Meiosis and ascospore development in Cochliobolus heterostrophus.

Cochliobolus heterostrophus produces eight filiform ascospores per ascus, following meiosis and a postmeiotic mitosis. Early ascus development and nuclear divisions in C. heterostrophus resemble those of the prototypic Pyrenomycete Neurospora crassa. However, the two fungi differ in several important details owing to differences in ascus and ascospore shape, spindle pole body (SPB) behavior during spore delimitation, and ascospore development. In C. heterostrophus, the two spindles at meiosis II, and the four spindles at the postmeiotic mitosis are aligned irregularly, unlike the tandem or ladder rung-like orientation of spindles of N. crassa. Prior to ascospore delimitation, all eight nuclei reorient themselves and their SPB plaques migrate toward the base of the ascus. The SPB plaques facilitate demarcation of the lower end of each incipient ascospore. The filiform ascospores are uninucleate and unsegmented at inception but they become highly multinucleate, multisegmented, and helically coiled when mature. An account of ascus development, nuclear divisions, and ascospore delimitation and maturation is presented here and supported by a series of photomicrographs.     

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.     

Diverse programs of ascus development in pseudohomothallic species of Neurospora,Gelasinospora, and Podospora

Meiosis and ascospore development in the four-spored pseudohomothallic ascomycetes Neurospora tetrasperma, Gelasinospora tetrasperma, Podospora anserina, and P. fefraspora have been reexamined, highlighting differences that reflect independent origins of the four-spored condition in the different genera. In these species, as in the heterothallic eight-spored N. crassa, fusion of haploid nuclei is followed directly by meiosis and a postmeiotic mitosis. These divisions take place within a single unpartitioned giant cell, the ascus, which attains a length of >0.1 mm before nuclei are enclosed by ascospore walls. Two basically different modes underlie the delivery of opposite mating type nuclei into each of the four ascospores in the different genera. In N. tefrasperma on the one hand, the mating type locus is closely centromere-linked. Mating types therefore segregate at the first meiotic division. The second division spindles of N. tefrasperma overlap and are usually parallel to one another, in contrast to the their tandem arrangement in N. crassa. As a result, nonsister nuclei of opposite mating type are placed close together in each half-ascus and a pair is enclosed in each ascospore. In the Podospora and Gelasinospora species on the other hand, the second-division spindles are in tandem, with sister nuclei of opposite mating type associated as a pair in each half-ascus. It is established for P. anserina and inferred for P. fetraspora and G. fefrasperma that a single reciprocal crossing over almost always occurs in the mating type-centromere interval, ensuring that mating types segregate at the second meiotic division and that nuclei of opposite mating type are enclosed in each ascospore. Other differences are also seen that are less fundamental. Neurospora tetrasperma differs from the other species in the orientation of chromosomes and spindle pole body plaques at interphase (I.) Third-division spindles are oriented parallel to the ascus wall in Gelasinospora but across the ascus in Podospora and Neurospora. The two Podospora species differ from one another in nuclear behavior following mitosis in the young ascospores. In P. tefraspora, two of the four nuclei migrate into the tail cell, which degenerates, leaving one functional nucleus of each mating type. In P. anserina, by contrast, only one of the four nuclei moves into the tail cell, leaving the germinating ascospore with two functional nuclei of one mating type and one of the other. The pseudohomothallic condition with its heterokaryotic vegetative phase has significant consequences for both the individual organism and the breeding system. Genetic controls of development and recombination are complex. Inbreeding is not obligatory. © 1994 WiIey-Liss, Inc.     

Cytology of recessive sexual-phase mutants from wild strains of Neurospora crassa.

Wild-collected strains of Neurospora crassa harbor recessive mutations that are expressed in the sexual phase when homozygous. Thirty-two representative mutants that produced barren perithecia were examined cytologically. Six of these mutants failed to form asci. Of the remaining 26, chromosome pairing was disturbed in 12 and meiosis was disturbed at pachytene or diplotene in 5. Seven mutants showed normal meiosis I but then diverged from the normal sequence, and two showed perithecial beak abnormalities. In many mutants, ascus development and nuclear divisions continued after the initial defect, albeit abnormally. Nuclear divisions were often delayed, essentially uncoupling them from other ascus events such as the formation of enlarged spindle pole body plaques, ascospore wall membranes, and spore delimitation. All 32 mutants were recessive and none showed obvious morphological abnormalities during vegetative growth. This phenotype contrasts sharply with that of numerous laboratory-induced ascus mutants, which are frequently expressed pleiotropically in the vegetative phase and several are dominant in the sexual phase.     

