Monokaryotic Stage Research Articles

Cytoplasmic Mixing, Not Nuclear Coexistence, Can Explain Somatic Incompatibility in Basidiomycetes.

Nonself recognition leading to somatic incompatibility (SI) is commonly used by mycologists to distinguish fungal individuals. Despite this, the process remains poorly understood in basidiomycetes as all current models of SI are based on genetic and molecular research in ascomycete fungi. Ascomycete fungi are mainly found in a monokaryotic stage, with a single type of haploid nuclei, and only briefly during mating do two genomes coexist in heterokaryotic cells. The sister phylum, Basidiomycota, differs in several relevant aspects. Basidiomycete fungi have an extended heterokaryotic stage, and SI is generally observed between heterokaryons instead of between homokaryons. Additionally, considerable nuclear migration occurs during a basidiomycete mating reaction, introducing a nucleus into a resident homokaryon with cytoplasmic mixing limited to the fused or neighboring cells. To accommodate these differences, we describe a basidiomycete model for nonself recognition using post-translational modification, based on a reader-writer system as found in other organisms. This post-translational modification combined with nuclear migration allows for the coexistence of two genomes in one individual while maintaining nonself recognition during all life stages. Somewhat surprisingly, this model predicts localized cell death during mating, which is consistent with previous observations but differs from the general assumptions of basidiomycete mating. This model will help guide future research into the mechanisms behind basidiomycete nonself recognition.

Morphological and molecular characterization of a microsporidian parasite, Takaokaspora nipponicus n. gen., n. sp. from the invasive rock pool mosquito, Ochlerotatus japonicus japonicus

A new genus and species of Microsporidia, Takaokaspora nipponicus n. gen., n. sp. is described from Ochlerotatus japonicus japonicus (Theobald) and Ochlerotatus hatorii (Yamada) based on light microscope and ultrastructural morphology, developmental features, transmission cycles and comparative sequence analyses of the small subunit ribosomal DNA (SSU rDNA). The microsporidium is both vertically and horizontally transmitted, exhibits dimorphic development alternating between diplokaryotic and monokaryotic stages and produces two morphologically distinct spores, one in larvae and another in adult females. Horizontal transmission of infection to larval mosquitoes occurs via direct oral ingestion of uninucleate spores that are produced in vertically-infected larval hosts. Development in horizontally-infected hosts is diplokaryotic following karyokinesis of uninucleate schizonts and binary fission to produce small (4.3μm×2.0μm) membrane free, ovoid, binucleate spores that are confined to adult female reproductive tissues (ovariole sheath and oviducts). Vertical transmission of the microsporidium from adult females to larval progeny takes place via surface contamination of the egg (transovum). Microsporidian development in vertically-infected larvae is haplophasic with unpaired nuclei throughout, producing rosette-shaped sporogonial plasmodia contained within a thin non-persistent sporophorous vesicle and culminating in the formation of membrane free, uninucleate, conical spores (7.0μm×2.8μm). Development is confined to host fat body tissue which appears as swollen white masses in the thorax and selected segments of the abdomen causing larvae to appear abnormally distorted and results in death during the third and fourth instar stages. The SSU rDNA sequences obtained from the two morphologically identical microsporidia isolated from Oc. j. japonicus and Oc. hatorii were nearly identical and unique when compared with GenBank entries of all other mosquito-parasitic species. Phylogenetic trees constructed by Maximum Parsimony, Maximum Likelihood and bootstrap analyses using the Neighbor Joining search parameter yielded similar typologies. In each case, the novel microsporidium was the sister group to the clade containing Parathelohania species from Anopheles mosquitoes and the monotypic Novothelohania ovalae from Ochlerotatus caspius showing approximately 10–13% sequence divergence to those two genera providing strong support for establishment as a separate genus.

