Isolation and characterization of recessive sporeless mutants in the basidiomycete Coprinus cinereus

Summary Twenty-four sporeless mutants were isolated from an Amut Bmut strain (mutant in the incompatibility factors) of the basidiomycete Coprinus cinereus. All the sporeless mutations were recessive to the wild type. These mutants and a previously isolated recessive sporeless strain, N2-7 (Kanda and Ishikawa 1986) were crossed with a wildtype strain. An F1 random spore analysis indicated that sporulation deficiencies in these mutants were caused by single nuclear gene mutations. These mutations were all complementary to each other, thus twenty-five sporulation genes were identified. Five of them were linked to the A incompatibility factor. Cytological observations classified these mutants into the following four types according to the stage of the blockage: (1) meiosis stopped at meta-anaphase I; (2) meiosis was completed, but further basidial development did not occur; (3) basidial development stopped at the sterigma stage; (4) basidial development stopped at the prespore stage.     

Meiosis,spindle pole body cycle,and taxonomy of the heterobasidiomycetePachnocybe ferruginea

Meiosis and the meiotic spindle pole body cycle were studied electron microscopically in basidia of the heterobasidiomycetePachnocybe ferruginea. Spindle pole body splitting in prometaphase I and II, and intermeiotic and postmeiotic duplication were investigated in particular detail. During prophase, the spindle pole body consists of two three-layered discs connected by a middle piece. At late prophase I and again in prometaphase II, the discs contact the nuclear envelope. Then, the nuclear membrane at the contact area is separated from the non-contacted part of the nuclear envelope and finally disappears. Each disc nests into the nuclear opening of the otherwise intact nuclear envelope. The disc remains in the gap and generates a half spindle. At late metaphase I, a co-disc develops eccentrically within the parent disc. The co-disc detaches from the parent disc during interphase I and becomes one of the metaphase II spindle pole bodies. Co-discs are absent during the second division. A cap of endoplasmic reticulum encloses each disc during prophase I through anaphase I. In the second meiotic division, the caps covering the spindle pole bodies of one nucleus of the pair, are developed from the neighbouring nucleus. Spindle pole bodies ofP. ferruginea are similar to those of the rusts, and especially to those ofEocronartium muscicola andHelicobasidium mompa. Part 73 of the series Studies inHeterobasidiomycetes.     

Conditions for fruit body formation in the white rot basidiomycete Phanerochaete chrysosporium

In Phanerochaete chrysosporium fruit body formations is subject to strong catabolite repression by glucose in the presence of physiological levels of nitrogen. Walseth cellulose was found to be the best source of carbon for the induction of fruit body and consequent basidiospore synthesis. Ejected basidiospores collected from cultures grown under these conditions for two weeks are contaminated with neither conidia nor mycelial fragments and are therefore suitable for genetic analysis of recombination. Under conditions of nitrogen limitation, the glucose catabolite repression of fruit body synthesis was relieved. Exogenous adenosine 3,5-monophosphate but not other related nucleotides, also relieved glucose catabolite repression of fruit body formation.     

Meiosis in the aquatic fungusCatenaria allomycis

Summary Catenaria allomycis Couch (Blastocladiales) is an endobiotic fungal parasite primarily of species of the genusAllomyces. The life cycle ofC. allomycis contains both sexual and asexual phases. Synaptonemal complexes have been found in young developing resistant sporangia (RS) suggesting that meiosis occurs within the thick walled RS prior to syngamy. Ultrastructural evidence suggests that meiosis proceeds through pachytene in the developing RS and is arrested in diplotene of prophase I until the sporangia are induced to germinate at which time the meiotic process is completed. Quantitative nuclear counts in developing RS support the ultrastructural observations. Meiotic nuclei are characterized by polar fenestrae in the nuclear envelope and intranuclear plaque-like microtubule organizing centers (MTOC).Portion of a Ph.D. dissertation submitted by the senior author to the Graduate School, University of Georgia.     

