Ascus Development Research Articles

Podospora anserina bibliography n° 10 - Additions

Podospora anserina is a coprophilous fungus growing on herbivore dung. It is a pseudohomothallic species in which ascus development results, as in Neurospora tetrasperma but through a different process, in the formation of four large ascospores containing nuclei of both mating types.

Ultrastructural Studies of Ascosporogenesis in Ascobolus stictoideus (Pezizales, Ascomycetes)

Ascosporogenesis in Ascobolus stictoideus is studied and compared with that found in other members of Pezizales. Early phases of ascus development involving spore delimiting membranes from the plasmalemma and spore formation within the epiplasm are much like those of other species of Pezizales, with the exception of the appearance of organelles, such as pigmented bodies and net bodies, which are not found in other taxa. Primary spore walls are very thick and electron translucent. Opaque globular bodies, amorphous dense material derived from net bodies, and dense membrane‐bound bodies associated with the endoplasmic reticulum appear in the epiplasm and later play a role in spore wall formation. Dense granular bodies containing small particles are transported from the epiplasm into the perisporic sac along with pigmented bodies. These deeply opaque bodies increase in size, appear to coalesce, and are deposited onto the epispore layer of the spore. The phylogenetic significance of spore wall development is discussed in light of recent molecular data.

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.

Repeat-Induced Point Mutations inPad-1,a Putative RNA Splicing Factor fromNeurospora crassa,Confer Dominant Lethal Effects on Ascus Development

We describe the characterization of a gene,Pad-1,fromNeurospora crassawhich displays sequence characteristics of the RS class of hnRNA-binding proteins (hnRNP) and mRNA splicing factors. This is the first report of the isolation of a putative hnRNP gene fromN. crassa.PAD-1 showed 30% identity and 57% similarity to a protein, HCC1, which was isolated using autoantibodies from patients suffering from hepatocellular carcinoma. Both HCC1 and PAD-1 show amino acid sequence similarities to the human splicing factor, U2AF65. Mutations induced inPad-1by repeat-induced point (RIP) mutation show dominant effects on ascus and ascospore formation, a novel phenotypic class of RIP mutants. A mutant isolated from thePad-1RIP cross displayed a severe vegetative growth defect and dominant effects on ascus development, indicating thatPad-1is essential for both asexual and sexual development.

A Putative Rhamnogalacturonase Required for Sexual Development of Neurospora crassa

In previous work, the asd-I (ascus development) gene of the filamentous fingus Neurospora crassa was identified as a gene expressed preferentially during the sexual cycle and shown to be essential for normal sexual development. The asd-I gene has been sequenced and further characterized. It contains two introns, the first of which is in-frame and inefficiently or differentially spliced. The predicted ASD-I protein has extensive homology with rhamnogalacturonase B of Aspergillus aculeatus, which cleaves the backbone within the ramified hairy regions of pectin. In homozygous asd-I crosses, sexual development is initiated and large numbers of normal-sized asci are formed. Ascospore delineation does not occur, however, and no sexual progeny are produced. As most asd-I asci contain eight nuclei, the two meiotic divisions and subsequent mitotic division typical of normal crosses seem to occur, but the haploid nuclei are not partitioned into ascospores. In wild-type crosses, the ASD-I protein is present in large amounts in croziers and young asci, but it is only faintly detectable in more mature asci containing developing ascospores. Models to explain the possible role of a rhamnogalacturonase in sexual development are presented.