CHROMOSOME NUMBERS IN THE HYPOCREALES. II. ASCUS DEVELOPMENT IN NECTRIA CINNABARINA

The cytology of ascus development in Nectria cinnabarina was investigated with the orceinsmear technique, from crozier formation to ascospore maturation. At prophase I synapsis occurs while the chromosomes are still contracted, and the nucleus passes through dictyotene, a diffuse stage rarely seen in plants. A haploid complement of five chromosomes has been precisely determined. The first two divisions in the ascus constitute meiosis, and the third (mitotic) is followed by ascospore delimitation. A fourth division takes place in the ascospore, which is subsequently divided by a septum into two uninucleate cells. Of all species of Nectria thus far investigated N. cinnabarina is the only species in which additional nuclear divisions in the ascus do occur, accounting for the multinucleate condition in the ascospore cells. The bearing that this distinctive nuclear condition has on phylogeny and evolution in the Hypocreales is discussed.     

Six decades of Neurospora ascus biology at Stanford

Ascus is the largest cell in the entire life cycle of Neurospora; it is where the transient diploid nucleus undergoes meiosis and a postmeiotic mitosis. The eight haploid nuclei are then sequestered into eight linearly ordered ascospores. Dodge's pioneering work on Neurospora and its simple nutritional requirements inspired Beadle and Tatum of Stanford University to use N. crassa for their landmark demonstration that individual genes specify enzymes. McClintock visited Stanford in 1944, and showed that meiosis and chromosome behaviour in Neurospora are similar to those of higher eukaryotes. Most of the subsequent Neurospora ascus biology work was carried out in David Perkins' laboratory at Stanford from 1960–2007. Since 1974, I have extensively used an iron-haematoxylin staining procedure, the DNA-specific fluorochrome acriflavine, and GFP-tagged genes for visualizing meiotic chromosome behaviour and gene silencing during ascus and ascospore development. Our recent discovery of meiotic silencing, and the availability of genome sequence and GFP-tagged genes will no doubt pave the way for molecular analysis of complex processes during ascus development.     

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.     

A study of the fine structure of ascosporogenesis in Saccobolus kerverni

Summary Observations of ascospore fromation in KMnO₄-fixed Saccobolus kerverni apothecia with the electron microscope reveal the following sequence. Ascus formation is preceded by the development of croziers whose fine structure differs little from that of vegetative hyphae. Following fusion of the two nuclei in the ascus mother cell, the resultant ascus elongates, and two large vacuoles appear, first below and later above the fusion nucleus. These vacuoles soon occupy dominant positions at the tip and bottom of the ascus and assume a flocculent appearance. Nuclear blebbing occurs during meiosis, mitosis, and the subsequent spore delimitation process in the central cytoplasmic portion of the ascus. Each spore initial is surrounded by two membranes, the plasma and investing membranes, between which the spore wall is deposited in two layers, an inner primary wall and an outer secondary wall. Following primary wall deposition the spores clump; secondary wall deposition begins outside the primary wall at the places where the spores are contiguous. Interdigitation of these walls and disappearance of the investing membranes in the sutures lead to the envelopment of all eight ascospores in a common secondary wall. A flocculent material in the epiplasmic vacuoles aggregates around the mature spore balls.Based on a portion of a dissertation presented to the Faculty of the Graduate School of the University of Texas in partial fulfillment of the requirements for the degree of Doctor of Philosophy.     

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.     

