Septal Pore Caps Research Articles

Septal-pore-associated structures of Hysterangium clathroides and Hysterangium nephriticum (Hysterangiales, Basidiomycota, Fungi)

The hyphae of Basidiomycota and Ascomycota are divided into compartments by the cross-walls (septa). In mushroom-forming fungi, members of subphylla Pezizomycotina O.E. Erikss. & Winka (Ascomycota) and Agaricomycotina Doweld (Basidiomycota), the septa are associated with specific intracellular structures, which play a role in maintaining the integrity of hyphae in case of injury but also in cell differentiation (Webster & Weber 2007, Moore et al. 2011). In basidiomycetes, these structures may also be involved in certain intracellular processes and the formation of sporocarps (Van Peer et al. 2010, Moore et al. 2011). In this fungal group they are the reticulum-connected and -derived membranous structures called septal pore caps (SPC) or parenthesomes, associated with a dolipore (Moore 1985, 1996, van Driel 2007). The pores may also be occluded by a range of materials of different form. The septal-pore-associated structures may differ considerably between species of different evolutionary lineages. This quality was used as a differentiating factor in the taxonomy at both species and higher-order levels (Lutzoni et al. 2004, McLaughlin et al. 2009).

Extremely thin layer plastification for focused-ion beam scanning electron microscopy: an improved method to study cell surfaces and organelles of cultured cells.

Since the recent boost in the usage of electron microscopy in life-science research, there is a great need for new methods. Recently minimal resin embedding methods have been successfully introduced in the sample preparation for focused-ion beam scanning electron microscopy (FIB-SEM). In these methods several possibilities are given to remove as much resin as possible from the surface of cultured cells or multicellular organisms. Here we introduce an alternative way in the minimal resin embedding method to remove excess of resin from two widely different cell types by the use of Mascotte filter paper. Our goal in correlative light and electron microscopic studies of immunogold-labelled breast cancer SKBR3 cells was to visualise gold-labelled HER2 plasma membrane proteins as well as the intracellular structures of flat and round cells. We found a significant difference (p < 0.001) in the number of gold particles of selected cells per 0.6 μm2 cell surface: on average a flat cell contained 2.46 ± 1.98 gold particles, and a round cell 5.66 ± 2.92 gold particles. Moreover, there was a clear difference in the subcellular organisation of these two cells. The round SKBR3 cell contained many organelles, such as mitochondria, Golgi and endoplasmic reticulum, when compared with flat SKBR3 cells. Our next goal was to visualise crosswall associated organelles, septal pore caps, of Rhizoctonia solani fungal cells by the combined use of a heavy metal staining and our extremely thin layer plastification (ETLP) method. At low magnifications this resulted into easily finding septa which appeared as bright crosswalls in the back-scattered electron mode in the scanning electron microscope. Then, a septum was selected for FIB-SEM. Cross-sectioned views clearly revealed the perforate septal pore cap of R. solani next to other structures, such as mitochondria, endoplasmic reticulum, lipid bodies, dolipore septum, and the pore channel. As the ETLP method was applied on two widely different cell types, the use of the ETLP method will be beneficial to correlative studies of other cell model systems and multicellular organisms.

Fungal evo–devo: organelles and multicellular complexity

Peroxisome-derived Woronin bodies of the Ascomycota phyla, and the endoplasmic reticulum (ER)-derived septal pore cap (SPC) of the Basidiomycota, are both fungal organelles that prevent cytoplasmic bleeding when multicellular hyphal filaments are wounded. Analysis of Woronin body constituent proteins suggests that these organelles evolved in part through gene duplication and co-opting of non-essential genes for new functions, indicating that new organelles can arise through typical evolutionary mechanisms. Interestingly, clades possessing the Woronin body and SPC also produce the largest and most complex multicellular fungal reproductive structures. Certain Woronin body and SPC mutants have defects in growth and development, suggesting functions beyond cellular wound healing. I argue that studying these specialized systems will help to reveal the basis for fungal diversity and provide general principles for co-evolution of organelles and multicellular complexity.

The septal pore cap is an organelle that functions in vegetative growth and mushroom formation of the wood‐rot fungus Schizophyllum commune

Mushroom-forming basidiomycetes colonize large areas in nature. Their hyphae are compartmentalized by perforated septa, which are usually covered by a septal pore cap (SPC). Here, we describe, for the first time, the composition and function of SPCs using the model system Schizophyllum commune. The SPC of S. commune was shown to consist of a proteinaceous matrix covered by a lipid membrane. The matrix was demonstrated to define the ultrastructure of the SPC and to consist of two main proteins, Spc14 and Spc33. Gene spc14 encodes a protein of 86 amino acids, which lacks known domain, signal or localization sequences. Gene spc33 encodes a 239 and a 340 amino acid variant. Both forms contain a predicted signal anchor that targets them to the ER. Immuno-localization showed the presence of Spc33 in the SPC but not in ER. From this and previous reports it is concluded that the SPC is derived from this organelle. Inactivation of spc33 resulted in loss of SPCs and the inability to close septa. The latter may well explain why vegetative growth and mushroom formation were severely reduced in strains in which spc33 was inactivated.