Cellular interactions between biotrophic fungal pathogens and host or nonhost plants

Biotrophic fungi, such as the rust fungi and the powdery mildew fungi, represent a distinct and economically important group of plant pathogens. Studies of nonhost interactions with Uromyces vignae (cowpea rust fungus, monokaryotic stage) or Erysiphe cichoracearum (plantain powdery mildew fungus) suggest that for both types of biotrophs, penetration through the epidermal wall commonly fails in association with wall-associated defense responses, in part elicited by breakdown products of the plant cell wall. Data suggest that extracellular hydrogen peroxide plays a primary role in this penetration failure, acting either as a signaling agent or as a necessary adjunct to some other defense response within the wall. Protoplast involvement, but not necessarily transcription or translation, is necessary for these wall-associated responses, and they can be reduced by reducing the adhesion between the plant cell wall and the plasma membrane. The latter observation suggests that this adhesion is important for at least some types of defensive signaling between the wall and the cell contents. In host or nonhost plants, Erysiphe species increase this adhesion and wall-associated responses occur even in susceptible plants. However, in its host species, U. vignae locally reduces this adhesion around the penetration site and, as an apparent consequence, no wall-associated responses can be detected. Thus, this latter system is ideal for studying postpenetration cellular and molecular changes in resistant or susceptible host cells without the confounding effect of responses to the trauma of cell wall penetration. Such studies indicate that not only is penetration failure in nonhost plants determined while the fungus is growing through the epidermal cell wall, but that it is at this stage of infection in host species that cellular responses are initiated that determine whether the plant cell will become susceptible to infection or exhibit the hypersensitive response.

Ultrastructure of intercellular hyphae and haustoria of the monokaryotic stage of Puccinia lagenophorae

During the aecial infection (monokaryotic stage) of Puccinia lagenophorae, the intercellular hyphae grow densely in the mesophyll and vascular tissues of Senecio vulgaris leaves. An amorphous material is deposited between intercellular hyphae and host cell wall and may act as adhesive material. Intercellular hyphae are characterized by the presence of perforate, partial and complete septa. These hyphae contain one nucleus per compartment and glycogen besides the usual organelles. This stage of infection is characterized by filamentous haustoria. A penetration peg is formed as a continuation of the haustorial mother cell. The haustoria are characterized by a narrow proximal part, which is enclosed by a thick collar and a wide distal part enclosed by a matrix (extrahaustorial matrix), which is an extension of the host cell wall and bound by an invaginated host plasma membrane (extrahaustorial membrane). The haustorium contains a single nucleus, mitochondria, ribosomes, vesicles, endoplasmic reticulum and vacuoles. The most striking feature of this stage is that intercellular hyphae and haustoria of the fungus heavily invade vascular tissue of the host leaf

Involvement of reactive oxygen species in the response of resistant (hypersensitive) or susceptible cowpeas to the cowpea rust fungus.

A previous study had indicated that scavengers of reactive oxygen species (ROS) delayed cell death (the hypersensitive response (HR)) triggered in epidermal cells of intact, resistant, cowpea (Vigna unguiculata (L.) Walp) leaves by the monokaryotic stage of the cowpea rust fungus (Uromyces vignae Barclay race 1). This HR had been monitored by cell autofluorescence, which occurs after protoplast collapse. In the present study, when cytoplasmic disorganization was used to monitor cell death more directly, ROS-scavengers, superoxide dismutase, catalase, horseradish peroxidase, and desferal-Mn(IV) had no effect on HR development. Cytological staining for superoxide or hydrogen peroxide generation also did not reveal the presence of ROS before or during the early stages of the HR, but did, as in the previous study, suggest a role in the autofluorescence and browning of invaded cells that occur following protoplast collapse. Staining of plant mitochondria with nitroblue tetrazolium, possibly attributable to increased dehydrogenase activity but not superoxide generation, occurred transiently around invasion hyphae (monokaryotic stage of the fungus) or haustoria (dikaryotic stage) of the fungus as they entered a cell in the susceptible or resistant cultivar. Around invasion hyphae in epidermal cells in resistant plants, this staining diminished as cytoplasmic streaming stopped, and gradually disappeared as cell death progressed. These data are consistent with other evidence that rust fungi initially negate non-specific defensive responses in both resistant and susceptible cells as part of the establishment of biotrophy. They also suggest that the HR in the cowpea-cowpea rust fungus pathosystem is not triggered by an oxidative burst.