Morphogenesis of Ascospores in Saccharomyces cerevisiae

Ultrastructural changes associated with ascospore formation in Saccharomyces cerevisiae were investigated by using freeze-etching and thin-sectioning techniques. The first nuclear division (meiosis I) is indicated by the appearance of spindle fibers within the nucleus. The nucleus subsequently elongates and eventually assumes a barbell shape; the second nuclear division (meiosis II) occurs before nuclear separation. The spindle fibers involved in meiosis II appear to be oriented perpendicular to those observed in meiosis I. A discrete bilaminar structure (forespore wall) progressively delineates each ascospore nucleus and encloses cytoplasmic material including mitochondria and endoplasmic reticulum. The forespores then elongate, close off, and become separated from the ascus cytoplasm by membranes. The ascospores assume a spherical shape as spore coat material is laid down; the latter stages of ascospore formation are characterized by thickening of the ascospore wall and disintegration of the ascus cytoplasm. No structures which could be identified as chromosomes were observed.     

The fine structure of ascospore shape and development inCeratocystis fimbriata

Ascospore development inCeratocystis fimbriata Ell. & Halst. commenced in an eight-nucleate ascus. A single vesicle formed along the periphery of the ascus from fragments of ascospore delimiting membranes, surrounded all eight nuclei and eventually invaginated, first forming pouches with open ends, then finally enclosing each of the eight nuclei in a separate sac, thus delimiting ascospores. Pairing of the ascospores followed and brim formation occurred at the contact area between two ascospores. Osmiophilic bodies contributed to the formation of brim-like appendages by fusing to the ascospore walls. Additional brims were observed at opposite ends of the ascospores giving them a double-brimmed appearance.Abbreviations AV ascus vesicle - DM delimiting membrane - EV electron translucent bodies - G granules - M mitochondria - N nucleus - OB osmiophilic bodies - PMV plasmamembrane vesicles - PW primary wall - SW secondary wall     

Meiotic silencing in the homothallic fungus Gibberella zeae

The homothallic ascomycete fungus Gibberella zeae is an important pathogen on major cereal crops. The objective of this study was to determine whether meiotic silencing occurs in G. zeae. Cytological studies demonstrated that GFP and RFP-fusion proteins were not detected during meiosis, both in heterozygous outcrosses and homozygous selfings. The deletion of rsp-1, a homologue used for studies on meiotic silencing of Neurospora crassa, triggered abnormal ascospores from selfing, but outcrosses between the mutant and wild-type strain resulted in some ascospores with mutant phenotype (low occurrence of ascus dominance). When the ectopic mutants that carried an additional copy of rsp-1 were selfed, they primarily produced ascospores with normal shape but a few ascospores (0.23 %) were abnormal, in which both endogenous and ectopically integrated genes contained numerous point mutations. The ectopic mutants showed low occurrence of ascus dominance in outcrosses with strains that carried the wild-type allele. Approximately 10 % of ascospores were abnormal but all of the single-ascospore isolates produced normal-shaped ascospores from selfing. However, no ascus dominance was observed when the mutants were outcrossed with a sad-1 deletion mutant, which lacks the putative RNA-dependent RNA polymerase essential for meiotic silencing in N. crassa. All results were consistent with those generated from an additional gene, roa, required for ascospore morphogenesis. This study demonstrated that G. zeae possesses a functional meiotic silencing mechanism which is triggered by unpaired DNA, as in N. crassa.     

