Programmed ascospore death in the homothallic ascomycete Coniochaeta tetraspora

Immature asci of Coniochaeta tetraspora originally contain eight uninucleate ascospores. Two ascospore pairs in each ascus survive and mature, and two die and degenerate. Arrangement of the two ascospore types in individual linear asci is what would be expected if death is controlled by a chromosomal gene segregating at the second meiotic division in about 50% of asci. Cultures originating from single homokaryotic ascospores or from single uninucleate conidia are self-fertile, again producing eight-spored asci in which four spores disintegrate, generation after generation. These observations indicate that differentiation of two nuclear types occurs de novo in each sexual generation, that it involves alteration of a specific chromosome locus, and that the change occurs early in the sexual phase. One, and only one, of the two haploid nuclei entering each functional zygote must carry the altered element, which is segregated into two of the four meiotic products and is eliminated when ascospores that contain it disintegrate. Fusion of nuclei cannot be random-a recognition mechanism must exist. More study will be needed to determine whether the change that is responsible for ascospore death is genetic or epigenetic, whether it occurs just before the formation of each ascus or originates only once in the ascogonium prior to proliferation of ascogenous hyphae, and whether it reflects developmentally triggered alteration at a locus other than mating type or the activation of a silent mating-type gene that has pleiotropic effects. Similar considerations apply to species such as Sclerotinia trifoliorum and Chromocrea spinulosa, in which all ascospores survive but half the spores in each ascus are small and self-sterile. Unlike C. tetraspora, another four-spored species, Coniochaetidium savoryi, is pseudohomothallic, with ascus development resembling that of Podospora anserina.     

Substructural studies on sporulation of Saccharomycopsis lipolytica

During sporulation of diploids from crosses between different strains of the yeast Saccharomycopsis (Candida) lipolytica irregular numbers of ascospores per ascus have been observed. Using the serial section method it could be shown now by means of electron microscopy that in one-, two-, and three-spored asci unenclosed "naked" nuclei occur additionally to nuclei incorporated in mature spores. It was demonstrated that the production of less than four spores per ascus in this yeast is not the result of a lack of meiotic products but of the nonutilization of nuclei from meiosis. In 2--4 spored asci usually four products of meiosis in form of enclosed and free nuclei could be demonstrated which indicate a normal meiotic division. All ascospores derived from asci with different spore numbers are uninuclear. It is assumed that a defect in spore formation caused by structural changes of chromosomes or aneuploidy should give rise to the occurrence of non incorporated nuclei and spore irregularity. It was concluded that meiosis and spore formation in Saccharomycopsis lipolytica seem to represent parallel and coordinated processes which generally resemble those recorded for Saccharomyces cerevisiae and Hansenula species.     

Expression of Meiotic Drive Elements Spore Killer-2 and Spore Killer-3 in Asci of Neurospora Tetrasperma

It was shown previously that when a chromosomal Spore killer factor is heterozygous in Neurospora species with eight-spored asci, the four sensitive ascospores in each ascus die and the four survivors are all killers. Sk-2K and Sk-3K are nonrecombining haplotypes that segregate with the centromere of linkage group III. No killing occurs when either one of these killers is homozygous, but each is sensitive to killing by the other in crosses of Sk-2K x Sk-3K. In the present study, Sk-2K and Sk-3K were transferred by recurrent backcrosses from the eight-spored species Neurospora crassa into Neurospora tetrasperma, a pseudohomothallic species which normally makes asci with four large spores, each heterokaryotic for mating type and for any other centromere-linked genes that are heterozygous in the cross. The action of Sk-2K and Sk-3K in N. tetrasperma is that predicted from their behavior in eight-spored species. A sensitive nucleus is protected from killing if it is enclosed in the same ascospore with a killer nucleus. Crosses of Sk-2K x Sk-2S, Sk-3K x Sk-3S, and Sk-sK x Sk-3K all produce four-spored asci that are wild type in appearance, with the ascospores heterokaryotic and viable. The Eight-spore gene E, which shows variable penetrance, was used to obtain N. tetrasperma asci in which two to eight spores are small and homokaryotic. When killer and sensitive alleles are segregating in the presence of E, only those ascospores that contain a killer allele survive. Half of the small ascospores are killed. In crosses of Sk-2K x Sk-3K (with E heterozygous), effectively all small ascospores are killed. The ability of N. tetrasperma to carry killer elements in cryptic condition suggests a possible role for Spore killers in the origin of pseudohomothallism, with adoption of the four-spored mode restoring ascospore viability of crosses in which killing would otherwise occur.     