Leucine biosynthesis and its regulation in the basidiomycete Rhodosporidium toruloides

Complementation tests and enzyme analyses separated 29 leucine auxotrophs of the Basidiomycete Rhodosporidium toruloides into three groups, each deficient in one of the leucine biosynthetic enzymes. The following differences are suggested between the organization of the leucine pathway in R. toruloides and the Ascomycetes Saccharomyces cerevisiae and Neurospora crassa: (1) isopropylmalate, the product of the first enzymic reaction appears not to be an internal inducer of the later enzymes of the pathway; this is consistent with the apparent lack of mutants homologous to the leu3 class in N. crassa and S. cerevisiae; (2) as in S. cerevisiae, but unlike N. crassa, isopropylmalate synthase is under the control of a general cross pathway control system; (3) unlike S. cerevisiae, but like N. crassa, R. toruloides appears to possess only one gene encoding isopropylmalate synthase.Abbreviations IPM Isopropylmalate - EMS methanesulphonic acid, ethyl ester     

Cytology of teliospore germination and basidiospore formation inUromyces appendiculatus var.appendiculatus

Summary The cytology of teliospore germination and basidiospore formation inUromyces appendiculatus var.appendiculatus was characterized with light and fluoroscence microscopy. Meiosis of the diploid nucleus occurred in the metabasidium. The four haploid daughter nuclei migrated into the basidiospore initials where they underwent a post meiotic mitosis. Each basidiospore was delimited from the meatabasidium by a septum at the apex of the sterigma. Seventy-five percent of mature basidiospores were binucleate, 24.5% uninucleate, and 0.5% trinucleate. Mature, released basidiospores measured 16×9 m, were smooth-surfaced, and reniform to ovate-elliptical in shape.This study represents portion of a dissertation submitted by the senior author to the Faculty of Biology of the University of Constance in March, 1983, in partial fulfillment of the requirements for the degree of Doctor of Natural Sciences (Dr. rer. nat.).     

Molecular diversity of soil basidiomycete communities in northern-central New South Wales, Australia

Basidiomycete communities were profiled using terminal RFLP (TRFLP) and amplified ribosomal DNA restriction analysis (ARDRA) approaches at seven field sites under differing land use in northern-central New South Wales (NSW), Australia. TRFLP data indicated greater basidiomycete species richness at sites with natural vegetation. Sixty-seven basidiomycete ARDRA-types were detected. Various putatively ectomycorrhizal fungi were detected at all sites with native vegetation. Most ectomycorrhizal taxa had affinities to the genus Tomentella, while two Pisolithus taxa and putatively ectomycorrhizal Cantharellales taxa were also detected. Although soils under woodland or grassland communities supported a range of putatively saprotrophic taxa, only members of the Ceratobasidiales were detected in soils under agricultural land use. This study is the first investigation of fungal communities in soils of northern-central NSW, Australia.     

Sequential production of enzymes and basidiospore formation in fruiting bodies of Coprinus macrorhizus

The production of NADP-dependent glutamate dehydrogenase was initiated at the stage of first meiotic prophase in pileus cells but not in stipe cells of dikaryotic and monokaryotic fruiting bodies in Coprinus macrorhizus. The production of chitinase and glucanases assayed with laminarin and lichenan was observed after the completion of meiosis only in pileus cells. The light conditions that were effective for the delay or inhibition of cellular events in the pileus cells were also effective for the delay or inhibition of enzyme production. But all sporeless mutants tested, which were defective at the various stages of basidiospore formation, produced the normal levels of these enzymes. The results indicate that the sequential production of enzymes and cellular events leading to basidiospore formation in pileus cells are independent from each other.Abbreviation GDH_NADP NADP-dependent glutamate dehydrogenase     

Vanillate hydroxylase from the white rot basidiomycete Phanerochaete chrysosporium

A soluble enzyme fraction from Phanerochaete chrysosporium catalyzed the oxidative decarboxylation of vanillic acid to methoxy-p-hydroquinone. The enzyme, partially purified by ammonium sulfate precipitation, required NADPH and molecular oxygen for activity. NADH was not effective. Optimal activity was displayed between pH 7.5–8.5. Neither EDTA, KCN, NaN₃, nor o-phenanthroline (5 mM) were inhibitory. The enzyme was inducible with maximal activity displayed after incubation of previously grown cells with 0.1% vanillate for 30h.Abbreviations MHQ Methoxy-p-hydroquinone - GLC gas liquid chromatography - TMSi trimethylsilane - TLC thin layer chromatography     

