Roles for receptors, pheromones, G proteins, and mating type genes during sexual reproduction in Neurospora crassa

Here we characterize the relationship between the PRE-2 pheromone receptor and its ligand, CCG-4, and the general requirements for receptors, pheromones, G proteins, and mating type genes during fusion of opposite mating-type cells and sexual sporulation in the multicellular fungus Neurospora crassa. PRE-2 is highly expressed in mat a cells and is localized in male and female reproductive structures. Δpre-2 mat a females do not respond chemotropically to mat A males (conidia) or form mature fruiting bodies (perithecia) or meiotic progeny (ascospores). Strains with swapped identity due to heterologous expression of pre-2 or ccg-4 behave normally in crosses with opposite mating-type strains. Coexpression of pre-2 and ccg-4 in the mat A background leads to self-attraction and development of barren perithecia without ascospores. Further perithecial development is achieved by inactivation of Sad-1, a gene required for meiotic gene silencing. Findings from studies involving forced heterokaryons of opposite mating-type strains show that presence of one receptor and its compatible pheromone is necessary and sufficient for perithecial development and ascospore production. Taken together, the results demonstrate that although receptors and pheromones control sexual identity, the mating-type genes (mat A and mat a) must be in two different nuclei to allow meiosis and sexual sporulation to occur.     

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.     

Evidence for Mitochondrial DNA Polymorphism and Uniparental Inheritance in the Cellular Slime Mold Polysphondylium Pallidum: Effect of Intraspecies Mating on Mitochondrial DNA Transmission

Restriction fragment length polymorphisms (RFLPs) were used as markers to monitor mitochondrial inheritance in the cellular slime mold, Polysphondylium pallidum. When two opposite mating types (mat1 and mat2) of closely related strains were crossed, all the haploid progeny regardless of mating type inherited their mitochondrial DNA from the mat2 parent only. When opposite mating types from more distantly related strains were crossed, most of the progeny also inherited their mitochondrial DNA from the mat2 parent, but some inherited their mitochondrial DNA from the mat1 parent. In both cases however, the transmission of mitochondrial DNA was uniparental, since in every individual progeny only one type of mitochondrial DNA exists. Moreover, in crosses involving more distantly related strains all the progeny of a single macrocyst were shown to contain the same type of mitochondrial DNA. These findings are discussed in regard to mechanisms of transmission and the possible involvement of nuclear genes in the control of transmission of mitochondrial DNA in Polysphondylium.     

Life history and developmental processes in the basidiomycete Coprinus cinereus.

Coprinus cinereus has two main types of mycelia, the asexual monokaryon and the sexual dikaryon, formed by fusion of compatible monokaryons. Syngamy (plasmogamy) and karyogamy are spatially and temporally separated, which is typical for basidiomycetous fungi. This property of the dikaryon enables an easy exchange of nuclear partners in further dikaryotic-monokaryotic and dikaryotic-dikaryotic mycelial fusions. Fruiting bodies normally develop on the dikaryon, and the cytological process of fruiting-body development has been described in its principles. Within the specialized basidia, present within the gills of the fruiting bodies, karyogamy occurs in a synchronized manner. It is directly followed by meiosis and by the production of the meiotic basidiospores. The synchrony of karyogamy and meiosis has made the fungus a classical object to study meiotic cytology and recombination. Several genes involved in these processes have been identified. Both monokaryons and dikaryons can form multicellular resting bodies (sclerotia) and different types of mitotic spores, the small uninucleate aerial oidia, and, within submerged mycelium, the large thick-walled chlamydospores. The decision about whether a structure will be formed is made on the basis of environmental signals (light, temperature, humidity, and nutrients). Of the intrinsic factors that control development, the products of the two mating type loci are most important. Mutant complementation and PCR approaches identified further genes which possibly link the two mating-type pathways with each other and with nutritional regulation, for example with the cAMP signaling pathway. Among genes specifically expressed within the fruiting body are those for two galectins, beta-galactoside binding lectins that probably act in hyphal aggregation. These genes serve as molecular markers to study development in wild-type and mutant strains. The isolation of genes for potential non-DNA methyltransferases, needed for tissue formation within the fruiting body, promises the discovery of new signaling pathways, possibly involving secondary fungal metabolites.     