CYTOLOGY OF HELMINTHOSPORIUM TURCICUM AND ITS ASCIGEROUS STAGE,TRICHOMETASPHAERIA TURCICA

Knox- Davies , P. S., and J. G. Dickson . (U. Wisconsin, Madison.) Cytology of Helmintho sporium turcicum and its ascigerous stage, Trichometasphaeria turcica . Amer. Jour. Bot. 47(5) : 328—339. Illus. 1960.–The cells of the vegetative hyphae were generally multinucleate. Interphase nuclei resembled those of higher organisms, with a matrix of thread-like chromatin material surrounding a spherical nucleolus. “Beaked” nuclei frequently associated with anastomosing hyphae were interpreted as migrating nuclei. Nuclear division in the vegetative hyphae was rapid. Various division stages were distinguished but it was difficult to make accurate chromosome counts. The nucleoli were discarded at prophase or prometaphase and were reorganized in daughter nuclei at telophase. An outstanding feature of nuclear division was that all the nuclei in a cell divided simultaneously. Conidiophores and conidia were occasionally joined by wide cytoplasmic connections. They were multinucleate throughout their development. Mechanisms therefore exist for the perpetuation of heterokaryons through the conidium. Ascus development was studied in a hybrid between a dark and an albino isolate. Crozier formation was typical and nuclear fusion occurred in the young ascus. Four nuclear divisions were completed in the ascus before there was evidence of ascospore delimitation. Further nuclear division took place in the ascospores whose cells were multinucleate. The occurrence of less than 8 ascospores in an ascus appeared to follow degeneration of nuclei rather than the incorporation of a number of division-Ill nuclei in a single ascopore. Chromosome counts and irregularities in the appearance and behavior of nuclei and chromosomes in the asci indicate that aneuploidy occurs in Trichometasphaeria turcica. It is suggested that aneuploidy is a common phenomenon in the conidial stage of the fungus H. turcicum, and possibly also in other imperfect fungi.     

SPINDLES, SPINDLE PLAQUES, AND MEIOSIS IN THE YEAST SACCHAROMYCES CEREVISIAE (HANSEN)

The intranuclear spindle of yeast has an electron-opaque body at each pole. These spindle plaques lie on the nuclear envelope. During mitosis the spindle elongates while the nuclear membranes remain intact. After equatorial constriction there are two daughted nuclei, each with one spindle plaque. The spindle plaque then duplicates so that two side-by-side plaques are produced. These move rapidly apart and rotate so that they bracket a stable 0.8 µm spindle. Later, during mitosis, this spindle elongates, etc. Yeast cells placed on sporulation medium soon enter meiosis. After 4 hr the spindle plaques of the more mature cells duplicate, producing a stable side-by-side arrangement. Subsequently the plaques move apart to bracket a 0.8 µm spindle which immediately starts to elongate. When this meiosis I spindle reaches its maximum length of 3–5 µm, each of the plaques at the poles of the spindle duplicates and the resulting side-by-side plaques increase in size. The nucleus does not divide. The large side-by-side plaques separate and bracket a short spindle of about 1 µm which elongates gradually to 2 or 3 µm. Thus there are two spindles within one nucleus at meiosis II. To the side of each of the four plaques a bulge forms on the nucleus. The four bulges enlarge while the original nucleus shrinks. These four developing ascospore nuclei are partially surrounded by cytoplasm and by a prospore wall which originates from the cytoplasmic side of the spindle plaque. Eventually the spore nuclei pinch off and the spore wall closes. In some of the larger yeast cells this development is completed after 8 hr on sporulation medium.     

Cytological and genetic studies of the life cycle of Saccharomycopsis lipolytica

Summary In the alkane yeast Saccharomycopsis lipolytica (formerly: Candida lipolytica) the variability in the ascospore number is caused by the absence of a correlation between the meiotic divisions and spore wall formation. In four spored yeasts, after meiosis II, a spore wall is formed around each of the four nuclei produced by meiosis II. However, in the most frequently occurring two spored asci of S. lipolytica, the two nuclei are already enveloped by the spore wall after meiosis I due to a delay of meiosis II. This division takes place within the spore during the maturation of the ascus. In this case germination of the binucleate ascospore is not preceded by a mitosis. It follows that the cells of the new haploid clones are mononucleate. In the three spored asci, which occur rarely, only one nucleus is surrounded by a spore wall after meiosis I; the other nucleus undergoes meosis II before the onset of spore wall formation. The result is one binucleate and two mononucleate spores. In the one spored asci the two meiotic divisions occur within the young ascospore, i.e. spore wall formation starts immediately after development of the ascus. These cytological observations were substantiated by genetic data, which in addition confirmed the prediction that binucleate spores may be heterokaryotic. This occurs when there is a postreduction of at least one of the genes by which the parents of the cross differ. This also explains the high frequency of prototrophs in the progeny on non-allelic auxotrophs since random spore isolates are made without distinguishing between mono-and binucleate spores. The possibility of analysing offspring of binucleate spores by tetrad analysis is discussed. These findings enable us to understand the life cycle of S. lipolytica in detail and we are now in a position to start concerted breeding for strain improvement especially with respect to single cell protein production.     

