Dikaryotic Cells Research Articles

Trimorphomyces: a New Genus in the Tremellaceae

Trimorphomyces Bandoni and Oberw. gen. nov. (Tremellaceae) includes a single species, T. papilionaceus Bandoni and Oberw. sp. nov., which parasitizes Arthrinium sphaerospermum Fckl. The sporocarps are mainly or entirely conidial at first; conidia and basidia are mixed in mature basidiocarps. The basidia are cruciately-septate, stalked. Basidiospores germinate by repetition or by budding to form a haploid yeast phase. Blastic conidia are formed successively; terminal pairs develop synchronously and conjugate before release. The conidia are H-shaped and dikaryotic. The dikaryotic conidia germinate by forming paired buds and a dikaryotic yeast phase is initiated. Dikaryotic yeast cells are produced in the same manner as the parental conidia and are released as H-shaped, conjugated pairs. In the presence of Arthrinium sphaerospermum hyphae, dikaryotic conidia or dikaryotic yeast cells germinate by hyphal and haustorial formation.

Bifactorial Heterothallism and Vegetative Diploidy in Clitocybe Tabescens

The sexual incompatibility system and nuclear cycle of the root-rot fungus Clitocybe tabescens were investigated. Mating among monosporous progeny of a single fruiting body was clearly determined by bifactorial heterothallism. Compatible pairings of monosporous isolates were distinguishable from incompatible pairings on the basis of macroscopic colony morphology and presence or absence of clamp connections and dikaryotic cells. Dikaryotic cells isolated from compatible pairings, however, were unstable and always produced uninucleate, presumably diploid cells lacking clamp connections after prolonged culture without transfer. Diploidy in uninucleate cells was confirmed in another experiment. Compatible pairings of auxotrophic mutants derived from monosporous strains yielded uninucleate, prototrophic cells. The simplest explanation for the observed prototrophy is that complementation occurred within diploid nuclei. The diploid cells found in compatible pairings produced cultures similar in every respect to isolations of naturally occurring vegetative material of C. tabescens. The only other hymenomycete known to have a prolonged vegetative diploid phase is Armillaria mellea, a species which is closely related to C. tabescens. The possibility of interfertility between these two species was examined by pairing representative monosporous isolates. Isolates of C. tabescens were intersterile with all representatives of A. mellea tested. Clitocybe tabescens Scop. ex Bres. is a destructive phytopathogenic basidiomycete. This fungus is broadly distributed in temperate regions, but is especially common in the Southeastern United States where it is well-known for causing root rot in several species of trees and shrubs (8, 13). The fruiting bodies and rhizomorphs of C. tabescens are morphologically quite similar to those of Armillaria mellea (Vahl ex Fr.) Kummer (9) and, in fact, both species have been placed in the genus Armillariella by Singer (10). A difference between the two species of potential interest to experimental mycologists is that C. tabescens generally fruits readily in the laboratory (7, 9, 11), whereas A. mellea fruits sporadically at best under artificial conditions. Previous reports (1-6, 12) have shown that mating among haploid strains of A. mellea is determined by bifactorial heterothallism, a two-factor, multiallelic incompatibility system occurring commonly in the Hymenomycetes. Significantly, these studies also demonstrated that compatible matings of A. mellea haploids yield genetically stable, diploid (rather than dikaryotic) mycelia. Since A. mellea is the only hymenomycete presently known to have a persistent vegetative diploid phase in nature, I have examined the incompatibility system and vegetative ploidy of the closely related species C. tabescens in this study. Furthermore, because of the morphological similarity between C. tabescens and A. mellea, I have investigated the possibility of interbreeding between these two species. MATERIALS AND METHODS

Absence of differences in polysomal RNAs from vegetative monokaryotic and dikaryotic cells of the fungus Schizophyllum commune

Total polysomes, including free and membrane-bound polysomes, were isolated from the monokaryotic and dikaryotic mycelial cell types of the basidiomycete Schizophyllum commune grown under submerged conditions. Sucrose gradient centrifugation showed that these isolated polysomes had a size distribution as expected for polysomes functioning in vivo and thus contained intact mRNA. RNA preparations extracted from the polysomes were used to analyze the mRNA sets in the monokaryon and dikaryon. Saturation hybridization of single-copy DNA with a vast excess of polysomal RNA and cell-free translation of polysomal RNA in a wheat germ system followed by two-dimensional gel electrophoresis of the products did not reveal significant differences between the mRNA sets in both mycelial cell types. Moreover, it was found that the sequence complexities and coding properties of polysomal RNA and total (nuclear and cytoplasmic) RNA from S. commune are not detectably different. From these results and those obtained in previous studies it was concluded that the differentiation between monokaryotic and dikaryotic mycelial cell types as controlled by A and B incompatibility genes involves differential modification of polypeptides during or after mRNA translation.