Zygote formation and recombination between like mating types in the yeast Saccharomycopsis lipolytica by protoplast fusion

Summary Protoplasts from auxotrophic strains of the alkane yeast, Saccharomycopsis (Candida) lipolytica, will hybridize despite identity in mating type. Fusion products following regeneration and selection form stable prototrophic diploids, and recombinant progeny can be obtained either through the parasexual or the sexual cycle. These results confirm that mating type alleles of this yeast control only the initial steps in the mating sequence, cell recognition and agglutination, but not karyogamy and meiosis.     

A mating type-linked mutation that disrupts the uniparental inheritance of chloroplast DNA also disrupts cell-size control in Chlamydomonas.

An intriguing feature of early zygote development in Chlamydomonas reinhardtii is the active elimination of chloroplast DNA from the mating-type minus parent due presumably to the action of a zygote-specific nuclease. Meiotic progeny thus inherit chloroplast DNA almost exclusively from the mating-type plus parent. The plus-linked nuclear mutation mat3 prevents this selective destruction of minus chloroplast DNA and generates progeny that display a biparental inheritance pattern. Here we show that the mat3 mutation creates additional phenotypes not previously described: the cells are much smaller than wild type and they possess substantially reduced amounts of both mitochondrial and chloroplast DNA. We propose that the primary defect of the mat3 mutation is a disruption of cell-size control and that the inhibition of the uniparental transmission of chloroplast genomes is a secondary consequence of the reduced amount of chloroplast DNA in the mat3 parent.     

A homologue of the yeast SHE4 gene is essential for the transition between the syncytial and cellular stages during sexual reproduction of the fungus Podospora anserina.

The Podospora anserina cro1 gene was identified as a gene required for sexual sporulation. Crosses homozygous for the cro1-1 mutation yield fruiting bodies which produce few asci due to the formation of giant plurinucleate cells instead of dikaryotic cells after fertilization. This defect does not impair karyogamy, but meioses of the resultant polyploid nuclei are most often abortive. Cytological studies suggest that the primary defect of the mutant is its inability to form septa between the daughter nuclei after each mitosis, a step specific for normal dikaryotic cell divisions. The cro1-1 mutant would thus be unable to leave the syncytial vegetative state while abiding by the meiotic programme. cro1-1 also shows defects in ascospore germination and growth rate. GFP-tagging of the CRO1 protein reveals that it is a cytosolic protein mainly expressed at the beginning of the dikaryotic stage and at the time of ascospore maturation. The CRO1 protein exhibits significant similarity to the SHE4 protein, which is required for asymmetric mating-type switching in budding yeast cells. Thus, a gene involved in asymmetric cell divisions in a unicellular organism plays a key role at the transition between the syncytial (vegetative) state and the cellular (sexual) state in a filamentous fungus.     

Mating-type heterokaryosis and selfing in Cryphonectria parasitica

Selfing in the chestnut blight fungus, Cryphonectria parasitica, occurs by two different genetic mechanisms. Most self-fertile isolates of C. parasitica are heterokaryotic for mating type, and the progeny from selfing segregate for mating type. Further, we resolved mating-type (MAT) heterokaryons into homokaryons of both mating types by isolating uninucleate asexual spores (conidia). However, because ascospore progeny, with rare exceptions, are not MAT heterokaryons, C. parasitica must lack a regular mechanism to maintain heterokaryosis by selfing. We hypothesize that heterokaryon formation may occur either because of recurrent biparental inbreeding, or by mating-type switching, possibly one involving some kind of parasexual process. The second mechanism found for selfing in C. parasitica occurred less frequently. Three single-conidial isolates (MAT-1 and MAT-2) selfed and produced progeny that did not segregate for mating type. It is currently not known if meiosis occurs during ascospore formation by this mechanism.     

