Proteins from conidial rodlet preparations of Neurospora crassa were solubilized in trifluoroacetic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized rodlets revealed a predominant protein of approximately 7 kDa. This protein was absent from preparations of N. crassa cultures carrying the eas mutation. The protein was purified by reverse-phase high-performance liquid chromatography and the N-terminal amino acid sequence of the purified protein was found to be identical to an internal portion of the deduced amino acid sequence of eas. Comparison of the sequences indicates a 29-amino-acid leader which is cleaved to generate the mature protein.
A spontaneous suppressor mutant, hah, which suppressed the colonial growth of adenylyl cyclase mutants (cr-1) was isolated. The morphology of cr-1 hah was filamentous, but slightly different from that of wild type on solid medium. The hah strain formed many high aerial hyphae, but did not form any conidia. The expression levels of an adenylyl cyclase gene, nac, in both hah and cr-1 hah were much higher than those in wild type or in cr-1. The level of cAMP in cr-1 was very low but returned to close to the wild-type level in the cr-1 hah suppressed strain, whereas that in the strain carrying hah alone was similar to or a little higher than that in wild type. The hah gene was located 13.3 map units from in1 on the right arm of the linkage group V. The hah mutation was recessive and allele specificity of hah for the suppression of cr-1 mutations was weak.
Detection and localization of myosin immunoanalogue protein in the yeast Candida albicans were achieved by immunoblotting, indirect immunofluorescence assay, and immunoelectron microscopy. A polypeptide with an M(r) about 110,000, from cytosolic extract and insoluble fraction in the corresponding membrane pellet, was reacted with polyclonal and monoclonal antibodies raised against vertebrate muscle myosin. This protein was located by immunofluorescence and immunoelectron microscopy in the cell cortex along the plasmalemma, in the cytoplasm, and in the septum corresponding to bud scar region situated between the yeast-mother cell and the bud.
We used nucleotide sequences of the small subunit ribosomal genes (SSU rDNA) to examine evolutionary relationships of apothecial ascomycetes (division Ascomycota; class Discomycetes sensu), commonly known as the cup fungi. The apothecial ascomycetes include both lichen-forming and free-living fungi. We sequenced the SSU rDNA from representatives of 10 fungal genera from four orders: Pezizales (Ascobolus lineolatus, Morchella elata agg., Peziza badia); Leotiales (Leotia lubrica, Sclerotinia sclerotiorum); Caliciales (Calicium tricolor, Mycocalicium albonigrum, Sphaerophorus globosus); and Lecanorales (Lecanora dispersa, Porpidia crustulata). Of these, C. tricolor, S. globosus, L. dispersa, and P. crustulata are lichen-forming fungi. Based on parsimony analyses of approximately 1750 aligned nucleotides of their SSU rDNA, we determined a most parsimonious tree (MPT). This hypothesis suggests that the apothecial ascomycetes are a paraphyletic assemblage, basal to other groups of filamentous ascomycetes including representatives of the perithecial fungi and cleistothecial fungi. The most parsimonious tree produced using this dataset supported the monophyly of the orders Pezizales, Leotiales, and Lecanorales. However, there was no support for monophyly of the representative Caliciales; S. globosus had affinities with members of the Lecanorales. This phylogenetic hypothesis recognizes Pezizales as basal and supports Nannfeldt's hypothesis (1932) of a primitive apothecial ascomata with subsequent evolution of perithecial and cleistothecial forms. This MPT provides a foundation for understanding evolution of the ascomycetous fungi.
Staben, C. 1995. Resistance to azole drugs in Neurospora crassa. Experimental Mycology 19: 163-165. Neurospora crassa was susceptible to azole drugs: ketoconazole (MIC 1 μg/ml), fluconazole (MIC 5 μg/ml), and SCH39304 (MIC 5 μg/ml). Mutants of N. crassa resistant to ketoconazole were selected and genetically characterized. The seven characterized resistance mutations represented at least four genetic loci. Some mutants, but not all, were also resistant to fluconazole and to SCH39304.
His-Asp-Glu-Leu (HDEL)-bearing proteins were quantified in different endoplasmic reticulum (ER) subcompartments of Saccharomyces cerevisiae and the plant parasite Uromyces viciae-fabae by immuno-electron microscopy (immuno-EM). In both fungi, the immunogold labeling of these proteins within the ER was three times greater than within the nuclear envelope. In U. viciae-fabae, the ER in germinating uredospores differed from the ER in fungal structures produced within the plant, e.g., haustoria. In haustoria, the cisternal ER differentiated large tubular-vesicular complexes (TVC). TVC contained higher levels of HDEL-bearing proteins than ordinary ER cisternae. ELISA readings also indicated an increased concentration of these proteins in isolated haustoria compared to germinating uredospores. In S. cerevisiae, the ER was differentiated into cortical and internal regions. Immuno-EM revealed that labeling of the binding protein (BiP) was lower in the ER of the cell cortex. Heat shock increased BiP signals, but the relative distribution within the ER did not change. Our results suggest that ER subcompartments can be differentiated by immunogold labeling of proteins with a retention signal. In special cases, such as in the parasitic phase of rust fungi, these proteins accumulate to higher levels in ER subcompartments, probably as a response to plant-induced stress.