Meiosis in NEUROSPORA CRASSA. I. the Isolation of Recessive Mutants Defective in the Production of Viable Ascospores

A scheme has been devised for efficient isolation of recessive meiotic mutants of Neurospora crassa. These mutants were detected by their reduced fertility or by the abortion of ascospores. Their isolation involved the selection and screening of the strains arising from ascospores disomic (n + 1) for linkage group I (LG I), which bears the mating-type locus. These strains are self-fertile heterokaryons that contain two types of haploid nuclei of opposite mating types (A + a). Selfings of these strains are homozygous for genes on all linkage groups except LGI and therefore allow the expression of recessive mutants with an altered sexual cycle. Using this selection procedure, three classes of mutants were detected. In one class, mutants had an early block in perithecial development (class I), and in another mutants had altered perithecia, but apparently unaltered fertility (class III). No recessive mutants were observed and all mutants tested (eight of class I and two of class III) were expressed only when used as the maternal parent. A third mutant class displayed normal production of perithecia, but defective formation of asci (class IIA), or black ascospores (class IIB). Four of 13 class IIA mutants were analyzed, and two of them [asc(DL131) and asc (DL400)] were definitely recessive analysis of 10 of 13 class IIB mutants disclosed six recessive, mutually complementing mutants: ase(DL95), asc(DL243), asc(DL711), asc(DL879), asc(DL917m) and asc(DL961). Mutants asc(DL95), asc(DL243) and the previously studied mei-1 mutant (Smith 1975) complemented one another in crosses, but did not recombine. These may be alleles of the same gene, or they may comprise a gene cluster.     

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.     

A chromosome rearrangement in Neurospora that produces segmental aneuploid progeny containing only part of the nucleolus organizer

In translocation T(ILVL)OY321 of Neurospora crassa a distal portion of the nucleolus organizer chromosome, including ribosomal DNA sequences and the nucleolus satellite, is interchanged with a long terminal segment of IL. When OY321 is crossed by Normal sequence, one-fourth of the meiotic products are segmental aneuploids that contain two copies of the long IL segment and that are deficient for the distal portion of the organizer. Each such product forms a nucleolus and is viable. The complementary aneuploid products are deficient for the IL segment and are therefore inviable. — In crosses of OY321xOY321, each product is capable of making two nucleoli; nucleoli formed by the separated nucleolus organizer parts usually fuse, but most 8-spored asci contain some nuclei in which two separate nucleoli can be seen. One nucleolus is then terminal on its chromosome while the second is interstitial and somewhat smaller. — In crosses of OY321 x Normal, half of the meiotic products are capable of making two nucleoli. However, only about 15% of 8-spored asci have one or more nuclei containing separate nucleoli. At pachytene and later in prophase I, the single fusion nucleolus is associated with three bivalent chromosome segments. Each nucleus of every ascus contains at least one nucleolus, even in asci where some nuclei display two nucleoli. — Crosses of Aneuploid x Normal are usually semibarren, producing a reduced number of ascospores, some of which are inviable. Some aneuploid cultures become fully fertile by reverting to a quasinormal sequence lacking a satellite. In some crosses of Aneuploid x Normal, individual asci may show at prophase I either complete loss, partial loss, or pycnosis of the translocated IL segment. This observation of pycnosis suggests chromosome inactivation. — Growth from aneuploid ascospores is initially slow, but can accelerate to the wild-type rate.     