Reconstructing the evolution of agarics from nuclear gene sequences and basidiospore ultrastructure

Traditional classifications of agaric fungi involve gross morphology of their fruit bodies and meiospore print-colour. However, the phylogeny of these fungi and the evolution of their morphological and ecological traits are poorly understood. Phylogenetic analyses have already demonstrated that characters used in traditional classifications of basidiomycetes may be heavily affected by homoplasy, and that non-gilled taxa evolved within the agarics several times. By integrating molecular phylogenetic analyses including domains D1–D3 and D7–D8 of nucLSU rDNA and domains A–C of the RPB1 gene with morphological and chemical data from representative species of 88 genera, we were able to resolve higher groups of agarics. We found that the species with thick-walled and pigmented basidiospores constitute a derived group, and hypothesize that this specific combination of characters represents an evolutionary advantage by increasing the tolerance of the basidiospores to dehydration and solar radiation and so opened up new ecological niches, e.g. the colonization of dung substrates by enabling basidiospores to survive gut passages through herbivores. Our results confirm the validity of basidiospore morphology as a phylogenetic marker in the agarics.     

Isoprenylated cyclohexanoids from the basidiomycete Hexagonia speciosa

Nine oxygenated cyclohexanoids, speciosins L-T (1-9) as well as a 5H-furan-2-one metabolite, 5′-O-acetylaporpinone A (10), together with known analogs, speciosins A, B, D, E, F, I and K (11-17), and aporpinone A (18), were isolated from a scale-up cultures of the basidiomycete Hexagonia speciosa. Their structures were elucidated by analysis of spectroscopic data, including 1D and 2D NMR. Speciosin B (12) showed significant cytotoxicity against several tumor cell lines with IC₅₀ values in the range 0.23-3.30 μM.     

Meiosis and fertility of interspecific hybrids betweenPhaseolus vulgaris L. andP. acutifolius A. Gray

Summary Meiosis and fertility of interspecific hybrids obtained from reciprocal crosses between Phaseolus vulgaris and P. acutifolius were examined. Bivalents as well as univalents were found at Metaphase I. The majority of the microsporocytes had four or more univalents and the average was 6.3 univalents per cell. The average number of lagging chromosomes at Anaphase I was 2.3 per cell and the most frequent chromosome distribution at late Anaphase I was 10–12. The lower than expected number of lagging chromosomes as compared with the number of univalents at Metaphase I suggests the possible occurrence of precocious separation of bivalents. The male fertility as measured by pollen stainability was 17%, however, the frequency of pollen germination in selfing was 3.5%. Upon selfing of the interspecific hybrids, no dividing embryos were found even though 7 and 26% of the ovules were fertilized at 12 hours and four days after pollination. In backcrosses to P. vulgaris (male), 6 and 20% of the ovules were fertilized and 0 and 4% of the ovules contained dividing embryos at the same sampling times. When P. acutifolius was the male parent, respective values were 8 and 31% for fertilization and 0 and 13% for ovules with dividing embryos. The frequencies of backcross embryos recovered at 14–26 days were in agreement with the frequencies of dividing embryos at four days. The ability to obtain backcross plantlets demonstrates the feasibility to further utilize interspecific hybrids for the improvement of P. vulgaris Technical paper No. 5311 of the Oregon Agricultural Experiment Station. Research was supported by the Oregon Agricultural Experiment Station, the Science and Education Administration of the U.S. Department of Agriculture under Grant 5901-0410-8-0028-0 from the Competitive Research Grants Office, the Research Council of Oregon State University (NIH Biomedical Research Support Grant RR 07079) and the Processor Research Council of Oregon, A.R. and C T.S. are respectively supported by an African Graduate Fellowship from the African-American Institute and a fellowship from the National Science Council of the Republic of China     

