Mutational analysis of the [Het-s] prion analog of Podospora anserina. A short N-terminal peptide allows prion propagation.
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The het-s locus is one of nine known het (heterokaryon incompatibility) loci of the fungus Podospora anserina. This locus exists as two wild-type alleles, het-s and het-S, which encode 289 amino acid proteins differing at 13 amino acid positions. The het-s and het-S alleles are incompatible as their coexpression in the same cytoplasm causes a characteristic cell death reaction. We have proposed that the HET-s protein is a prion analog. Strains of the het-s genotype exist in two phenotypic states, the neutral [Het-s*] and the active [Het-s] phenotype. The [Het-s] phenotype is infectious and is transmitted to [Het-s*] strains through cytoplasmic contact. het-s and het-S were associated in a single haploid nucleus to generate a self-incompatible strain that displays a restricted and abnormal growth. In the present article we report the molecular characterization of a collection of mutants that restore the ability of this self-incompatible strain to grow. We also describe the functional analysis of a series of deletion constructs and site-directed mutants. Together, these analyses define positions critical for reactivity and allele specificity. We show that a 112-amino-acid-long N-terminal peptide of HET-s retains [Het-s] activity. Moreover, expression of a mutant het-s allele truncated at position 26 is sufficient to allow propagation of the [Het-s] prion analog. (+info)
The Mla (powdery mildew) resistance cluster is associated with three NBS-LRR gene families and suppressed recombination within a 240-kb DNA interval on chromosome 5S (1HS) of barley.
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Powdery mildew of barley, caused by Erysiphe graminis f. sp. hordei, is a model system for investigating the mechanism of gene-for-gene interaction between large-genome cereals and obligate-fungal pathogens. A large number of loci that confer resistance to this disease are located on the short arm of chromosome 5(1H). The Mla resistance-gene cluster is positioned near the telomeric end of this chromosome arm. AFLP-, RAPD-, and RFLP-derived markers were used to saturate the Mla region in a high-resolution recombinant population segregating for the (Mla6 + Mla14) and (Mla13 + Ml-Ru3) resistance specificities. These tightly linked genetic markers were used to identify and develop a physical contig of YAC and BAC clones spanning the Mla cluster. Three distinct NBS-LRR resistance-gene homologue (RGH) families were revealed via computational analysis of low-pass and BAC-end sequence data derived from Mla-spanning clones. Genetic and physical mapping delimited the Mla-associated, NBS-LRR gene families to a 240-kb interval. Recombination within the RGH families was at least 10-fold less frequent than between markers directly adjacent to the Mla cluster. (+info)
Secretion of cryparin, a fungal hydrophobin.
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Cryparin is a cell-surface-associated hydrophobin of the filamentous ascomycete Cryphonectria parasitica. This protein contains a signal peptide that directs it to the vesicle-mediated secretory pathway. We detected a glycosylated form of cryparin in a secretory vesicle fraction, but secreted forms of this protein are not glycosylated. This glycosylation occurred in the proprotein region, which is cleaved during maturation by a Kex2-like serine protease, leaving a mature form of cryparin that could be isolated from both the cell wall and culture medium. Pulse-chase labeling experiments showed that cryparin was secreted through the cell wall, without being bound, into the culture medium. The secreted protein then binds to the cell walls of C. parasitica, where it remains. Binding of cryparin to the cell wall occurred in submerged culture, presumably because of the lectin-like properties unique to this hydrophobin. Thus, the binding of this hydrophobin to the cell wall is different from that of other hydrophobins which are reported to require a hydrophobic-hydrophilic interface for assembly. (+info)
Cryphonectria hypovirus 3, a virus species in the family hypoviridae with a single open reading frame.
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Isolate Grand Haven (GH) 2 is a naturally occurring isolate of the chestnut blight fungus, Cryphonectria parasitica, that is greatly reduced in virulence due to the presence of a double-stranded RNA virus. Unlike many other virus-infected, hypovirulent isolates, GH2 is not substantially reduced in pigmentation, conidiation, or laccase expression compared to its virus-free counterpart. The dsRNA genome of the GH2 virus was cloned, sequenced, and compared to hypovirulence-associated viruses of the family Hypoviridae. GH2 dsRNA is considerably smaller than previously characterized members of the family, 9.8 kb compared to 12.5-12.7 kb for other members. The genome organization of GH2 dsRNA reflected the substantial difference in genome size. Like other members of the family, one strand contained a poly(A)(+) tail at the 3' end and a long sequence with several minicistrons at the 5' end of the same strand. Only a single open reading frame (ORF) of 8622 nucleotides was predicted from deduced translations of the poly(A)(+)-containing strand, however. This contrasts with the two-ORF structures of previously characterized members. Analysis of the deduced ORF of GH2 dsRNA revealed putative proteinase, RNA polymerase, and helicase domains similar to those previously identified in confirmed members of the virus family Hypoviridae. GH2 dsRNA was more distantly related to Cryphonectria hypovirus (CHV) 1-EP713 and CHV2-NB58 than the latter two were to each other but has features in common with each of those viruses. We propose that the GH2 virus be included in this taxon as a member of the genus Hypovirus, representing a strain of a new species, CHV3. (+info)
Phylogenetic relationships among ascomycetes: evidence from an RNA polymerse II subunit.