Expression of Two Closely Linked Hydrophobin Genes ofCoprinus cinereusIs Monokaryon-Specific and Down-Regulated by theoid-1Mutation

A protein with characteristic properties of a fungal hydrophobin (CoH1) was isolated from the monokaryotic stage of the basidiomyceteCoprinus cinereus. A cosmid clone containing the corresponding gene (coH1) was identified using a cDNA probe derived by RT–PCR. Hybridization and sequence analysis identified a second gene,coH2, just 4.1 kb downstream ofcoH1 encoding a hydrophobin (CoH2) with 64% sequence identity. BothcoH1 andcoH2 are subject to developmental regulation. They are expressed in vegetative monokaryotic cells but not in the asexual oidia produced on the surface of monokaryons. Transcripts of the genes were barely detected in dikaryotic mycelium and were absent from fruit bodies. Loss of aerial growth due to a mutation known asoid-1was correlated with lack of both hydrophobins.

Callose deposition during the interaction between cowpea (Vigna unguiculata) and the monokaryotic stage of the cowpea rust fungus (Uromyces vignae).

The interaction between the race 1 of the cowpea rust fungus (Uromyces vignae Barclay) and the resistant cowpea (Vigna unguiculata (L.) Walp.) ev. Queen Anne is characterized by the deposition of callose around intracellular fungal structures. Ultrastructural examination of the early stages of infection by basidiospores of the fungus revealed two types of deposits induced by intracellular hyphae: a non-callose collar at the penetration site and a callose encasement around the invasion hypha. The callose encasement was developed from the site where the fungus encountered the inside of the plant cell wall and was separated from the fungus by the extrahyphal membrane and an extension of plant plasma membrane. The incidence of encasements was reduced in plants treated with inhibitors of transcription (actinomycin D), protein synthesis (cycloheximide), protein glycosylation (tunicamycin) and microfilament polymerization (cytochalasin E). Inhibitors of Golgi-associated vesicle transfer (monensin, brefeldin A) and anti-microtubule agents (colchicine, oryzalin) had no effect. When the fungus was killed by heat treatment in either the resistant or the susceptible cultivar, callose was deposited at various locations along the fungus, mostly in the extrahyphal matrix. The data suggest that unlike the extrahaustorial membrane surrounding D-haustoria of this fungus, the extrahyphal membrane is capable of generating callose. Since this process does not normally occur in the susceptible or the resistant cv. even when callose is deposited in the latter by regions of the plasma membrane not associated with the fungus, we conclude that the deposition of callose by the extrahyphal membrane is inhibited by the living fungus.

Plant nuclear migrations as indicators of critical interactions between resistant or susceptible cowpea epidermal cells and invasion hyphae of the cowpea rust fungus

SUMMARYLiving epidermal cells of cowpea (Vigna unguiculata (L.) Walp.) were examined by contrast‐enhanced video microscopy during penetration by invasion hyphae of the monokaryotic stage of the cowpea rust fungus (Uromyces vignae Barclay race 1). In resistant or susceptible host cvs, the plant nucleus migrated to the penetration site before the fungus had fully penetrated the plant wall and, at sites of unsuccessful infection, remained during the formation of a callose‐containing papilla. Nuclear migration was also induced by applying hemicellulase, but not H2O2, to localized sites of wall damage. Hemicellulase‐induced migration was inhibited by calcium chelators and a protein kinase inhibitor, but not catalase. In both resistant and susceptible cvs, the plant nucleus migrated away from successful infection sites at about the time that the fungal penetration peg made contact with the plant plasma membrane, and the epidermal cell showed no further cytological responses to the growth of the fungal intra‐epidermal vesicle. In the susceptible cv., the nucleus migrated back to the fungus when the latter initiated tip growth. Inhibitors of transcription or translation did not affect this migration and only slightly reduced fungal growth. In the resistant cv. in which the invaded cell exhibited a hypersensitive response (HR), the plant nucleus changed its appearance before the cessation of cytoplasmic streaming and usually did not migrate to the fungus, even if the latter initiated tip growth. Nuclear DNA cleavage usually followed the subsequent cessation of cytoplasmic streaming. Treatments that delayed cell death and increased fungal growth also increased the frequency of nuclear migration to the fungus. It is argued that these and other data suggest that U. vignae negates nonspecific, penetration‐induced, defence responses upon entering cells of both susceptible and resistant cultivars. The results also suggest that effects on the plant nucleus are one of the earliest signs of the HR in this system, often preceding the cessation of cytoplasmic streaming and detectable changes in plasma membrane permeability.