Use of Neurospora spore killer strains to obtain centromere linkage data without dissecting asci

Use of a centromere-linked Spore killer gene Sk reduces manyfold the labor involved in obtaining tetrad data that would otherwise require ordered dissection of intact linear eight-spored asci. Heterozygous crosses are made for Spore killer (SkK X SkS) and for markers to be tested. In such crosses only SkK ascospores survive. The four viable (SkK) and four aborted (SkS) ascospores of each ascus are ejected from the perithecium as a physically disordered group. The four surviving SkK ascospores of individual asci are germinated and scored. SkK segregates from SkS at the first meiotic division. If both marker alleles are represented in the surviving products, they must therefore have segregated from one another at the second division. Four-spore (Fsp) genes have been used to eliminate one postmeiotic nuclear division, so that only two ascospores per ascus need to be scored. The Spore killer method has been useful for mapping closely linked genes in centromere regions, for identifying genes that are far out on chromosome arms, for obtaining information on meiotic crossing-over, and for comparing linkages in different species.     

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.     

Abnormal ascospore morphology in the bud mutant of Neurospora tetrasperma

A recessive ascospore mutant of Neurospora tetrasperma, named bud, was isolated from a wild-collected heterokaryotic strain with four different nuclear components. bud segregates as a single mendelian gene. When bud is homozygous, meiosis is apparently normal but postmeiotic events are not. Abnormal orientation of spindles at the postmeiotic mitosis often results in failed pair-wise association of nuclei and their irregular distribution along the length of the ascus prior to spore delimitation. Consequently, many asci cut out more than four ascospores; some contain no nuclei while others contain more than two. The most dramatic effect of bud is on ascospore delimitation itself. Many ascospores are irregularly shaped and are often interconnected, because of incomplete spore delimitation. Ascospores also show one or two lobes or bud-like extensions of varying sizes. Over 75% of ascospores from bud x bud remain white or tan and are inviable. The interaction of bud with a dominant Eight-spore mutant (E) was examined in both heterozygous and homozygous crosses. When both bud and E are heterozygous, bud has no effect on ascospore delimitation or on the phenotype of E because bud is recessive; many asci produce 5-8 ascospores just as in E x E(+). And when bud is homozygous and E is heterozygous, ascospore delimitation is less affected than when E is absent. Moreover, when both bud and E are homozygous, the effect on ascospore development is less extreme than when E is homozygous singly.     

Sexual diploids of Aspergillus nidulans do not form by random fusion of nuclei in the heterokaryon

The sexual stage of Aspergillus (Emericella) nidulans consists of cleistothecia containing asci, each with eight ascospores. The fungus completes the sexual cycle in a homokaryotic or a heterokaryotic mycelium, respectively. The common assumption for the last 50 years was that different nuclear types are not distinguishable when sexual development is initiated. When cultured on a medium limited for glucose supplemented with 2% sorbitol, sexual development of A. nidulans is slowed and intact tetrads can be isolated. Through tetrad analysis we found that unlike haploid nuclei fuse preferentially to the prezygotic diploid nucleus. When heterokaryons are formed between nuclei of different genetic backgrounds, then recombinant asci derived from opposite nuclei are formed exclusively. Strains in the same heterokaryon compatibility group with moderate differences in their genetic backgrounds can discriminate between the nuclei of a heterokaryon and preferentially form a hybrid diploid nucleus, resulting in 85% recombinant tetrads. A. nidulans strains that differ at only a single genetic marker fuse the haploid nuclei at random for formation of diploid nuclei during meiosis. These results argue for a genetically determined "relative heterothallism" of nuclear recognition within a heterokaryon and a specific recruitment of different nuclei for karyogamy when available.     