Hyalopycnis blepharistoma: a pycnidial basidiomycete

Morphology and development of Hyalopycnis blepharistoma (Berk.) Seeler are discussed. The presence of infrequent clamp connections, dikaryotic hyphal cells and conidia, and the colour reaction with diazonium blue B all suggest that H. blepharistoma is a basidiomycete. However, failure to observe a basidial stage precludes assignment to known orders of basidiomycetes.

Frequency and diversity of RNA sequences in different cell types of the fungus Schizophyllum commune.

In the basidiomycete Schizophyllum commune an RNA complexity of 13,500 average-sized (1100 nucleotides) RNA sequences was found by means of RNA excess hybridization to complementary DNA. 600 abundant poly(A)-containing RNA sequences were present in about 100 copies per cell, whereas the rare 13,000 poly(A)-containing RNA sequences were present in about 5 copies per cell on average. Homologous and heterologous cDNA hybridizations to poly(A)-rich RNAs revealed some quantitative, but no significant qualitative differences between the monokaryotic and the dikaryotic cell type. Heterologous hybridization and subsequent hybridizations of remaining single-stranded cDNA to poly(A)-rich RNAs from both cell types showed qualitative differences amounting to 2-3% of the rare sequences in the two cell types. However, from the results of similar hybridizations with total RNAs, i.e. including non-polyadenylated RNA, it could be calculated that the RNA sequences in monokaryons and dikaryons are at least 99% the same. Identity of RNA sequences in monokaryons and dikaryons was also inferred by comparison of the patterns of polypeptides synthesized by translation of total RNA in cell-free systems.

Direct studies of dikaryotization in Schizophyllum commune. II. Behavior and fate of multikaryotic hyphase.

The growth, duplication and fate of multikaryotic hyphae bearing true clamp connections, as derived from compatible matings of Schizophyllum commune, were studied by phase contrast microscopy. The nuclei (N) of multikaryotic apices maintained a near central position during hyphal growth. True clamp connection formation occurred with near synchronous mitosis followed by septal synthesis across the clamp neck and main hyphal axis. Nuclear progeny after mitosis in a hexakaryon included 6 N in the apex, 1 N in the clamp and 5 N in the penultimate cell; the solitary nucleus in the clamp later entered the penultimate cell. Similar events occurred for clamp connection formation and mitosis in the trikaryon, quadrikaryon or pentakaryon, whether in the apex or primary branches. Nuclear content of the multikaryotic apex (2 N through 10 N) had no apparent effect on the rate of individual hyphal growth. Reduction of the nuclear number in a trikaryon occurred by long-term entrapment of a solitary nucleus in the clamp and subsequent outgrowth of the dikaryotic penultimate cell. Occasionally, more than one nucleus became entrapped in the clamp cell. The ephemeral nature of the multikaryon was indicated by the fact that older cultures appeared to be exclusively dikaryotic hyphae at the colony periphery.