How the Cytoskeleton Recognizes and Sorts Nuclei of Opposite Mating Type during the Sexual Cycle in Filamentous Ascomycetes

In heterothallic filamentous ascomycetes, two nuclei of opposite mating type must recognize one another in a plurinucleate cell to form a pair prior to karyogamy. In pseudohomothallic species, two nuclei of opposite mating type must also pair after meiosis to form a binucleate spore. We have examined the cytoskeletal involvement in nuclear pairings by immunofluorescence and drug disruption, using heterothallic and pseudohomothallic species, as well as species without defined mating type (homothallic). Nuclei of species with defined mating type have spindle pole bodies which react with chromatin stains; those of homothallic species do not. The reactivity is seen only in interphase, not during nuclear divisions; thus, the DNA concerned is nuclear and not organellar. From light and immunofluorescence microscopy, the DNA is located at the nuclear face of the spindle pole body (SPB). We suggest that the DNA-SPB association may be involved in the recognition of self and nonself between nuclei of opposite mating types. Nuclei which cooperate in cell formation during ascus development or sporulation are placed in close proximity by the arrangement of spindles during the division preceeding cell formation; after division, each nuclear pair remains linked by intertwined microtubule asters. Nuclear pairs must migrate before binucleate spore formation. Drug disruptions established that actin-myosin interaction was the most important cytoskeletal factor in normal spore production. The ascomycete SPB shows unexpected flexibility in form and location during development. Prior to sporulation the outer plaque shows extensive modification in size and orientation. The modified portion detaches from the nucleus and acts as a cortical microtubule organizing center, while the rest of the spindle pole body remains at the nucleus.

Neurospora tetrasperma bibliography

Dodge's discoveries of pseudohomothallism and of heterokaryosis were both accomplished using Neurospora tetrasperma, which remained his favorite species. In the decades following Dodge's work, studies involving N. tetrasperma were continued in only a few laboratories, notably those of Alfred Sussman, Branch Howe, Adrian Srb, and Robert Metzenberg. The species has now begun to receive increased attention, both for comparative molecular-evolutionary studies and for studies that take advantage of certain unique features. Nuclear, mitochondrial, and plasmid DNA's have been used to explore phylogenetic relationships. Work is progressing on ascus development, recombination, vegetative incompatibility, and mating type. The following list is a first attempt to bring together the scattered literature on N. tetrasperma. (A few entries are also included that deal with pseudohomothallism generally.) The search has not been exhaustive. Please call any nontrivial omissions to my attention.

Cytoskeleton interactions in the ascus development and sporulation of Sordaria macrospora

ABSTRACT The organization of actin during meiosis and sporulation in the ascus of the filamentous ascomycete Sordaria macrospora was determined by immunofluorescence without removal of the cell wall. Actin is present as a dense cortical network of microfilaments (MF) and plaques, a perinuclear shell of actin in prophase I of meiosis, and a complex array of MF involved in alignment of prespore nuclei and closure of spore cell membranes. The relationship of actin to the previously examined microtubule system of the ascus was determined by double-label immunofluorescence. The cytoskeletal inhibitors nocodazole, cytochalasin D and 2,3-butanedione monoxime were used to examine the roles of actin and myosin in ascus development. Microfilament and microtubule arrays are interdependant; disruption of one network results in abnormalities in the other. Both microfilaments and actin-myosin interaction are required for separation and migration of duplicated spindle pole bodies, septation and sporulation.

Ascospore development in Ophiostoma piceae

The development of ascoma, ascus, and ascospore in Ophiostoma piceae was studied ultrastructurally and compared with that of other Ascomycetes. Ascospore delimitation commenced with the formation of double delimiting membranes in the ascus. The ascospore wall, consisting of the primary and secondary walls, was deposited between these membranes. The elongate ascospores of O. piceae differed from other species having similarly shaped ascospores, with respect to the shape and arrangement of asci in the ascoma, the number of wall layers of the ascospore, and formation of the secondary wall. Asci in O. piceae are spindle shaped, arranged along the periphery of the ascomatal wall. The ascospores have three layered walls, whereas some other species in Ceratocystis s.l. also with elongated ascospores probably have only two wall layers. Key words: Ophiostoma, elongated ascospores, sheaths, centrum.

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.