Features of the rapid cell death induced in cowpea by the monokaryon of the cowpea rust fungus or the monokaryon-derived cultivar-specific elicitor of necrosis

Rapid cell death induced in the resistant cowpea cultivar, Dixie Cream, by the monokaryotic stage of race 1 of the cowpea rust fungus was delayed by treatments with Superoxide dismutase, mannitol, catalase and the lipoxygenase inhibitor salicyl hydroxamic acid. These treatments also delayed cell death caused by a cultivar-specific elicitor of necrosis present in intercellular washing fluids from monokaryon-infectcd susceptible plants. The data suggest that active oxygen species, hydrogen peroxide and lipid peroxidation may be involved in both elicitor- and fungal-induced cell death, and in both cases, treatments were effective when applied close to the onset of the first cytological signs of necrosis. Pre-inoculation treatment of cv. Dixie Cream with heat, or prenecrosis treatments with the protein synthesis inhibitor blasticidin S or abscisic acid, also delayed fungal-induced cell death, but heat was the only treatment of all those tested that resulted in a slight increase in fungal growth. Cell death was also delayed by damaging or killing the fungus at various stages before the normal onset of necrosis suggesting the necessity for sustained fungal metabolism. All of these data, and the time-course of elicitor-induced necrosis, are consistent with the hypothesis that the elicitor is the primary cause of cell death in this incompatible plant-fungal interaction. However, the data also suggest that cell death may not be the primary cause of the cessation of fungal growth.

Carbohydrates on the surface of urediniospore‐ and basidiospore‐derived infection structures of heteroecious and autoecious rust fungi

summaryNine flucirescein isothiocyanate‐labelled lectins with affinity towards different carbohydrates were used to probe the surface carbohydrates of infection structures in vitro, derived from urediniospores and basidiospores of an autoecious rust species, Uromyces viciae‐fabae (Pers. J Schroet., and of a heteroecious rust species, Uromyces rumicis (Schum.) Wint. Lectin binding was quantified by measuring fluorescence photometrically.All lectins bound in a characteristic pattern to the infection structures of the respective spore types of each rust fungus. Differences were especially obvious between those infection structures normally produced inside leaf tissue, namely the urediniospore‐derived substomatal vesicles with infection hyphae, and the basidiospore‐derived intraepidermal vesicles. The dikaryotic stage and the monokaryotic stage of the heteroecious fungus differed mainly in their affinity for the leotins from Bandeira simplicifolin and Lotus tetragonulobus.A statistical analysis comparing the binding of the lectins to infection structures of Two rust fungi suggested that cell surface composition is determined by nuclear condition. The monokaryotic stages of both rust fungi have a higher degree of similarity than the dikaryotic and monokaryotic stages of the same rust fungus.