Ultrastructural aspects of ascosporulation in Arthroderma quadrifidum (=Trichophyton terrestre).

The ultrastructural features of developing and mature ascospores were delineated after mating Arthroderma quadrifidum on pablum cereal agar. Incipient ascospores each contained a granulated nucleus bounded by a nuclear envelope while presumptive ascospore cytoplasm was bounded by a double membrane and resided in glycogen-rich epiplasm of the ascus. Mature ascospores contained nuclei and mitochondria while the ascus epiplasm still retained abundant inclusions. The ascospore wall demonstrated the presence of heterogeneous material between the plasmalemma and the outer spore membrane which appeared smooth.     

A novel role of Dma1 in regulating forespore membrane assembly and sporulation in fission yeast

In fission yeast Schizosaccharomyces pombe, a diploid mother cell differentiates into an ascus containing four haploid ascospores following meiotic nuclear divisions, through a process called sporulation. Several meiosis-specific proteins of fission yeast have been identified to play essential roles in meiotic progression and sporulation. We report here an unexpected function of mitotic spindle checkpoint protein Dma1 in proper spore formation. Consistent with its function in sporulation, expression of dma1(+) is up-regulated during meiosis I and II. We showed that Dma1 localizes to the SPB during meiosis and the maintenance of this localization at meiosis II depends on septation initiation network (SIN) scaffold proteins Sid4 and Cdc11. Cells lacking Dma1 display defects associated with sporulation but not nuclear division, leading frequently to formation of asci with fewer spores. Our genetic analyses support the notion that Dma1 functions in parallel with the meiosis-specific Sid2-related protein kinase Slk1/Mug27 and the SIN signaling during sporulation, possibly through regulating proper forespore membrane assembly. Our studies therefore revealed a novel function of Dma1 in regulating sporulation in fission yeast.     

Division spindle and centrosomal plaques during mitosis and meiosis in some Ascomycetes

The behaviour of the division spindle and centrosomal plaques is described in four species of Ascomycetes (Ascobolus immersus, Ascobolus stercorarius, Podospora anserina and Podospora setosa) studied by light and electron microscopy. Two unique features of the kinetical apparatus were observed: presence of centrosomal plaques and intranuclear location of the spindle. In all types of mitoses (mycelium, crosier and postmeiotical mitosis) the apparatus is structurally identical to that found in meiosis. The centrosomal plaques, present in all divisions, are always contiguous with the nuclear envelope and never show centrioles similar to those commonly found in Metazoa and Protozoa. During metaphase and anaphase the plaque is constituted of two zones situated on each side of the nuclear envelope: an electron opaque outer zone and inner one less opaque in which most of the microtubules end. In Podospora the outer zone appears in sections as consisting of two dark layers separated by a clear one. Two dispositions of plaques are possible: either they are entirely contiguous with the nuclear envelope (Ascobolus) or only partially so, the remainder being perpendicular to the nuclear envelope (Podospora). — The localisation of the plaques in the ascus was determined by light and electron microscopy. The nuclear envelope was shown to remain intact during division. It was possible to observe that the sporal wall of each spore originated from the same unique double membrane formed in the ascus during the meiotic second division and postmeiotical mitosis. This fact is of genetical interest for the study of morphological and physiological characters of the spores.     

Ascospore linearity in the four-spored ascus ofNeurospora tetrasperma

The four-spored ascus ofNeurospora tetrasperma is linearly ordered, i.e. the order of the ascospores within the linear ascus directly reflects preceding meiotic events. This conclusion is based upon the finding of only two types of arrangements of homokaryotic ascospores in asci showing second division segregation and the failure to find any of the other four theoretically possible types of homokaryotic arrangements. The data are also consistent with the regular occurrence of nuclear passing at both the second and third meiotic divisions during ascus development. This work was supported by Public Health Service Grant GM 10672. Supported in part by Public Health Service Training Grant 5-T1-GM-767-05.