Bending of DNA segments with Saccharomyces cerevisiae autonomously replicating sequence activity, isolated from basidiomycete mitochondrial linear plasmids

Summary Previous studies have indicated that DNA bending is a general structural feature of sequences (ARSs) from cellular DNAs of yeasts and nuclear and mitochondrial genomic DNAs of other eukaryotes that are capable of autonomous replication in Saccharomyces cerevisiae. Here we showed that bending activity is also tightly associated with S. cerevisiae ARS function of segments cloned from mitochondrial linear DNA plasmids of the basidiomycetes Pleurotus ostreatus and Lentinus edodes. Two plasmids, designated pLPO2-like (9.4 kb), and pLPO3 (6.6 kb) were isolated from a strain of P. ostreatus. A 1029 by fragment with high-level ARS activity was cloned from pLPO3 and it contained one ARS consensus sequence (A/T)TTTAT(A/G)TTT(A/T) indispensable for activity and seven dispersed ARS consensus-like (10/11 match) sequences. A discrete bent DNA region was found to lie around 500 by upstream from the ARS consensus sequence (T-rich strand). Removal of the bent DNA region impaired ARS function. DNA bending was also implicated in the ARS function associated with a 1430 by fragment containing three consecutive ARS consensus sequences which had been cloned from the L. edodes plasmid pLLE1 (11.0 kb): the three consecutive ARSs responsible for high-level ARS function occurred in, and immediately adjacent to, a bent DNA region. A clear difference exists between the two plasmid-derived ARS fragments with respect to the distance between the bent DNA region and the ARS consensus sequence(s).     

Basidiospore formation in monokaryotic fruiting bodies of a mutant strain of Coprinus macrorhizus

The process of basidiospore formation in a mutant strain Fis^c of Coprinus macrorhizus, a heterothallic species of Basidiomycete, which forms monokaryotic fruiting bodies was examined. A single nucleus in a young basidium divided mitotically and two daughter nuclei were fused subsequently. The fused nucleus then divided meiotically forming four basidiospores on a basidium. The typical chromosome behaviours in the first meiotic prophase were observed. Synaptonemal complexes were observed in a basidium at the first meiotic prophase. A continuous illumination of fruiting bodies was effective to arrest meiosis in monokaryotic fruiting bodies at the particular stage of meiotic division.     

Mitosis in the basidiomycete fungusTulasnella araneosa

Summary This is a report of a light and electron microscopic study of mitosis in the basidiomycetous fungusTulasnella araneosa. The study employs serial section analyses of nuclei preselected with fluorescence microscopy. It is the first such study of nuclear division in theTulasnellaceae and the first of conjugately dividing nuclei in basidiomycetous hyphal segments lacking clamp connections. Mitosis inT. araneosa is unusual in that the spindle pole body (SPB) develops asymmetrically; the SPB middle piece is large and transversely curved; and the nuclear envelopes of adjacent late anaphase nuclei fuse. Analyses of mitotic characteristics used for phylogenetic purposes indicate that, of the many characters available, only SPB characteristics are presently valuable. Available evidence indicates that the SPB ofT. araneosa is more different from that ofUredinales than it is from representatives of the other four orders ofBasidiomycotina that have been thoroughly studied.     

Gene cloning and transformation in the basidiomycete fungus Coprinus cinereus: Isolation and expression of the isocitrate lyase gene (acu-7)

Summary The cloned isocitrate lyase structural gene of Aspergillus nidulans (acuD) was shown to hybridize under reduced stringency conditions to unique sequences in genomic DNA digests of the basidiomycete fungus Coprinus cinereus. A gene library of C. cinereus was constructed in the lambda replacement vector L47 and screened for sequences hybridizing to the A. nidulans gene. A recombinant phage was isolated which contained the hybridizing sequence on a 5.6-kb BamHI fragment. This fragment was subcloned into pUC13 to give plasmid pHIONA1 and shown to contain a functional C. cinereus isocitrate lyase gene (acu-7) by transformation of an acu-7 mutant. Direct selection for Acu⁺ transformants was not possible because of the toxicity of the acetate selection medium. Acu⁺ transformants were obtained as cotransformants by transforming an acu-7 trp-1 double mutant, having mutations in both the isocitrate lyase and tryptophan synthetase structural genes, with a plasmid containing the trp-1 gene and either pHIONA1 or the original lambda clone. Up to 47.5% of the selected Trp⁺ transformants were cotransformed to Acu⁺. A physical analysis of 40 Acu⁺ transformants showed that the acu-7 gene had integrated at non-homologous and often multiple sites in the genome. Meiotic stability of the integrated gene was demonstrated by genetic crosses.     

