Fungal cannons: explosive spore discharge in the Ascomycota

The ascomycetous fungi produce prodigious amounts of spores through both asexual and sexual reproduction. Their sexual spores (ascospores) develop within tubular sacs called asci that act as small water cannons and expel the spores into the air. Dispersal of spores by forcible discharge is important for dissemination of many fungal plant diseases and for the dispersal of many saprophytic fungi. The mechanism has long been thought to be driven by turgor pressure within the extending ascus; however, relatively little genetic and physiological work has been carried out on the mechanism. Recent studies have measured the pressures within the ascus and quantified the components of the ascus epiplasmic fluid that contribute to the osmotic potential. Few species have been examined in detail, but the results indicate diversity in ascus function that reflects ascus size, fruiting body type, and the niche of the particular species.     

The mechanism of ascus firing – Merging biophysical and mycological viewpoints

The actively discharging ascus is the unique spore-bearing cell that is responsible to dispatch spores into the atmosphere. From a physical perspective, this type of ascus is a sophisticated pressure gun that reliably discharges the spores at an extremely high velocity, without breaking apart. We identify four essential steps in discharge of spores whose order and timing may vary across species. First, asci that fire are mature, so a cue must be present that prevents discharge of immature spores and signals maturity. Second, pressure within the ascus serves to propel the spores forward; therefore a mechanism should be present to pressurize the ascus. Third, in ostiolate fruiting bodies (e.g. perithecia), the ascus extends through an opening to fire spores into the air. The extension process is a relatively unique aspect of the ascus and must be structurally facilitated. Fourth, the ascus must open at its tip for spore release in a controlled rupture. Here we discuss each of these aspects in the context of understanding the process of ascus and fruiting body function. While there is great diversity among fungi, we focus on discharge in a few model species, and then discuss how this framework may vary in other fungi. Our goal is to tie the physiological and molecular studies of ascus function with concepts in engineering that dictate structure.     

Sporulation in single-spore isolates from amitrole-induced multispored asci of Saccharomyces cerevisiae.

Amitrole treatment causes multispored ascus production by cells of a yeast strain whose asci normally contain two diploid spores. Single spores were isolated from asci containing two to eight spores and their ability to germinate was determined. Cells in colonies grown from single spores sporulated in the same manner as the parent strain indicating that amitrole had not induced meiotic division in the developing asci.     

Induction of multispored asci in two-spored strains of Saccharomyces cerevisiae by amitrole.

Although growth of two yeast strains characterized by consistent production of two diploid spores per ascus was inhibited in complex presporulation media containing amitrole, a fraction of the cells produced were able to form asci with more than two spores after transfer to acetate sporulation medium. Cells grown in a defined presporulation medium containing amitrole did not acquire this ability. The increase in spore numbers per ascus is attributed either to the induction by amitrole in growth medium of cells with more than one nucleus or to the restoration of normal meioses in the multispored asci.     

Yeast Mutants That Produce a Novel Type of Ascus Containing Asci Instead of Spores

Tetraploid yeast cells lacking BFR1 or overexpressing an essential gene BBP1 produce a novel type of ascus that contains asci instead of spores. We show here that the asci within an ascus likely arise because a/α spores undergo a second round of meiosis. Cells depleted of Bbp1p or lacking Bfr1p are defective in a number of processes such as nuclear segregation, bud formation, cytokinesis and nuclear spindle formation. Furthermore, deletion of BFR1 or overexpression of BBP1 leads to an increase in cell ploidy, indicating that Bfr1p and Bbp1p play roles in both the mitotic cell cycle and meiosis. Bfr1p and Bbp1p interact with each other in a two hybrid assay, further suggesting that they might form a complex important for cell cycle coordination.     

Preferential Ascus Discharge during Cross Maturation in SORDARIA BREVICOLLIS

Crosses involving spore color mutants of Sordaria brevicollis all showed a decline in the frequency of second division asymmetric asci (2:2:2:2's) as the cross matured. This decline was due to the preferential maturation and/or discharge of these asci. The proportion of spindle overlap and recombinational asci within the group did not change as shown by ascus dissection. The preferential discharge was also found to occur in two-point crosses where the asci did not contain wild-type spores.     

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.     