Biparental Inheritance of Non-Mendelian Gene Markers in CHLAMYDOMONAS MOEWUSII

The first two non-Mendelian gene mutations to be identified in Chlamydomonas moewusii are described. These putative chloroplast gene mutations include one for resistance to streptomycin (sr-nM1) and one for resistance to erythromycin (er-nM1). In one- and two-factor reciprocal crosses, usually over 90% of the germinating zygospores transmitted these mutations and their wild-type alternatives from both parents (biparental zygospores); the remaining zygospores transmitted exclusively the non-Mendelian markers of the mating-type "plus" parent. Among the biparental zygospores, a strong bias in the transmission of non-Mendelian alleles from the mating-type "plus" parent was indicated by an excess of meiotic and postmeiotic mitotic progeny that were homoplasmic for non-Mendelian alleles from this parent compared to those that were homoplasmic for the non-Mendelian alleles from the mating-type "minus" parent. At best, weak linkage was detected between the sr-nM1 and er-nM1 loci. Non-Mendelian, chloroplast gene markers in Chlamydomonas eugametos and Chlamydomonas reinhardtii showed a predominantly uniparental mode of transmission from the mating-type "plus" parent in crosses performed under the same conditions used for the C. moewusii crosses.     

Isogenic Strains of PHYCOMYCES BLAKESLEEANUS Suitable for Genetic Analysis

The progeny of crosses between wild-type strains of Phycomyces usually do not exhibit all of the expected genotypes from meiosis. By backcrossing, we have isolated a new (+) mating-type strain, A56, which is nearly isogenic with the (-) wild-type NRRL1555 commonly used in Phycomyces research. Tetrad analysis of the backcrosses shows that meiosis becomes more regular as the parental (+) and (-) strains become more isogenic. In our two-factor crosses with unlinked markers, the regularity of meiosis is measured as the percent of reciprocal ditypes plus tetratypes in the progeny. We have shown that this percentage increases from about 15% for crosses between nonisogenic parents to 90% in the eighth backcross. The results indicate that routine, reliable recombination analyses are possible in P. blakesleeanus.     

Cloning the Mating Types of the Heterothallic Fungus Podospora Anserina: Developmental Features of Haploid Transformants Carrying Both Mating Types

DNAs that encode the mating-type functions (mat+ and mat-) of the filamentous fungus Podospora anserina were cloned with the use of the mating-type A probe from Neurospora crassa. Cloning the full mat information was ascertained through gene replacement experiments. Molecular and functional analyses of haploid transformants carrying both mating types lead to several striking conclusions. Mat+ mat- strains are dual maters. However, the resident mat information is dominant to the mat information added by transformation with respect to fruiting body development and ascus production. Moreover, when dual mating mat+ mat- strains are crossed to mat+ or mat- testers, there is strong selection, after fertilization, that leads to the loss from the mat+ mat- nucleus of the mat information that matches that of the tester. Finally, the mat locus contains at least two domains, one sufficient for fertilization, the other necessary for sporulation.     

Alternation of nuclear phase in the filamentous basidiomycete,Helicobasidium mompa

Variation in the number of nuclei and cellular ploidy were observed in eight strains ofHelicobasidium mompa. The basidiospores, single-spore isolates and field-isolated strains were all dikaryons. The cellular ploidy, which was assessed by analyzing the fluorescence emitted by DAPI-stained nuclei, was unstable: monokaryotic strains derived from the original dikaryotic strains by successive subcultures were mainly tetraploid, although the original dikaryon was in most cases diploid. On the other hand, a dikaryotic strain derived by treatment with benomyl was haploid. These results suggest that diploid dikaryon is a normal nuclear phase ofH. mompa in nature, and the alternation of ploidy may be due to a feature of the mating system of this fungus.     