Lamboy, J. S., Staples, R. C., and Hoch H. C. 1995. Superoxide dismutase: A differentiation protein expressed in Uromyces germlings during early appressorium development. Experimental Mycology 19, 284-296. Germlings of the bean rust fungus Uromyces appendiculatus detect penetration sites on the surface of the host leaf by thigmosensing topographical features. Within 2-4 min after the apex of a urediospore germ tube encounters the cuticular lip of a stomate, the germling ceases polarized growth and begins to swell over the aperture. The mechanism by which the cells detect topographical signals is not understood; however, previous experiments indicated that the initiation process does not involve de novo gene expression. In order to detect posttranslational modifications, the protein profiles of induced and noninduced germlings were compared at the earliest stages of appressorium formation, and a 21-kDa differentiation protein was identified by a shift in isoelectric point. The N-terminal amino acid sequence exhibited homology with superoxide dismutase (SOD), and antibodies to a synthetic peptide fragment of the respective sequence recognized cooper/zinc isozymes of SOD in electroblots of native gels. Electroelution of the active enzyme bands and separation by SDS-PAGE indicated that the 21-kDa protein is a component of a tetrameric 85-kDa SOD.
A culture of Aspergillus nidulans (FGSC 359) was gradually adapted for growth in media containing up to 2 M NaCl or was exposed to a salt shock with 2 M NaCl. The intracellular glycerol level increased by about 7.9-fold in salt-adapted and 2.4-fold in salt-shocked cultures when compared to the unadapted culture. The biosynthetic pathway involved in the accumulation of glycerol was investigated under long-term salt adaptation and short-term salt shock. Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) was induced 1.4-fold in salt-shocked but not in salt-adapted cultures. An alternate enzymatic pathway involving glycerol dehydrogenase (NADP(+)-dependent) utilizing dihydroxyacetone (DHA) and/or DL-glyceraldehyde (DL-GAD) was induced by NaCl. DHA-dependent glycerol dehydrogenase activity was induced about 6.3-fold in salt-adapted and 1.35-fold in salt-shocked cultures, while DL-GAD-dependent activity was induced about 6.1-fold in salt-adapted and 1.2-fold in salt-shocked cultures. However, the level of glycerol dehydrogenase activity with DL-GAD as substrate was 7% of the DHA-dependent activity. We conclude that a salt-inducible NADP(+)-dependent glycerol dehydrogenase activity electrophoretically indistinguishable from previously described glycerol dehydrogenase I results in glycerol accumulation in salt-stressed A. nidulans.
Gunde-Cimerman, N., and Cimerman, A. 1995. Pleurotus fruiting bodies contain the inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase-lovastatin. Experimental Mycology 19, 1-6. In the fruiting bodies of the fungus Pleurotus ostreatus, also called the oyster mushroom, we found a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase—lovastatin. The appearance of the inhibitor during the development of fruiting bodies was followed and lovastatin determined in the vegetative mycelium, in the primordia, as well as in different parts of sporocarps of different sizes. Less lovastatin was found in stipes as compared to pili or in mature stages in the lamellae and basidiospores.
The large subunit (28S) of the ribosomal DNA repeat of Morchella, Verpa, and Disciotis and a closely related genus (Gyromitra) was enzymatically amplified via the polymerase chain reaction. Restriction fragment length polymorphisms were found among the lines investigated and used to infer phylogenetic relationships. More variability was observed toward the 5' end than toward the 3' end of the 28S rRNA gene. The RFLP data were used to assemble a phylogenetic tree for the taxonomic group. Based on the RFLP data three black Morchella species isolates differed by approximately 0.5, 1.0, and 1.5%, respectively, from all other isolates in the Morchellaceae examined in this study. Gyromitra gigas, used as an outgroup, had approximately 6.2% difference from all members of the Morchellaceae. In some cases more genetic variation was observed intraspecifically than between putative species. Additionally, the hypothesis that Morchella is composed of only a few (possibly three) polymorphic species was supported by our findings.