Preferential Occurrence of Nonsister Spores in Two-Spored Asci of SACCHAROMYCES CEREVISIAE: Evidence for Regulation of Spore-Wall Formation by the Spindle Pole Body

Yeast cells subjected to a reversible thermal arrest of meiosis yielded progressively fewer spores per ascus as the arrest was extended. Dissection of two-spored asci by a newly developed method that prevents selection of false asci revealed that the spores were not a random sample of the haploid meiotic products. Most, if not all, pairs of spores contain nonsister products of the reductional division. Electron microscopic examination of the meiotic cells revealed the cytological basis for this bias. All four spindle pole bodies (SPBs) present at the second meiotic division normally gain a structural modification (the outer plaque) upon which the initiation of the prospore wall occurs. In the formation of a two-spored ascus, only one spindle pole body on each meiosis II spindle was so modified. These observations suggest that the morphogenesis of spores is regulated at meiosis II by limiting the number of SPBs gaining the outer plaque. The enhancement of spore yield upon addition of fresh medium suggests that this morphogenetic regulation responds more directly to nutrient deprivation arising during the thermal arrest, rather than to elevated temperature per se.     

紫红曲有性阶段的研讨

紫红曲因在其菌丝体及孢子生长过程中,具有合成某些药用及色素物质功能而被重视。这些物质在东方国家中早被用于医疗及食物染色方面。本文在阐明该菌闭襄壳内数目众多的子囊孢子系来自多数子囊,其子囊壁在发育早期即已消失。具8个子囊孢子的单一子囊的确时有发生,所以红曲属名拉丁文原意“单一子囊”,虽然有时极易被置凝,但基本是正确的。此菌之另一显著特性为一次性结合可产生多数产囊丝钩及发育后的闭囊壳。此现象导致孢子形成以子囊孢子数目远超过分生孢子。     

A novel phenotype of eight spores asci in deletants of the prion-like Rnq1p in Saccharomyces cerevisiae

We report that a null rnq1 mutation in the yeast RNQ1 (YCL028w) prion-like gene of so far unknown function produces the doubling of spores in the asci. This phenotype is possibly due to the lack of inhibition by Rnq1p of an additional mitotic division during ascus formation. This novel phenotype termed "octopus asci" could be similar to prion [PIN+] phenotype.     

STRUCTURE AND DEVELOPMENT OF CLEISTOIODOPHANUS CONGLUTINATUS GEN. & SP. N. (ASCOBOLACEAE)

Cleistoiodophanus represents a new coprophilous genus of the tribe Iodophaneae in the Ascobolaceae (Pezizales). The only species thus far discovered, C. conglutinatus, is described and illustrated. Aspects of its cytological development are described from cultures obtained from apothecia found on sheep dung near Gainesville, Florida. Plasmogamy occurs in acogonial coils, two or three cells of which give rise to ascogenous hyphae. Ascogonia are quickly enclosed by vegetative hyphae and the ascocarp continues in a cleistohymenial development. Unlike Iodophanus and related genera, the excipulum remains intact even after spore maturation and the asci push through the epihymenial regions to release spores. The asci are characteristically thickened at their apices, diffusely amyloid, and somewhat saccate. The asci are predominantly 8-spored, but have been found with four or 16 spores per ascus. A previously undescribed Oedocephalum imperfect stage was induced in culture.     

Rare occurrence of the tetratype tetrads in Saccharomycodes ludwigii.

Genetic data suggesting the absence of crossover in Saccharomycodes ludwigii have been described. Tetrad data obtained from 888 asci from 60 pairs of genes with 22 genetic markers showed the absence of tetratype asci, except for 5 asci in which a single pair of alleles showed tetratype segregation to the other genetic markers in each ascus. Spore arrays in the linear asci showed that the + - + - and + - - + (or - + + -) asci occurred at almost equal frequencies. The two coherent spores at each end of an ascus were always marked with different alleles of a gene.     

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.     

Oxylipin-coated hat-shaped ascospores of Ascoidea corymbosa

We previously implicated 3-hydroxy oxylipins and ascospore structure in ascospore release from enclosed asci. Using confocal laser scanning microscopy on cells stained with fluorescein-coupled, 3-hydroxy oxylipin-specific antibodies, we found that oxylipins are specifically associated with ascospores and not the vegetative cells or ascus wall of Ascoidea corymbosa. Using gas chromatography--mass spectrometry the oxylipin 3-hydroxy 17:0 could be identified. Here, we visualize for the first time the forced release of oxylipin-coated, hat-shaped ascospores from terminally torn asci, probably through turgor pressure. We suggest that oxylipin-coated, razor-sharp, hat-shaped ascospore brims may play a role in rupturing the ascus to affect release.     