Levels of the ubiquitin ligase substrate adaptor MEL-26 are inversely correlated with MEI-1/katanin microtubule-severing activity during both meiosis and mitosis

The MEI-1/MEI-2 microtubule-severing complex, katanin, is required for oocyte meiotic spindle formation and function in C. elegans, but the microtubule-severing activity must be quickly downregulated so that it does not interfere with formation of the first mitotic spindle. Post-meiotic MEI-1 inactivation is accomplished by two parallel protein degradation pathways, one of which requires MEL-26, the substrate-specific adaptor that recruits MEI-1 to a CUL-3 based ubiquitin ligase. Here we address the question of how MEL-26 mediated MEI-1 degradation is triggered only after the completion of MEI-1's meiotic function. We find that MEL-26 is present only at low levels until the completion of meiosis, after which protein levels increase substantially, likely increasing the post-meiotic degradation of MEI-1. During meiosis, MEL-26 levels are kept low by the action of another type of ubiquitin ligase, which contains CUL-2. However, we find that the low levels of meiotic MEL-26 have a subtle function, acting to moderate MEI-1 activity during meiosis. We also show that MEI-1 is the only essential target for MEL-26, and possibly for the E3 ubiquitin ligase CUL-3, but the upstream ubiquitin ligase activating enzyme RFL-1 has additional essential targets.     

Evidence for strong inter- and intracontinental phylogeographic structure in Amanita muscaria, a wind-dispersed ectomycorrhizal basidiomycete

A growing number of molecular studies show that many fungi have phylogeographic structures and that their distinct lineages are usually limited to different continents. As a conservative test of the extent to which wind-dispersed mycorrhizal fungi may exhibit phylogeographic structure, we chose to study Amanita muscaria, a host-generalist, widespread, wind-dispersed fungus. In this paper, we document the existence of several distinct phylogenetic species within A. muscaria, based on multilocus DNA sequence data. According to our findings, A. muscaria has strong intercontinental genetic disjunctions, and, more surprisingly, has strong intracontinental phylogeographic structure, particularly within North America, often corresponding to certain habitats and/or biogeographic provinces. Our results indicate that the view of A. muscaria as a common, widespread, easily identifiable, ecologically plastic fungus with a wide niche does not correctly represent the ecological and biological realities. On the contrary, the strong associations between phylogenetic species and different habitats support the developing picture of ecoregional endemisms and relatively narrow to very narrow niches for some lineages.     

Meiosis,SC-formation,and karyotype structure in diploidPaeonia tenuifolia and tetraploidP. officinalis

The meiosis of the diploidPaeonia tenuifola and the allotetraploidP. officinalis was studied after conventional methanol/acetic acid-fixation and synaptonemal complex (SC) spreading. Meiosis inP. tenuifolia (2n = 10) is normal with five bivalents in metaphase I, and the SCs in pachytene show regular features. InP. officinalis (2n = 4x = 20) univalents, bivalents and multivalents are found in metaphase I. The SCs reveal several abnormalities: a high number of unpaired lateral elements, partner exchanges between three and four lateral elements, loops and lateral element thickenings. These characteristics are compared with the situations found in other polyploid and hybrid species. It is noteworthy that the abnormalities in meiosis ofP. officinalis are not reflected in its somatic karyotype. Its features were analysed after silver staining and fluorescent staining with chromomycin and compared with those ofP. tenuifolia. Synaptonemal Complex Spreading in Plants2; for part1 see Pl. Syst. Evol.154, 129–136 (1986).