Fernández, Murray, P., and Passeron, S. 1995. The soluble aminopeptidase system from Saccobolus platensis. Characterization of a new glutamate aminopeptidase. Experimental Mycology 19, 214-222. The aminopeptidase pattern from the fungus Saccobolus platensis was investigated by ion-exchange chromatography fractionation of the soluble proteins. The chromogenic p-nitroanilide derivatives of nine different amino acids were used as substrates. Apart from the previously characterized major alanine aminopeptidase (P. Fernádez Murray, A. Samela, and S. Passeron, 1992. Exp. Mycol. 16, 279-290), two new minor aminopeptidase activities were found: one mainly a proline aminopeptidase and a second highly specific for the hydrolysis of the chromogenic derivative of glutamate. This last activity was subjected to ammonium sulfate fractionation and successive phenyl-Sepharose, DEAE-Sephacel, and Sephacryl S-200 HR column chromatography. A highly purified enzyme fraction was obtained. This new glutamate aminopeptidase had a molecular weight of 22 kDa and an optimum pH range of 7.2-8.0 and was inhibited by o-phenanthroline and bestatin.
Dithiothreitol (DTT) extraction of N-acetylglucosaminidase and trehalase from intact Candida albicans ATCC 10261 cells was monitored as an index of cell envelope porosity during N-acetylglucosamine-induced morphogenesis. Trehalase, which is secreted into the cell envelope during starvation and bud-formation, displayed similar extraction kinetics in starved, germ tube-forming, and bud-forming cells, indicating that the mother cell wall remains largely unchanged during morphogenic outgrowth and that the porosity of bud and mother cell walls is similar. N-acetylglucosaminidase, which is secreted specifically during morphogenesis, was released eightfold more rapidly from germ tube-forming than bud-forming cells, reflecting major differences in porosity between bud and germ tube. In addition, by assaying DTT extracts and extracted cell residues, it was found that the total extracellular N-acetylglucosaminidase activity increased 2- to 2.5-fold during DTT treatment. Thus, DTT unmasks a cryptic form of N-acetylglucosaminidase. The cryptic activity was associated with the cell wall fraction.
Amir, R., Steudle, E., Levanon, D., Hadar, Y., and Chet, I. 1995. Turgor changes in Morchella esculenta during translocation and sclerotial formation. Experimental Mycology 19, 129-136. Turgor pressure was measured during six stages of growth and pseudosclerotial formation in Morchella esculenta indirectly (by thermocouple psychrometer) and directly (by cell pressure probe). The fungus was grown on a split plate, enabling separation between mycelium growing on defined medium (water potential -0.5 MPa) and sclerotia which formed on glucose noble agar (water potential -2.1 MPa). Under these conditions, nutrients were translocated from the mycelium to the developing sclerotia. Direct turgor potential measurements showed that the gradient between the mycelium and the sclerotia increases during sclerotial development (reaching a maximum of 0.53 MPa), thereby suggesting that translocation is a turgor-driven mass flow. During sclerotial development, the turgor potential in the peripheral tips of the sclerotial hyphae must be high enough to bring about the growth of the numerous hyphae, which comprise the sclerotium, and simultaneously low enough in the primary hyphae, which carry the stream of nutrients, to attract translocation from the mycelium. Since sclerotial hyphae are too small for direct measurement by cell pressure probe, a psychrometer was used, revealing high turgor in the sclerotial tissue (1.2 MPa) during selerotial development. Direct measurement in the primary hyphae at this time gave a value of 0.7 MPa. Taken together, these measurements indicate the presence of a turgor gradient inside the sclerotial tissue, from the primary hyphae to the peripheral cells. The present study is the first to make use of a cell pressure probe to measure turgor gradients in a fungus during translocation followed by sclerotial morphogenesis.
Balaji, B., Poulin, M. J., Vierheilig, H., and Piché Y. 1995. Responses of an arbuscular mycorrhizal fungus, Gigaspora margarita, to exudates and volatiles from the Ri T-DNA-transformed roots of nonmycorrhizal and mycorrhizal mutants of Pisum sativum L Sparkle. Experimental Mycology 19, 275-283. Transformed root cultures were established from the nonmycorrhizal (Myc-) and mycorrhizal (Myc+) Pisum sativum L Sparkle mutants to study the biochemical factors necessary for initiating and maintaining the arbuscular mycorrhizal (AM) symbiosis. Root exudates produced by both the Myc- and the Myc+ mutants inhibited the hyphal growth of Gigaspora margarita, whereas root volatiles from these mutants stimulated the hyphal growth significantly in the precolonization stage. Carbon dioxide is the principal volatile compound necessary for the elongation of hyphae from both the Myc- and the Myc+ transformed roots. The addition of quercetin, a flavonol compound, to the medium with a Myc- mutant enriched with an optimal CO2 improved hyphal elongation and spreading as previously reported but did not cause Myc- roots to become mycorrhizal. These results suggest that the root factors may stimulate or inhibit AM fungal growth and that they do not determine the mycorrhizal nature of P. sativum Sparkle mutants.
