demonstrated by showing that their effectiveness in transforming for mating type is limited to recipient strains possessing an A allele different from the one encoded by the cloned sequences. Transforming DNA is active in trans, suggesting that A encodes a diffusible product. Restriction mapping shows that A1 and A4 are coded in the same physical region of the genome, but within a subregion that contains extensive sequence divergence. In addition, Southern analyses show that there is only one copy of A1 or A4 per haploid genome, and that they do not cross-hybridize to one another or to any of the other A alleles. A1 and A4 were subcloned as 2.8 and 1.2 kb fragments, respectively, retaining in transformation all the mating-type activity demonstrated of the original cosmids.     

Sex-linked transcriptional divergence in the hermaphrodite fungus Neurospora tetrasperma

In the filamentous ascomycete Neurospora tetrasperma, a large (approx. 7 Mbp) region of suppressed recombination surrounds the mating-type (mat) locus. While the remainder of the genome is largely homoallelic, this region of recombinational suppression, extending over 1500 genes, is associated with sequence divergence. Here, we used microarrays to examine how the molecular phenotype of gene expression level is linked to this divergent region, and thus to the mating type. Culturing N. tetrasperma on agar media that induce sexual/female or vegetative/male tissue, we found 196 genes significantly differentially expressed between mat A and mat a mating types. Our data show that the genes exhibiting mat-linked expression are enriched in the region genetically linked to mating type, and sequence and expression divergence are positively correlated. Our results indicate that the phenotype of mat A strains is optimized for traits promoting sexual/female development and the phenotype of mat a strains for vegetative/male development. This discovery of differentially expressed genes associated with mating type provides a link between genotypic and phenotypic divergence in this taxon and illustrates a fungal analogue to sexual dimorphism found among animals and plants.     

The genetic structure of the A mating-type locus of Lentinula edodes

The Shiitake mushroom, Lentinula edodes (Berk.) Pegler is a tetrapolar basidiomycete with two unlinked mating-type loci, commonly called the A and B loci. Identifying the mating-types in shiitake is important for enhancing the breeding and cultivation of this economically-important edible mushroom. Here, we identified the A mating-type locus from the first draft genome sequence of L. edodes and characterized multiple alleles from different monokaryotic strains. Two intron-length polymorphism markers were developed to facilitate rapid molecular determination of A mating-type. L. edodes sequences were compared with those of known tetrapolar and bipolar basidiomycete species. The A mating-type genes are conserved at the homeodomain region across the order Agaricales. However, we observed unique genomic organization of the locus in L. edodes which exhibits atypical gene order and multiple repetitive elements around its A locus. To our knowledge, this is the first known exception among Homobasidiomycetes, in which the mitochondrial intermediate peptidase (mip) gene is not closely linked to A locus.     

Agouti-Related Maturation and Tissue Distribution of oc-Melanocyte Stimulating Hormone in Wild-Type (AwJ/AwJ) and Mutant (Ay/a,a/a) Mice

Because of ectopic overproduction of agouti protein, yellow alleles (A^y and A^vy) of the murine agouti gene may secondarily modulate the synthesis, maturation (i.e., acetylation), and/or tissue deployment of α-Melanocyte Stimulating Hormone (MSH). We used HPLC to test the hypothesis that A^y/a mice exhibit altered concentrations of desacetyl-, monoacetyl-, and diacetyl-α-MSH in pituitaries, sera, and telogen hair bulbs when compared to black (a/a) mice. We also used RIA to measure total MSH in those same tissues of A^y/a, a/a, and white-bellied agouti (A^wJ/A^wJ) mice (Strain C57BL/6J). We found no evidence that A^y/a mice possessed an imbalance of des-, mono-, and diacetylated α-MSH species. However, radioimmunoassay (RIA) analyses of total MSH suggest that wild-type agouti mice (A^wJ/A^wJ) exhibited significantly decreased (P < 0.05) tissue levels of total α-MSH in pituitaries, sera, and regenerating hair bulbs when compared to those of mutant A^y/a and a/a mice.     

Characterization of the expression of a desiccation-responsive rd29 gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants

Summary We characterized the expression of genes that correspond to a cDNA clone, RD29, which is induced by desiccation, cold and high-salt conditions in Arabidopsis thaliana. Northern analysis of desiccation-induced expression revealed a two-step induction process. Early induction occurs within 20 min and secondary induction occurs 3 h after the start of desiccation. Exogenous abscisic acid (ABA) induces RD29 mRNA within 3 h. Two genes corresponding to RD29, rd29A and rd29B, are located in tandem in an 8 kb region of the Arabidopsis genome and encode hydrophilic proteins. Desiccation induces rd29A mRNA with two-step kinetics, while rd29B is induced only 3 h after the start of desiccation. The expression of both genes is stimulated about 3 h after application of ABA. It appears that rd29A has at least two cis-acting elements, one involved in the ABA-associated response to desiccation and the other induced by changes in osmotic potential. The -glucuronidase (GUS) reporter gene driven by the rd29A promoter was induced at significant levels by desiccation, cold, high-salt conditions and ABA in both transgenic Arabidopsis and tobacco. Histochemical analysis of GUS activity revealed that the rd29A promoter functions in almost all the organs and tissues of vegetative plants during water deficiency.     

Structure and function of a mating-type gene from the homothallic species Neurospora africana

The homothallic Neurospora species, N. africana, contains sequences that hybridize to the A but not to a mating-type sequences of the heterothallic species N. crassa. In this study, the N. africana mating-type gene, mt A-1, was cloned, sequenced and its function analyzed in N. crassa. Although N. africana does not mate in a heterothallic manner, its mt A-1 gene functions as a mating activator in N. crassa. In addition, the N. africana mt A-1 gene confers mating type-associated vegetative incompatibility in N. crassa. DNA sequence analysis shows that the N. africana mt A-1 open reading frame (ORF) is 93% identical to that of N. crassa mt A-1. The mt A-1 ORF of N. africana contains no stop codons and was detected as a cDNA which is processed in a similar manner to mt A-1 of N. crassa. By DNA blot and orthogonal field agarose gel electrophoretic analysis, it is shown that the composition and location of the mating-type locus and the organization of the mating-type chromosome of N. africana are similar to that of N. crassa.