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In an effort to establish a suitable alternative to the widely used 18S rRNA system for molecular systematics of fungi, we examined the nuclear gene RPB2, encoding the second largest subunit of RNA polymerase II. Because RPB2 is a single-copy gene of large size with a modest rate of evolutionary change, it provides good phylogenetic resolution of Ascomycota. While the RPB2 and 18S rDNA phylogenies were highly congruent, the RPB2 phylogeny did result in much higher bootstrap support for all the deeper branches within the orders and for several branches between orders of the Ascomycota. There are several strongly supported phylogenetic conclusions. The Ascomycota is composed of three major lineages: Archiascomycetes, Saccharomycetales, and Euascomycetes. Within the Euascomycetes, plectomycetes, and pyrenomycetes are monophyletic groups, and the Pleosporales and Dothideales are distinct sister groups within the Loculoascomycetes. We confirm the placement of Neolecta within the Archiascomycetes, suggesting that fruiting body formation and forcible discharge of ascospores were characters gained early in the evolution of the Ascomycota. These findings show that a slowly evolving protein-coding gene such as RPB2 is useful for diagnosing phylogenetic relationships among fungi. (+info)
Pulmonary infection caused by Gymnascella hyalinospora in a patient with acute myelogenous leukemia.
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We report the first case of invasive pulmonary infection caused by the thermotolerant ascomycetous fungus Gymnascella hyalinospora in a 43-year-old female from the rural midwestern United States. The patient was diagnosed with acute myelogenous leukemia and treated with induction chemotherapy. She was discharged in stable condition with an absolute neutrophil count of 100 cells per microliter. Four days after discharge, she presented to the Cancer Clinic with fever and pancytopenia. A solitary pulmonary nodule was found in the right middle lobe which was resected by video-assisted thoracoscopy (VATHS). Histopathological examination revealed septate branching hyphae, suggesting a diagnosis of invasive aspergillosis; however, occasional yeast-like cells were also present. The culture grew a mold that appeared dull white with a slight brownish tint that failed to sporulate on standard media. The mold was found to be positive by the AccuProbe Blastomyces dermatitidis Culture ID Test (Gen-Probe Inc., San Diego, Calif.), but this result appeared to be incompatible with the morphology of the structures in tissue. The patient was removed from consideration for stem cell transplant and was treated for 6 weeks with amphotericin B (AmB), followed by itraconazole (Itr). A VATHS with biopsy performed 6 months later showed no evidence of mold infection. In vitro, the isolate appeared to be susceptible to AmB and resistant to fluconazole and 5-fluorocytosine. Results for Itr could not be obtained for the case isolate due to its failure to grow in polyethylene glycol used to solubilize the drug; however, MICs for a second isolate appeared to be elevated. The case isolate was subsequently identified as G. hyalinospora based on its formation of oblate, smooth-walled ascospores within yellow or yellow-green tufts of aerial hyphae on sporulation media. Repeat testing with the Blastomyces probe demonstrated false-positive results with the case isolate and a reference isolate of G. hyalinospora. This case demonstrates that both histopathologic and cultural features should be considered for the proper interpretation of this molecular test and extends the list of fungi recognized as a cause of human mycosis in immunocompromised patients. (+info)
Microascus cinereus (Anamorph scopulariopsis) brain abscess in a bone marrow transplant recipient.
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We report the first documented case of brain abscess due to the dematiaceous fungus Microascus cinereus, an organism common in soil and stored grain. M. cinereus was isolated from brain abscess material from a bone marrow transplant recipient. The patient responded well to treatment by amphotericin B lipid complex, itraconazole, and a craniotomy but later died from secondary complications caused by graft-versus-host disease. (+info)
Autumnal biomass and potential productivity of salt marsh fungi from 29 degrees to 43 degrees north latitude along the United States Atlantic Coast.
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It has been established that substantial amounts of fungal mass accumulate in standing decaying smooth cordgrass (Spartina alterniflora) marshes in the southeastern United States (e.g., in standing decaying leaf blades with a total fungal organic mass that accounts for about 20% of the decay system organic mass), but it has been hypothesized that in marshes farther north this is not true. We obtained samples of autumnal standing decaying smooth cordgrass from sites in Florida to Maine over a 3-year period. The variation in latitude could not explain any of the variation in the living fungal standing crop (as determined by ergosterol content) or in the instantaneous rates of fungal growth (as determined by acetate incorporation into ergosterol at a standard temperature, 20 degrees C), which led to the conclusion that the potential levels of fungal production per unit of naturally decaying grass are not different in northern and southern marshes. Twenty-one percent of the variation in the size of the living fungal standing crop could be explained by variation in the C/N ratio (the higher the C/N ratio the smaller the fungal crop), but the C/P ratio was not related to the size of the fungal crop. Instantaneous rates of fungal growth were negatively related to the size of the living fungal crop (r = -0.35), but these rates were not correlated with C/nutrient ratios. The same two predominant species of ascomycetes (one Phaeosphaeria species and one Mycosphaerella species) were found ejecting ascospores from standing decaying smooth cordgrass blades at all of the sites examined from Florida to Maine. (+info)