Host specificity versus plurivority in barley leaf rusts and their microcyclic relatives

The parasitic relations involving Puccinia hordei , heteroecious Uromyces rusts on Hordeum , and microcyclic Uromyces species on members of the Liliaceae and Amaryllidaceae are reported; the latter are putatively correlated with the heteroecious Uromyces rusts. In P. hordei , the dikaryotic stage is restricted to Hordeum spp. and the monokaryotic stage is compatible with Ornithogalum spp. Dipcadi erythraeum and Leopoldia eburnea . The latter stage is less parasitically specialized than the dikaryotic one. In heteroecious Uromyces rusts, uredinia and telia are formed on H. bulbosum, H. spontaneum and H. marinum but their monokaryotic stage radiates to a broader range of hosts, namely Bellevalia spp., Scilla spp., Muscari parviflorum and L. eburnea . Apparently, in contrast to P. graminis , the barley rusts have biogenetically radiated from the main to the alternate hosts. The microcyclic Uromyces species, presumably correlated with the heteroecious Uromyces rusts, are narrowly adapted to their source hosts in the genera Bellevalia, Scilla, Muscari and Pancratium . Only Urginea undulata and D. erythraeum are congenial with more than one microcyclic species, and L. eburnea is compatible with all tested rust species. It is noteworthy that all hosts with broader receptivity have originated in regions with less than 100 mm annual rainfall. The significance of this phenomenon and of the congeniality of L. eburnea with all listed rust species is discussed.

Infection and subsequent patterns of development of the pycnial-aecial stage of Puccinia poarum on Tussilago farfara and effect of the fungicide, oxycarboxin, on fungal growth

A histological study of infection and patterns of development of the monokaryotic stage of Puccinia poarum on Tussilago farfara leaves has been made in the presence and absence of the carboxylic acid anilide fungicide, oxycarboxin. Penetration of epidermal cells by basidiospores was direct, and such penetrated cells developed a mycelial ‘coil’ or ‘aggregation’ from which inter- and intracellular hyphae emerged. Early in infection, high proportions of penetrated cells showed intracellular hyphae in close proximity to enlarged host nuclei which contained enlarged nucleoli. Later in infection, nuclei in the centre of the pustule began to degenerate and nuclei of penetrated cells at the margin of the colony showed less association with intracellular hyphae. Three main patterns or types of development were observed depending on the timing of dikaryotization. Type 1 had a short period of pycnial formation and sporulation, followed by immediate dikaryotization, giving rapid aecial formation and sporulation; Type 2, a longer period of pycnial production and sporulation then, after dikaryotization, rapid aecial maturity from preformed protoaecia; Type 3, only pycnial production and sporulation, and protoaecial formation with no dikaryotization. Irrespective of developmental type, mean colony growth remained linear. Treatment of infected leaves with 800 μg/g oxycarboxin decreased mean growth, and prevented or delayed maturation of the pustules of P. poarum. However, the effect was variable; some pustules were completely killed while others were apparently little changed. Possible reasons for such effects are discussed.

An ultrastructural comparison of monokaryotic and dikaryotic haustoria formed by the fusiform rust fungus Cronartium quercuum f.sp. fusiforme

Haustoria formed by the monokaryotic stage of Cronartium quercuum f. sp. fusiforme in Pinus taeda differed from those of the dikaryotic phase in Quercus rubra. Monokaryotic (M) haustorial walls were continuous with the walls of relatively undifferentiated haustorial mother cells. The septate M-haustorial neck and expanded M-haustorial body were separated from the invaginated host plasmalemma by a sheath which was continuous with the host cell wall. Collars encasing sheaths were infrequently observed. Dikaryotic (D) haustoria were morphologically similar to M-haustoria; however, they differed in several respects when examined with TEM. The D-haustorial mother cell wall was thickened at the penetration site but a reduction in the number of wall layers occurred between the thickened portion of the mother cell and the D-haustorium. A darkly staining neckband was present in the wall of the nonseptate D-haustorial neck but was lacking in the M-haustorium. An extensive sheath separated the invaginated host plasmalemma from the D-haustorial wall distally from the neckband. However, the sheath was separated from the D-haustorial wall and from the host cell wall by an invaginated host plasmalemma doubled in the haustorial neck region proximally from the neckband.