What is a bona fide mating-type gene? Internuclear complementation of mat mutants in Podospora anserina

In the heterothallic ascomycete Podospora anserina, the mating-type locus is occupied by two mutually exclusive sequences termed mat+ and mat–. The mat+ sequence contains only one gene, FPR1, while the mat– sequence contains three genes: FMR1, SMR1 and SMR2. Previous studies have demonstrated that FPR1 and FMR1 are required for fertilization. Further analyses have led to the hypothesis that mat+ and mat– genes establish a mat+ and mat– nuclear identity, allowing recognition between nuclei of opposite mating type within the syncytial cells formed after fertilization. This hypothesis was based on the phenotypes of strains bearing mutations in ectopic mat genes. Here we present an analysis of mutations in resident mat– genes which suggests that, unlike FMR1 and SMR2, SMR1 is not involved in establishing nuclear identity. In fact, mutations in these two genes impair nuclear recognition, leading to uniparental progeny, while mutations in SMR1 block the sexual process, probably at a step after nuclear recognition. The nuclear identity hypothesis has also been tested through internuclear complementation tests. In these experiments, the mat– mutants were crossed with a mat+ strain carrying the wild-type mat– genes. Our rationale was that internuclear complementation should not be possible for nuclear identity genes: the relevant genes should show nucleus-restricted expression, and diffusion of their products to other nuclei should not occur. This test confirmed that SMR1 is not a bona fide mat gene since it can fulfill its function whatever its location, in either a mat? or a mat+ nucleus, and even when present in both nuclei. SMR2, but not FMR1, behaves like a nuclear identity gene with respect to internuclear complementation tests. A model is proposed that tentatively explains the ambiguous behaviour of the FMR1 gene and clarifies the respective functions of the three mat– proteins.     

Influence of activated<Emphasis Type="Italic"> A</Emphasis> and<Emphasis Type="Italic"> B</Emphasis> mating-type pathways on developmental processes in the basidiomycete<Emphasis Type="Italic"> Coprinus cinereus</Emphasis>

The A and B mating type pathways in Coprinus cinereus monokaryons can be activated by transformation with cloned genes from strains of compatible mating types. The presence of heterologous A mating-type genes (Aon) induces production of submerged chlamydospores, hyphal knots and sclerotia in cultures kept in the dark. Upon illumination of transformants of certain strains (218), fruiting body primordia may develop that arrest before karyogamy. Furthermore, formation of aerial spores (oidia) is repressed by the action of A mating type genes in the dark, but light overrides this repression. Heterologous B mating type genes enhance the effects of the A genes on developmental processes, and partially repress the negative action of light on A-mediated regulation of development. Most notably, A-induced fruiting occurs more efficiently and earlier when the B mating type pathway is also active (Bon). However, activation of the B pathway alone is not sufficient to induce fruiting. Unlike A-activated transformants, A+ B-activated transformants of monokaryon 218 form mature fruiting bodies. Therefore, the B genes control fruiting body maturation at the stage of karyogamy. Basidia within the fruiting bodies that were analysed contained four spores in a typical post-meiotic arrangement. In the absence of an activated A mating type pathway, B mating type genes cause deformation and hyperbranching of vegetative hyphae, a reduction in aerial mycelium, and invasion of the agar substrate - a phenotype resembling the "flat" phenotype known from B-activated Schizophyllum commune strains. B-activated transformants usually show enhanced production of chlamydospores and hyphal knots, but maturation of sclerotia is variably efficient. Activation of the B mating type pathway in monokaryons blocked acceptance of nuclei, but not activation of the A mating type pathway.     

Three Genes Which Affect Founding of Aggregations in Polysphondylium Pallidum

PN6024 is an extraordinary mutant strain of the cellular slime mold Polysphondylium pallidum, characterized by having defects in many unlinked genes. New strains with altered development appeared spontaneously as aberrant clones of PN6024. Genetic crosses using the macrocyst sexual cycle were used to show that PN6030 (a clone like PN6024 in phenotype) carries mutations at two loci, emm and hge, whereas PN6031 (a clone of altered morphology) carries in addition a mutation at a third locus, mgt. hge and possibly mgt are linked to the mating type locus mat. The relatively high frequency of recombination between mat and hge is strong evidence that meiosis precedes macrocyst germination. The mutant genes themselves are of interest. A major effect of the emm-1 mutation is to remove the requirement for light to trigger aggregation. hge-1 greatly reduces the frequency of aggregation, whereas mgt-1 greatly increases it under standard conditions. None of these mutations interrupts later development leading to stalks and spore cells. It is hypothesized that all three genes act on steps immediately preceding the differentiation of the founder cells which initiate aggregation.