Metschnikowia kunwiensis comb. nov., the teleomorph of Candida kunwiensis

The teleomorph of Candida kunwiensis Hong, Bae, Herzberg, Titze, Lachance, Metschnikowia kunwiensis, is described. Repeated attempts to obtain ascospore formation succeeded using modified V8 sporulation media and extended incubation times. The asci are ovoid, with only a small protrusion caused by the spore(s). The species is diplontic, possibly homothallic, with one or two ascospores per ascus. Aside from having atypical ovoid asci, the acicular shape of the spores is characteristic of the genus Metschnikowia. The type strain is CBS 9676(T).     

A constitutive, heat shock-activated neutral trehalase occurs in Schizosaccharomyces pombe in addition to the sporulation-specific acid trehalase

Trehalase was studied in Schizosaccharomyces pombe cells growing vegetatively on minimal medium and in sporulating cultures. Acid trehalase activity, measured at pH 4.2, was absent in vegetative cells and occurred only in asci, indicating that this activity represented the sporulation-specific trehalase reported previously. In contrast, neutral trehalase, measured at pH 6.0, was constitutively present in vegetative cells during the exponential and stationary growth phase as well as in asci. In vegetative cells, neutral trehalase did not sediment with cell walls, suggesting a cytoplasmic localization. Its activity increased ten-fold when growing cells were subjected to heat treatment of 2 h. Neutral trehalase from heat-treated cells had a pH optimum of 6.0 and was almost completely inhibited by 3 mM ZnCl2. Acid trehalase activity could be measured in intact asci, indicating that it is localized in the ascus cell walls, while neutral trehalase was not detectable in intact asci and appeared to be present primarily in the walls of ascospores and in the ascus epiplasm.     

Formation of spore ornamentation in two Sphaerophorus species

Two different ways of achieving a spore ornamentation have been demonstrated in Sphaereophorus , belonging to the Caliciales. In S. globosus the ornamentation is formed within the ascus by an external secondary spore wall in an ontogenetic process with several unique features. In S. murrayi the ornamentation is formed at a late stage, when the spores have been released from the asci. Carbonaceous material formed among the asci and paraphyses is added to the surface of the primary wall, and a very irregular ornamentation is formed. The name Sphaerophorus murrayi Ohlsson is validated.     

印度块菌子囊果内部解剖结构的扫描电镜观察

利用扫描电镜对成熟印度块菌子囊果的内部结构进行了观察,以系统揭示其子囊果内部组织特征,为块菌属的分类以及块菌属真菌子囊果的生理研究奠定基础。观察结果进一步证明,成熟印度块菌子囊果横切面上的白色迷宫状脉络是由不育的侧丝构成,而暗脉则是被侧丝缠绕并包裹着的可育的菌丝组织,即产孢组织,这些白色脉络和暗脉就构成了印度块菌子囊果横切面上迷宫状的纹脉;产孢组织中,可观察到正在发育的大大小小的子囊被缠绕在一起的大量产囊丝与侧丝包裹着,形成密密麻麻微小的类似蜂巢状结构;子囊孢子游离于子囊中,成熟子囊孢子表面有刺状纹饰,刺的顶端有小弯钩。单个子囊内含的子囊孢子大小与其内含的子囊孢子数目有关,子囊内所含的子囊孢子越多,子囊孢子就越小。     

Genetic variation in the orientation of nuclear spindles during the development of asci in Neurospora

The effect of a single mutant gene, peak-2, in Neurospora crassa is to alter the ascus from one in which the eight spores are linearly arranged to one in which the eight spores are arranged non-linearly. The same gene transferred to N. tetrasperma determines an ascus similar to that of the mutant N. crassa but in striking contrast to the normal ascus for N. tetrasperma, which includes four heterokaryotic spores, linearly arranged. A comparative cytological study has been made on asci from both species, including material from zygotes that are homozygous normal, heterozygous for peak-2, and homozygous for peak-2. The observed morphogenetic differences are closely related to differences in nuclear spindle orientation, which in turn are correlated with the geometry of the cell. The observations are shown to bear on the basis of pseudohomothallism in N. tetrasperma. Speculations on the importance of the cell wall in relation to spindle orientation are presented. Differences of expression of peak-2 in the different species are discussed.