Characterization of DNA from the Basidiomycete Schizophyllum commune

We have examined the whole-cell DNA of homokaryons and dikaryons of Schizophyllum commune by physical methods and reassociation kinetics. DNA with a single-strand length of 2800 bases was routinely isolated with urea—phosphate buffers and hydroxyapatite. Melting profiles indicate the bulk of the DNA to consist of a 57 ± 1.22% guanine plus cytosine content (GC) with a small amount of DNA melting at temperatures indicative of 22.1% GC. Analytical ultracentrifugations in CsCl gradients revealed a single band with buoyant density 1.718 g/ml equating to 59% GC. On the other hand, cesium chloride isopycnic centrifugation in the preparative ultracentrifuge disclosed a light satellite (ϱ = 1.687 g/ml, 27% GC) constituting about 2% of the whole-cell DNA. Analyses of the kinetics of reassociation of the whole-cell DNA by three different methods estimate the DNA of an S. commune homokaryotic cell to be 8.6 times that of an Escherichia coli cell (mean, 3.6 × 10 7 nucleotide pairs, NTP; range of experimental values 3.1 − 4.1 × 10 7 NTP). The dikaryotic cell would contain approximately twice this amount of DNA. We detect no other differences between the DNA of homokaryotic and dikaryotic cells as described within the limits of these methods. The reassociation data are best described with a single unique component accounting for 80–90% of the DNA and with a single repetitive fraction of 10 ± 2% of whole-cell DNA. Some ambiguity surrounds the repetition frequency and sequence complexity of the repetitive fraction because it does not reassociate well as a separate fraction (i.e., it does not reassociate with the anticipated rate constant, nor does it approach completion of the reaction) after being isolated by denaturation, reassociation, and isolation on hydroxyapatite.

Genetic versatility of incompatibilty in plants

Abstract Flowering plants have at least 11 different self-incompatibility systems; most, if not all, are oppositional systems in which an incompatible reaction occurs between like alleles in pollen and style. Higher fungi have complementary systems in which compatibility is due to the presence of unlike alleles in the mating cells. This is shown to be a natural consequence of the contrast between haploid pollen entering a diploid style in a plant, and a dikaryotic cell presenting one nucleus to enter a haploid monokaryotic cell of a fungus. From the genetic dominance, co-dominance, and negative complementation (competition), the polymeric state of the S gene product is deduced as: a polymer in pollen and a monomer in the style of gametophytic systems, with the exception of a pollen co-polymer in grasses and a pollen monomer or co-polymer in Tradescantia paludosa; in the multiallelic sporophytic system a polymer is presumed in both pollen and style. Four-gene systems are discussed and evidence is given for...

INTERNUCLEAR GENETIC TRANSFER IN VEGETATIVE DIKARYONS OF SCHIZOPHYLLUM COMMUNE: I. DI-MON MATING ANALYSIS

A series of hemi-compatible dikaryon x monokaryon (di-mon) matings was designed to determine whether there was any genetic interaction between the dikaryotic nuclei. One of the nuclei in each dikaryon was known to carry a recessive gene (mnd) that promoted the development of an abnormal growth form, mound. Dikaryons containing both mnd( +) and mnd nuclei produced mosaic colonies that consisted of three distinct kinds of hyphae: mound, fruiting body, and vegetative (devoid of any multihyphal structure). When dikaryotic hyphae from each of these morphological regions were used in di-mon matings, the genetic and developmental characteristics of the selected nuclear types were examined in the resulting derived dikaryons. The results showed that fruiting-body and vegetative cells contained the expected mnd and mnd(+) nuclei. Dikaryotic mound hyphae, however, contained only mnd nuclei. In a manner as yet unresolved, but clearly dependent on the presence of the mnd allele, the mnd( +) allele of a wild nucleus was altered to, or acquired, the mnd allele. A number of hypotheses were considered to explain the genetic event(s) that generates [mnd + mnd*] dikaryotic cells from [mnd(+) + mnd] cells, but none was found to be entirely satisfactory.

SEXUAL INCOMPATIBILITY FACTORS AND SOMATIC RECOMBINATION IN SCHIZOPHYLLUM COMMUNE

Two sets of diploid cultures of S. commune were observed for sectoring due to haploidization or recombination. Each set consisted of compatible and common-AB diploids otherwise almost isogenic. One of the sets included two compatible diploids with a large proportion of dikaryotic cells. The sectors were isolated and analysed for evidence of aneuploidy and frequent crossing over to determine whether they arose via mitotic or meiotic-like events. It was found that the recombination process in both common-AB and compatible diploids was predominantly mitotic. However, the compatible diploids which developed a high frequency of dikaryotic components gave some evidence of meiotic-like activity. Thus, compatible mating-type factors are necessary for dikaryosis, but not sufficient in themselves to produce it. In compatible mycelia where dikaryosis does occur, meiotic-like recombination may also occur. It is proposed that both lapse into the dikaryotic state, and meiotic-like recombination was induced by different genes under control of the incompatibility factors. Dikaryosis and meiosis are thus seen as tandem phenomena, neither causal of the other but both induced by action of compatible mating-type factors.

