Clamp connection

Abstract: Sexual development in the mushroom Coprinus cinereus is under the control of the A and B mating-type loci, both of which must be different for a compatible, dikaryotic mycelium to form between two parents. The A genes, encoding proteins with homeodomain motifs, regulate conjugate division of the two nuclei from each mating partner and promote the formation of clamp connections. The latter are hyphal configurations required for the maintenance of the nuclear status in the dikaryotic phase of basidiomycetes. The B genes encode pheromones and pheromone receptors. They regulate the cellular fusions that complete clamp connections during growth, as well as the nuclear migration required for dikaryosis. The AmutBmut strain (326) of C. cinereus, in which both A- and B-regulated pathways are constitutively activated by mutations, produces, without mating, dikaryon-like, fertile hyphae with clamp connections. In this study we isolated and characterized clampless1-1 (clp1-1), a mutation that blocks clamp formation, an essential step in A-regulated sexual development, in the AmutBmut background. A genomic DNA fragment that rescues the clp1-1 mutation was identified by transformations. Sequencing of the genomic DNA, together with RACE experiments, identified an ORF interrupted by one intron, encoding a novel protein of 365 amino acids. The clp1-1 mutant allele carries a deletion of four nucleotides, which is predicted to cause elimination of codon 128 and frameshifts thereafter. The clp1 transcript was normally detected only in the presence of the A protein heterodimer formed when homokaryons with compatible A genes were mated. Forced expression of clp1 by promoter replacements induced clamp development without the need for a compatible A gene combination. These results indicate that expression of clp1 is necessary and sufficient for induction of the A-regulated pathway that leads to clamp development.

Abstract: Septins are conserved, cytoskeletal GTPases that contribute to cytokinesis, exocytosis, cell surface organization and vesicle fusion by mechanisms that are poorly understood. Roles of septins in morphogenesis and virulence of a human pathogen and basidiomycetous yeast Cryptococcus neoformans were investigated. In contrast to a well-established paradigm in S. cerevisiae, Cdc3 and Cdc12 septin homologues are dispensable for growth in C. neoformans yeast cells at 24 degrees C but are essential at 37 degrees C. In a bilateral cross between septin mutants, cells fuse but the resulting hyphae exhibit morphological abnormalities, including lack of properly fused specialized clamp cells and failure to produce spores. Interestingly, post-mating hyphae of the septin mutants have a defect in nuclear distribution. Thus, septins are essential for the development of spores, clamp cell fusion and also play a specific role in nuclear dynamics in hyphae. In the post-mating hyphae the septins localize to discrete sites in clamp connections, to the septa and the bases of the initial emerging spores. Strains lacking CDC3 or CDC12 exhibit significantly reduced virulence in a Galleria mellonella model of infection. Thus, C. neoformans septins are vital to morphology of the hyphae and contribute to virulence.

Abstract: Prototrophic strains recovered from crosses between auxotrophic strains of the lignin-degrading basidiomycete Phanerochaete chrysosporium were induced to fruit. The progeny of most of these self-crosses were prototrophic, indicating that the nuclei of the original prototroph were wild-type recombinants rather than complementary heterokaryons and that the binucleate basidiospores of this organism are homokaryotic. Various wild-type strains were shown to have multinucleate cells lacking clamp connections and to possess a variable number of sterigmata per basidium. Colonies arising from single conidia of various wild-type strains were all capable of producing fruit bodies and basidiospores. In addition, single basidiospores from three wild-type strains all produced fruit bodies and basidiospores. Nonfruiting as well as fruiting isolates were obtained from single basidiospores of five other wild-type strains. Basidiospores from these fruiting isolates always yielded colonies that fruited, again indicating that the spores are homokaryotic. Nonfruiting isolates from the same strain did not produce basidiospores when allowed to form a heterokaryon, implying that these isolates do not represent mating types. All this evidence indicates that P. chrysosporium has a primary homothallic mating system. In addition to fruiting and nonfruiting phenotypes, basidiospores from strain OGC101, a derivative of ME-446, gave rise to colonies which did not grow on cellulose (Cel−). The fruiting, nonfruiting, and Cel− phenotypes differed from each other and from the parental wild-type strain in a variety of characteristics, including growth, conidiation, and evolution of 14CO2 from 14C-side chain-labeled lignin, indicating that strain OCG101 is a heterokaryon.

Abstract: The presence of clamp connections on hyphae and the development of fruiting bodies in culture are primary characters which allow identification of the basidiomyceteSchizophyllum communein cases of human infection. The diagnostic problems presented by a nonclamped, nonfruiting isolate from a dense mass in the right upper lobe of the lung in a female with a past history of pulmonary tuberculosis and diabetes are described. Several features of the isolated fungus, including rapid growth rate and white, dense, cottony colonies, tolerance to the fungicide benomyl at a concentration of 10 mg/ml, and susceptibility to cycloheximide at 400 mg/ml, suggested that it might be a basidiomycete. Transmission electron microscopy showed the presence of a dolipore septum with perforate pore cap characteristic of fungi in the classHolobasidiomycetes. However, species identification remained elusive until compatibility tests with known single-basidiospore isolates confirmed the identification of the sterile lung isolate as S. commune. Sequence analysis of the 5 internal transcribed spacer region of ribosomal DNA further supported conspecificity. Fungus ball of the lung, in which a mass of fungal mycelium growsinapreexistingcavity,occursinpatientswithunderlying pulmonary disorders such as tuberculosis, previous infections caused by systemic fungi, recurrent bacterial pneumonia, lung abscess, or sarcoidosis. The infection is not generally diagnosed by sputum culture since the fungal elements are walled off and are not expelled, but hemoptysis is a commonfinding. Most cases involve species of Aspergillus, most commonly A. fumigatus ,o rScedosporium apiospermum (Pseudallescheria boydii)(10).Reportsofinfectioncausedbybasidiomycetesare rare, and to our knowledge, no basidiomycete has been reported from a case of fungus ball. Other than members of the basidiomycetous yeast genera Cryptococcus and Trichosporon, probably the best-known basidiomycetous agent of infection is Schizophyllum commune. Reports involving the lung include a case of allergic bronchopulmonary mycosis (7) in an otherwise healthy female and repeated isolation ofS. communefrom the sputum of a patient with chronic lung disease (3). Other reports ofS. communeinfection include cases of meningitis (2), sinusitis (1, 8, 13), ulcerative lesions of the hard palate (12), andpossibleonychomycosis(9)inbothimmunocompetentand immunosuppressed hosts. In all of the cases reported to date, the fungus has been recognized in tissue and in culture by the presence of clamp connections on the hyphae and by the development of basidiocarps (sexual fruiting bodies) in culture. However,Kameietal.(7)suggestedthatdiagnosisofinfection causedbyS.communemaybedifficultbecauseof(i)thefailure to observe clamp connections on hyphae which otherwise appear similar to those ofAspergillus, (ii) the absence of fruiting body formation in the dark, and (iii) the possibility of identification only when the fungus is a dikaryon capable of producing basidiocarps. We present a case of necrotizing granuloma of the lung in a patient with a history of tuberculosis and diabetes in whom septate, nonclamped hyphae were observed in histopathologic sections. This report provides an example of the diagnostic problems presented by an isolate which is suspected of being a basidiomycete but which fails to form the characteristic macroscopic and microscopic structures which would allow its identification.