Schaap, P. J., de Groot, P. W. J., Müller, Y., van Griensven, L. J. L. D., and Visser, J. 1995. Molecular cloning and sequence of the cytoplasmic ribosomal protein S15a gene from Agaricus bisporus. Experimental Mycology 19, 160-162. We have isolated an Agaricus bisporus cDNA which encodes an open reading frame of 130 amino acids. A comparison with the Genbank database shows that the deduced amino acid sequence of this open reading frame is highly homologous to the small subunit ribosomal proteins S15a of Brassica napus and Drosophila melanogaster and to the small subunit ribosomal proteins S24 of Strongylocentrotus purpuratus and Saccharomyces cerevisiae .
Amir, R., Levanon, D., Hadar, Y., and Chet, I. 1995. Factors affecting translocation and sclerotial formation in Morchella esculenta. Experimental Mycology 19, 61-70. Morchella esculenta was grown on square split plates, forming sclerotia on one side and mycelium on the other. After the fungus ceased to colonize and before sclerotial initials appeared, [14C]3-O-methyl glucose was added to the edge of the plate on the mycelial side. The effect of various activities in the mycelium (source) and sclerotia (sink) on sclerotial formation and translocation were examined using inhibitors and water potential changes of the media. Sodium azide or cycloheximide applied separately to both sides inhibited both sclerotial formation and translocation, showing that processes in the source and sink depend on metabolic activities as well as protein synthesis. The use of nikkomycin inhibited sclerotial formation, without affecting translocation to the sclerotia. Since the hyphal tips swelled and burst, the translocated compounds were lost to the media. In a strain defective in sclerotial formation, used as a control, no translocation took place, showing that there is a connection between sclerotial formation and translocation. Reversal of the water potential gradient between the two media (lower on the mycelial side), reduced the formation of sclerotia and translocation to them. Translocation to Morchella sclerotia takes place via turgor driven mass flow, but is nevertheless affected by activities in both the source and the sink.
Kempken, F., Schreiner, C., Schörgendorfer, K., and Kück, U. 1995. A unique repeated DNA sequence in the cyclosporin-producing strain of Tolypocladium inflatum (ATCC 34921). Experimental Mycology 19, 305-313. Recombinant λ clones containing repeated DNA sequences were isolated from the cyclosporin A-producing fungus Tolypocladium inflatum (ATCC 34921) by differential hybridization with total fungal DNA and rDNA probes. From this survey 1% of the λ clones appeared to contain repeated sequences. Subsequent analysis led to the identification of a dispersed repetitive DNA element. It was named CPA element (cyclosporin production associated) and appears to be strain specific, since it is absent from other related strains or fungi. Hybridization with chromosomal restriction fragments indicates an equal distribution of the CPA element in the genome. The copy number was estimated to be between 20 and 30 per haploid genome. Sequence analysis of a 0.9-kb XhoI fragment from three copies of the CPA element revealed strong conservation of this sequence among all copies. A 200-bp region exhibits similarities to a repeated sequence from Zea diploperennis. The use of this DNA sequence as a molecular marker for identification of this cyclosporin-producing strain ATCC 34921 is discussed as is the relevance of repeated DNA sequences for rearrangements of fungal karyotypes.
Navarro, E., Sandmann, G., and Torres-Martı́nez, S. 1995. Mutants of the carotenoid biosynthetic pathway of Mucor circinelloides. Experimental Mycology 19, 186-190. We have isolated and characterized a number of mutants affected in the biosynthesis of β-carotene in the fungus Mucor circinelloides. Mutants were obtained after mutagenesis with N -methyl-N-nitro-N-nitrosoguanidine or ultraviolet radiation. Carotene analysis and determination of in vitro activity for synthesis of prenyl pyrophosphates confirm that we have obtained mutants for all enzymatic steps from farnesyl pyrophosphate to β-carotene and regulatory mutants affecting total production of carotenes and light regulation.
Davis, R. H., and Ristow, J. L. 1995. Osmotic effects on the polyamine pathway of Neurospora crassa. Experimental Mycology 19, 314-319. In bacteria, mammals, and certain plants, the induction of the polyamine synthetic enzyme, ornithine decarboxylase (ODC), and the accumulation of its product, putrescine, follows osmotic manipulations of cells. In at least some of these cases, this response is indispensable for survival. We wished to determine whether the polyamine pathway of Neurospora crassa was regulated in response to hyper- or hypoosmotic conditions. Unlike ODC of most other classes of organisms, the N. crassa enzyme and the accumulation of putrescine appears to be relatively indifferent to these conditions, either during sudden transitions or in steady-state. We conclude that other mechanisms of osmotic adjustment or tolerance have evolved in N. crassa and perhaps other fungi that obviate the need for putrescine accumulation.