Light microscopy of basidia, basidiospores, and nuclei in spores and hyphae of Filobasidiella neoformans (Cryptococcus neoformans).

Three hypha-forming strains of Cryptococcus neoformans were induced to form basidia and basidiospores. Light microscopy showed that basidia formed at the ends of terminal hyphal cells and were able to produce from a few to many basidiospores. The morphology of the sexual structures indicated that these strains belonged to the recently described perfect state of C. neoformans, Filobasidiella neoformans. The average dimensions of the basidiospores were 1.9 mum in width by 2.7 mum in length. Giemsa staining revealed that dikaryotic cells were formed in all three strains. Only one strain had both terminal and subterminal dikaryons, indicating functional clamp connections, whereas the two remaining strains had dikaryons restricted to the terminal cells. Basidiospores of two strains were mononucleate, and yeast cell clones derived from single basidiospores of these two strains were able to complete the sexual life cycle, thus indicating their primary homothallic nature.

Isolation and characterization of compatible diploids of Schizophyllum commune.

Common-AB diploids with several heterozygous biochemical markers were mated with appropriately marked haploid strains of S. commune in an effort to obtain compatible, common-A, and common-B diploid progeny with biochemical markers identical to those of the common-AB parent. The spores from these crosses were germinated on minimal medium. Five compatible diploids, but no common-A or common-B diploids, marked as desired, were isolated by this method. Two possessed some dikaryotic cells and two had many dikaryotic cells. One of the latter was shown to have peculiar behaviour associated with one of its B mating-type factors.

Recovery of high frequency of dikaryotic cells following ultraviolet irradiation of dikaryons of Schizophyllum commune

Recovery of high frequency of dikaryotic cells following ultraviolet irradiation of dikaryons of Schizophyllum commune

Recovery of high frequency of dikaryotic cells following ultraviolet irradiation of dikaryons of Schizophyllum commune

Recovery of high frequency of dikaryotic cells following ultraviolet irradiation of dikaryons of Schizophyllum commune

Cytological evidence for somatic diploidization in dikaryotic cells ofArmillariella mellea

Dikaryotic hyphae isolated from basidiocarps ofArmillariella mellea are unstable in aseptic culture and change into monokaryotic hypae. During monokaryotization the nuclei of a dikaryon fuse and fusion nucleus immediately divides resulting in two uninucleate cells, from which the monokaryotic mycelium originates. Similar fusion of two nuclei takes place in matings of compatible singlespore isolates. It is concluded that the resulting monokaryotic mycelium is diploid.

Genetic Status of Monokaryotic Variants of the Wheat Stem Rust Fungus Isolated from Axenic Culture

Summary: Cytological aspects of monokaryotic variants produced by axenic cultures of Puccinia graminis Pers. f.sp. tritici Erikss. & E. Henn. were studied. Mitotic nuclei of variants at the end of stage I of division (late prophase to metaphase) contained approximately the same number of chromatinic bodies as haploid nuclei in dikaryotic hyphae of the parental wild type. However, an examination of spores produced by pathogenic variants showed that the size of uredospores and teliospores, the number of nuclei per spore cell, and the size of nuclei in teliospores, were all suggestive of the monokaryotic variants being diploid rather than haploid. Diploidy was also consistent with the occasional breakdown of both non-pathogenic and pathogenic monokaryons to form dikaryotic somatic cells; hyphal anastomosis was observed within one such region of dikaryotic cells produced by a non-pathogenic variant. On balance, it is concluded that nuclei in both pathogenic and non-pathogenic monokaryotic variants are diploid, and that mitotic chromosome counts do not distinguish between haploid and diploid nuclei. Diploidy is discussed in relation to naturally occurring monokaryotic rust fungi and to somatic variation in populations of dikaryotic rusts.