Cytogenetic behavior of spore killer genes in neurospora

Crosses heterozygous and homozygous for Sk-1, Sk-2 and Sk-3 were examined by light microscopy. All three Spore killers behave similarly. In heterozygous killer x sensitive crosses, meiosis and ascospore development are normal until after the second postmeiotic mitosis when four of the eight ascospores in each ascus stop developing and degenerate. The four surviving ascospores carry the killer. Death of sensitives thus occurs only after killer and sensitive alleles, Sk^K and Sk^S, have segregated into separate ascospores. Homozygous killer x killer crosses do not show such a pattern of degeneration. Either all ascospores are normal or, if some fail to mature, they do not resemble the degenerating sensitive ascospores in heterozygous asci.——With Sk-2, it was shown that Sk^S nuclei do not abort when both Sk^K and Sk^S are present in the same ascospore. Mutants affecting ascus development were used to obtain large ascospores enclosing both Sk^K and Sk^S meiotic products in a common cytoplasm. Sk^S nuclei do not then undergo the degeneration that would be seen if they were sequestered into separate ascospores, and viable Sk^S progeny are recovered in undiminished numbers when the mixed multinucleate large ascospores are germinated. In a four-spored mutant, where each ascospore encloses a single nucleus following meiosis, degeneration of Sk^S ascospores nevertheless occurs, even though the third nuclear division is omitted. Cycloheximide and temperature treatments do not affect the expression of Sk^K.     

Ascus development in two temperature-sensitive four-spore mutants of Neurospora crassa

Two nonallelic Four-spore mutants are known in which ascospore walls enclose the four immediate products of meiosis rather than the normal eight products of a postmeiotic mitosis. Expression depends on temperature. The Four-spore phenotype is expressed when the developing asci are subjected either to high temperatures (25-30 degrees C) for Fsp-1 or to low temperatures (15-20 degrees C) for Fsp-2. Heterozygous Fsp-1 X Fsp-1+ crosses make eight-spored asci at 15-20 degrees C but produce many four-spored asci at 25 degrees C and mostly four-spored asci at 30 degrees C. Homozygous Fsp-1 X Fsp-1 crosses respond similarly to increasing temperature but make 40-50% four-spored asci even at 20 degrees C. Heterozygous Fsp-2 X Fsp-2+ crosses produce almost exclusively four-spored asci at 15 degrees C but a mixture of four- and eight-spored asci at 20, 25, and 30 degrees C. Homozygous Fsp-2 X Fsp-2 crosses make all four-spored asci at 15 and 20 degrees C and a mixture of four- and eight-spored asci at 25 and 30 degrees C. When both Fsp-1 and Fsp-2 are present in a cross, either homozygous or heterozygous, no asci contain more than four ascospores at any temperature. Limited temperature-shift experiments with Fsp-1 and Fsp-2 show that the sensitive period for Four-spore expression is sometime after meiotic prophase, possibly at interphase II.     

ami1, an orthologue of the Aspergillus nidulans apsA gene, is involved in nuclear migration events throughout the life cycle of Podospora anserina

The Podospora anserina ami1-1 mutant was identified as a male-sterile strain. Microconidia (which act as male gametes) form, but are anucleate. Paraphysae from the perithecium beaks are also anucleate when ami1-1 is used as the female partner in a cross. Furthermore, in crosses heterozygous for ami1-1, some crozier cells are uninucleate rather than binucleate. In addition to these nuclear migration defects, which occur at the transition between syncytial and cellular states, ami1-1 causes abnormal distribution of the nuclei in both mycelial filaments and asci. Finally, an ami1-1 strain bearing information for both mating types is unable to self-fertilize. The ami1 gene is an orthologue of the Aspergillus nidulans apsA gene, which controls nuclear positioning in filaments and during conidiogenesis (at the syncytial/cellular transition). The ApsA and AMI1 proteins display 42% identity and share structural features. The apsA gene complements some ami1-1 defects: it increases the percentage of nucleate microconidia and restores self-fertility in an ami1-1 mat+ (mat-) strain. The latter effect is puzzling, since in apsA null mutants sexual reproduction is quite normal. The functional differences between the two genes are discussed with respect to their possible history in these two fungi, which are very distant in terms of evolution.     