Genetic control of the sexual cycle in Neurospora

Over 200 mutants that affect sexual development have been isolated and analysed in Neurospora crassa . Mutational, recombinational, and molecular analyses on the mating type region show that the mating type genes A and a specify both the mating function and a vegetative (heterokaryon) incompatibility function. The two functions are inseparable by recombination but separable by a mutational event, as in a m 33 . An unlinked tol mutation suppresses the vegetative incompatibility associated with mating type genes A and a without affecting their mating type function. Mating type mutants (except a m 33 ), as well as male- and female-sterile mutants, are usually defective in haploid sexual functions or in early peritheical development. Many mutants that affect post-fertilization events produce normal-sized perithecia that are barren. Some mutants show distinct blocks during ascus development from karyogamy through ascospore formation, leading to complete or partial abortion of asci. Other mutants are variable in their expression, with development blocked at different stages from ascus to ascus, and a few asci capable of producing viable ascospores. Another category shows dramatic effects on ascus development without drastically reducing fertility. These mutations affect ascus or ascospore morphology, the size and number of ascospores, or ascospore pigmentation. Spore killer strains show meiotic drive, causing the death of ascospores not carrying the Spore killer allele. Although many applications have been found for specific mutants, as described in the text, their use for analysing the molecular mechanisms that underlie morphogenesis and development remain at best rudimentary. Judging from the recent history of developmental genetics in organisms such as Drosophila , the wide array of developmental mutations described for Neurospora in this review should be a valuable resource for future research into the control of sexual development.

Developmental regulation of proteins during fruit-body morphogenesis in Sordaria brevicollis

Specific morphogenetic events have been correlated with specific changes in polypeptide expression during ascomatal development in Sordaria brevicollis. Ascomatal morphogenesis was characterized using low-temperature SEM and light microscopy. Polypeptides were analysed by two-dimensional polyacrylamide gel electrophoresis. In vivo analyses involved isolating polypeptides from mycelia and fruit bodies and then examining them either unlabelled or after pulse-labelling with [35S]methionine. Polypeptides were also examined after in vitro translation of isolated mRNA. Of over 200 polypeptides detected after pulse-labelling, 31 were identified as being associated exclusively with sexual differentiation. Five of these polypeptides were specific to uncrossed protoperithecia; 9 were present in both protoperithecia and perithecia; and 17 were only detected in perithecia after crossing. Other changes in the level of polypeptide expression were also evident. The in vitro translation of mRNA showed some polypeptide expression during fruit-body morphogenesis to be under transcriptional control. None of the polypeptides detected have been identified but some were correlated with morphogenetic events which included (1) protoperithecium formation, (2) early perithecium development prior to neck emergence and ascus formation, (3) ascus and/or neck development, (4) ascospore formation, and (5) late stages of ascus or ascospore maturity. Cytochemical staining for laccase showed that this enzyme is subject to developmental regulation during fruit-body morphogenesis. Laccase activity was localized at sites of hyphal adhesion.

Ultrastructure of ascosporogenesis in Ophiostoma davidsonii

The ultrastructural development of asci, ascospores and centrum organization in Ophiostoma davidsonii , was examined. Mechanisms possibly involved in ascospore wall and sheath development, revealed differences between O. davidsonii and Ceratocystis moniliformis . Ascospore delimitation commenced with the formation of double delimiting membranes. The prismatic and spindle-shaped ascospores developed individually, not in pairs as in C. moniliformis and C. fimbriata . Asci of O. davidsonii were arranged in a cluster at the perithecium base whereas in C. moniliformis asci line the innermost surface, except the ostiole region.

Ascospore development in <i>Ceratocystis sensu lato</i> (Fungi): a review

Ceratocystis. Ceratocxstiopsis and Ophiostoma are important pathogens of trees and some agricultural crops and have recently been found on proteas and forest trees in South Africa. Taxonomic controversy exists regarding these genera and ultrastructural studies on the development of asci, uniquely shaped ascospores and centrum structure are inadequate. This rev iew summarises current knowledge of ascospore shape and development of the centrum obtained from light and electron microscope studies of Cemtocystis sensu lato. Important questions requiring further investigations are outlined. It is furthermore proposed that additional ultrastructural studies are required to clarify the current taxonomic disagreement in this group. Such studies could also identify relationships between these fungi and other Ascomycetes.