Cultural and nutritional studies on variant strains of the wheat stem rust fungus

Monokaryotic variants which arose spontaneously from axenic cultures of Puccinia graminis Pers. f. sp. tritici Erikss. & E. Henn were examined for stability during an extended period of maintenance by serial transfer. Six modes of spontaneous variation were observed: gradual changes in growth rate from generation to generation; ‘regression’, i.e. a rapid progressive decline in growth rate over three successive transfers; adoption of different patterns of senescence; formation of distinct morphological and pigmented sectors ; rare formation of small groups of dikaryotic cells, and on one occasion, formation of a stable dikaryotic variant. Some variants were compared for hyphal morphology and carbohydrate nutrition. All strains tested could utilize d -glucose, d -fructose, d -mannose, sucrose, raffinose, cellobiose, and soluble starch, but growth of some variants was very poor on d -mannitol, d -glucitol, d -arabitol, ribitol and d -ribose. Relative ability to degrade starch showed an inverse correlation with pathogenicity. Although spontaneous variation in cultures was evident on some occasions, there were extended periods of time when little variation was apparent. Variant strains, which are easier to handle in axenic culture than the wild-type, are put forward as convenient organisms for physiological and other studies on rust fungi.

Genotypic variation within a phenotype as a possible basis for somatic hybridization in rust fungi

It is generally considered that the production of new strains of rust by somatic hybridization cannot be adequately explained by hyphal anastomosis and nuclear exchange. Such a mechanism would account for the observed recombination if, as is proposed, the rust fungi in their dikaryophase carry multiple haploid genotypes within any one phenotype. Such hidden variation could arise through whole chromosome exchange between the nuclei of a dikaryotic cell. Possible ways of generating genotypic variation within a phenotype are suggested by the results of recent investigations of Puccinia graminis f. sp. tritici in axenic culture.

Control of arabitol formation in Schizophyllum commune development

Dikaryotic cells of S. commune synthesized polyols throughout the life cycle when grown on glucose, cellobiose, or cellulose. Basidiospores contained arabitol and mannitol which were depleted during germination. The mannitol content of the young germlings rose to normal levels within a day; arabitol accumulation remained depressed for 5 to 7 days and then returned to normal levels characteristic of vegetative cells. Individual homokaryons differed in their production of intracellular polyols, which, unlike germlings, remained constant with cultural age. Homokaryon (str. 699) produced low levels of arabitol but high levels of glycerol while another homokaryon (str. 845) was the reverse. Mixtures of these homokaryons as well as the dikaryon (699×845) produced arabitol and glycerol levels intermediate between the parent homokaryons. High concentrations of glucose did not change the nature of the polyols produced. Arabitol formation could be induced prematurely in germlings or elevated in the dikaryon by growth on acetate or ethanol. Both homokaryons responded to growth on acetate with elevated arabitol production; acetate induction of arabitol formation was repressed in all types of cells if glucose were added simultaneously with acetate. Maltose, cellobiose, and trehalose also stimulated arabitol formation in young germlings, suggesting that glucose repression was the cause of decreased arabitol formation in basidiospore germlings. There was no correlation between the formation of arabitol and the derepression of isocitrate lyase or change in specific activities of alkaline and acid phosphatase in germlings grown on various carbon sources.

Dikaryotization In Clitocybe Truncicola

SUMMARY The process of dikaryotization in Clitocybe truncicola Peck was examined by phase-contrast microscopy. The first dikaryotic cells do not arise directly from the two homokaryotic cells that have fused but from homokaryotic cells some distance from the place of fusion. The intervening homokaryotic cells are severely disrupted and they appear to act as channels for the migrating, compatible nucleus. After a time, this nucleus ceases its migration and forms a dikaryotic association with one of the resident nuclei. From the cell containing this pair of nuclei the first dikaryotic hypha often arises. The first few conjugate divisions in this hypha are often marked by abnormal clamp connections and/or nuclear distributions. Eventually, however, the dikaryon stabilizes and later divisions are normal. During the past 45 years several partial descriptions of the cytology of dikaryotization in a number of different species of tetrapolar basidiomycetes have appeared. Both the methods used and the results reported differ radically. Lehfeldt (1923), Noble (1937) and Giesy and Day (1965) examined fixed and stained material. Buller (1931) and Lange (1966) examined living material, the former by light microscopy, the latter by phase-contrast microscopy. These accounts agree that the fusion of two compatible cells is the first step in dikaryotization; they disagree on the nature of the subsequent events. Lehfeldt (1923) found two distinct processes of dikaryotization among a group of tetrapolar basidiomycetes. In the Corticium serum type the first dikaryotic cells arise directly and presumably immediately