<Emphasis Type="Italic">Neurospora tetrasperma</Emphasis> crosses heterozygous for hybrid translocation strains produce rare eight-spored asci-bearing heterokaryotic ascospores

During ascogenesis in Neurospora, the ascospores are partitioned at the eight-nucleus stage that follows meiosis and a post-meiotic mitosis, and the ascospores that form in eight-spored asci are usually homokaryotic. We had previously created novel T ^Nt strains by introgressing four Neurospora crassa insertional translocations (EB4, IBj5, UK14-1, and B362i) into N. tetrasperma. We now show that crosses of all the T ^Nt strains with single-mating-type derivatives of the standard N. tetrasperma pseudohomothallic strain 85 (viz. T ^Nt a × 85A or T ^Nt A × 85a) can produce rare eight-spored asci that contain heterokaryotic ascospores, or ascospores with other unexpected genotypes. Our results suggest that these rare asci result from the interposition of additional mitoses between the post-meiotic mitosis and the partitioning of nuclei into ascospores, leading to the formation of supernumerary nuclei that then generate the heterokaryotic ascospores. The rare asci probably represent a background level of ascus dysgenesis wherein the partitioning of ascospores becomes uncoupled from the post-meiotic mitosis. Ordinarily, the severest effect of such dysgenesis, the production of mating-type heterokaryons, would be suppressed by the N. crassa tol (tolerant) gene, thus explaining why such dysgenesis remained undetected thus far.     

Suppressed recombination and a pairing anomaly on the mating-type chromosome of Neurospora tetrasperma

Neurospora crassa and related heterothallic ascomycetes produce eight homokaryotic self-sterile ascospores per ascus. In contrast, asci of N. tetrasperma contain four self-fertile ascospores each with nuclei of both mating types (matA and mata). The self-fertile ascospores of N. tetrasperma result from first-division segregation of mating type and nuclear spindle overlap at the second meiotic division and at a subsequent mitotic division. Recently, Merino et al. presented population-genetic evidence that crossing over is suppressed on the mating-type chromosome of N. tetrasperma, thereby preventing second-division segregation of mating type and the formation of self-sterile ascospores. The present study experimentally confirmed suppressed crossing over for a large segment of the mating-type chromosome by examining segregation of markers in crosses of wild strains. Surprisingly, our study also revealed a region on the far left arm where recombination is obligatory. In cytological studies, we demonstrated that suppressed recombination correlates with an extensive unpaired region at pachytene. Taken together, these results suggest an unpaired region adjacent to one or more paired regions, analogous to the nonpairing and pseudoautosomal regions of animal sex chromosomes. The observed pairing and obligate crossover likely reflect mechanisms to ensure chromosome disjunction.     

Genetic analysis of spore killing in the filamentous ascomycete Podospora anserina

In the present study, we analysed different Podospora anserina strains for their ability to induce spore killing and identified three new killer strains. Test crosses of killer strains with different sensitive strains revealed different second division segregation ratios suggesting an influence of the sensitive strain on the crossing-over frequency. In crosses of killer strain O with a sensitive strain, the frequency of two-spored asci was found to vary extremely from perithecium to perithecium. Furthermore, crosses of strain O with sensitive strain Us5 led to a significant proportion of asci containing an unexpected high number of surviving spores as the result of gene conversion. Finally, for the first time, we present data demonstrating that in a number of ascospores the killer and the corresponding sensitive allele is located in one individual nucleus. Mycelia derived from such ascospores display a "sensitive killer" phenotype. Crosses of these mycelia with a killer strain as well as with a sensitive strain result in spore killing. Strikingly, heterokaryotic spores containing the recombined "sensitive/killer" allele and a nucleus with a killer allele give rise to mycelia protected against spore killing during selfing.     

禾谷镰孢菌蛋白激酶FgCdc15功能的研究

细胞分裂是真核细胞生长发育的重要环节。本研究利用生物信息学的方法,通过使用酵母菌Cdc15蛋白激酶序列比对,发现禾谷镰孢菌中只存在一个Cdc15蛋白激酶。基因缺失的功能研究表明FgCDC15（FGSG_10381）基因敲除后,突变体菌落生长速度减慢,对小麦和玉米无致病力。FgCDC15基因敲除突变体产生的分生孢子外观形态正常,但隔膜数量变少,同时发现该基因在分生孢子阶段表达量最高。在有性生殖阶段,FgCDC15基因敲除突变体可产生极少量的子囊壳,但不产生子囊和子囊孢子。本文研究表明,禾谷镰孢菌蛋白激酶FgCdc15可能参与了有性和无性阶段的细胞分裂和生长,同时影响禾谷镰刀菌的致病毒力。