Cytology of the life-cycle of Morchella

Various stages of the morel life-cycle are studied cytologically. Photomicrographic evidence demonstrates that the average number of nuclei per cellular compartment in vegetative hyphae of Morchella is 10–15 and that hyphal fusions are quite frequent. The resting structures, the sclerotia, are actually pseudosclerotia which form from the repeated branching and enlargement of terminal hyphae from either primary (homokaryotic) or secondary (heterokaryotic) hyphae. Photomicrographs also depict the development of fruiting body primordia. Photomicrographs of ascus development demonstrate autogamy rather than de novo heterokaryon formation by hyphal fusion in the subhymenial layer of the fruiting body. For the first time a comprehensive life-cycle diagram of the morel is introduced.

Nonlinear Asci without Apical Pores in the Peak Mutant of Neurospora

Previous workers have shown that asci of the Neurospora crassa morphological mutant peak are swollen, and that ascospores are not linearly ordered. In the swollen asci, spindle orientations are abnormal at all three divisions but other events of meiosis and ascospore formation are apparently normal. In the present study, cytological features of ascus development in crosses involving peak are reported in greater detail. The swollen peak asci fail to differentiate apical pores, and mature ascospores are not discharged from the perithecia. The rare linear peak asci form normal apical pores, however. Ascus morphology is described in crosses of peak and two other dominant ascus mutants.

Presence of abnormal synaptonemal complexes in heterothallic species of Neurospora.

Synaptonemal complex abnormalities are frequent in reconstructed meiotic prophase nuclei of Neurospora crassa and Neurospora intermedia. Three kinds of synaptonemal complex anomalies were seen: lateral component splits, lateral component junctions, and multiple complexes. The anomalies apparently are formed during or after the pairing process, as they were not seen in the largely unpaired early zygotene chromosomes. Their presence at all the other substages from mid-zygotene to late pachytene indicates that they are not eliminated before the synaptonemal complex decomposes at diplotene. Abnormal synaptonemal complexes were seen in all 19 crosses of N. crassa and N. intermedia that were examined, including matings between standard laboratory strains, inversions, Spore killers, and strains collected from nature. The frequency of affected nuclei and degree of abnormality within a nucleus varied in different matings. No abnormalities were present in the homothallic species Neurospora africana and Neurospora terricola. Structural chromosome aberrations, introgression, and heterozygosity have been eliminated as causes for pairing disorder. The abnormal synaptonemal complexes seemingly do not interfere with normal ascus development and ascospore formation. The affected nuclei are not aborted during meiotic prophase, nor are they eliminated by abortion of mature asci. The abnormal meiocytes do not lead to aneuploidy, as judged by the low frequency of white ascospores in crosses between wild type strains that have many abnormalities. Thus, the abnormal synatonemal complexes do not appear to prevent chiasma formation between homologues.

Ultrastructure of ascus development in the teleomorph of Phoma arachidicola

Ascus development in the teleomorph of Phoma arachidicola was examined using light and electron microscopy. Ascus initials developed from pseudoparenchymatous elements in the pseudothecium and ascospores were delimited by a double-layered membrane produced independently of the plasmalemma. Ascospores were hyaline and became dark after discharge. No pseudoparaphyses were observed but threadlike strands, apparently remnants of the pseudoparenchymatous centrum compressed by developing asci, were interspersed among the asci. On the basis of these findings, the teleomorph of P. arachidicola cannot be accommodated in Didymella, Didymosphaeria or Mycosphaerella .

Ascus and crozier development in the Balansiae

The morphological development of the croziers and asci of four species of the Balansiae were examined and compared with other members of the order Clavicipitales to evaluate the potential usefulness of these developmental sequences as a taxonomic criterion at the family or subfamily level. Balansia epichloë, B. henningsiana, and Atkinsonella hypoxylon were very similar in all details of development. Myriogenospora atramentosa does differ in some characteristics, but is included in the single family Clavicipitaceae with the other species examined. Division of the order beyond the single family Clavicipitaceae is not supported by our data.

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.