Septal pore complex morphology in the Agaricomycotina ( Basidiomycota) with emphasis on the Cantharellales and Hymenochaetales

The ultrastructure of septa and septum-associated septal pore caps are important taxonomic markers in the Agaricomycotina ( Basidiomycota, Fungi). The septal pore caps covering the typical basidiomycetous dolipore septum are divided into three main phenotypically recognized morphotypes: vesicular-tubular (including the vesicular, sacculate, tubular, ampulliform, and globular morphotypes), imperforate, and perforate. Until recently, the septal pore cap-type reflected the higher-order relationships within the Agaricomycotina. However, the new classification of Fungi resulted in many changes including revision of existing and addition of new orders. Therefore, the septal pore cap ultrastructure of more than 325 species as reported in literature was related to this new classification. In addition, the septal pore cap ultrastructures of Rickenella fibula and Cantharellus formosus were examined by transmission electron microscopy. Both fungi have dolipore septa associated with perforate septal pore caps. These results combined with data from the literature show that the septal pore cap-type within orders of the Agaricomycotina is generally monomorphic, except for the Cantharellales and Hymenochaetales. It appears from the fungal phylogeny combined with the septal pore cap ultrastructure that the vesicular-tubular and the imperforate type both may have arisen from endoplasmic reticulum. Thereafter, the imperforate type eventually gave rise to the perforate septal pore cap-type.

Septal Pore Cap Protein SPC18, Isolated from the Basidiomycetous Fungus Rhizoctonia solani , Also Resides in Pore Plugs

The hyphae of filamentous fungi are compartmentalized by septa that have a central pore. The fungal septa and septum-associated structures play an important role in maintaining cellular and intrahyphal homeostasis. The dolipore septa in the higher Basidiomycota (i.e., Agaricomycotina) are associated with septal pore caps. Although the ultrastructure of the septal pore caps has been studied extensively, neither the biochemical composition nor the function of these organelles is known. Here, we report the identification of the glycoprotein SPC18 that was found in the septal pore cap-enriched fraction of the basidiomycetous fungus Rhizoctonia solani. Based on its N-terminal sequence, the SPC18 gene was isolated. SPC18 encodes a protein of 158 amino acid residues, which contains a hydrophobic signal peptide for targeting to the endoplasmic reticulum and has an N-glycosylation motif. Immunolocalization showed that SPC18 is present in the septal pore caps. Surprisingly, we also observed SPC18 being localized in some plugs. The data reported here strongly support the hypothesis that septal pore caps are derived from endoplasmic reticulum and are involved in dolipore plugging and, thus, contribute to hyphal homeostasis in basidiomycetous fungi.

Enrichment of perforate septal pore caps from the basidiomycetous fungus Rhizoctonia solani by combined use of French press, isopycnic centrifugation, and Triton X-100

Septal pore caps occur in many filamentous basidiomycetes located at both sides of the dolipore septum and are at their base connected to the endoplasmic reticulum. The septal pore cap ultrastructure has been described extensively by the use of electron microscopy, but its composition and function are not yet known. To enable biochemical and functional analyses in the future, we here describe an enrichment method for perforate septal pore caps from Rhizoctonia solani. Our method is based on the combined use of French press and isopycnic centrifugation, using a discontinuous sucrose gradient followed by a treatment with Triton X-100. Enrichment was monitored by the use of scanning electron microscopy and transmission electron microscopy. Using the same isolation method, smaller septal pore caps were isolated from two other basidiomycetes as well. Furthermore, we showed pore-occluding material co-purified with the septal pore caps. This observation supports the hypothesis that septal pore caps play a key role in the plugging process of the septal pores in filamentous basidiomycetes.

Septal pore apparatus and nuclear division of Auriscalpium vulgare

Ultrastructure of the septal pore apparatus and nuclear division of Auriscalpium vulgare (Russulales) was examined with freeze substitution and is presented for inclusion in the AFTOL Structural and Biochemical Database (http://aftol.umn.edu). Previously unreported septal characters for the Russulales (Agaricomycotina) were observed: Septa of the hymenophore had bell-shaped perforated septal pore caps that may extend along the septum and a zone of organelle exclusion surrounded the septal pore apparatus. Metaphase I of meiosis and metaphase of mitosis were similar. Globular spindle pole bodies with electron-opaque inclusions were set within polar fenestrae of the nuclear envelope. The nuclear envelope was mostly intact with occasional gaps. Fragments of endoplasmic reticulum were present near the spindle pole bodies but did not form a polar cap. Structural characters may distinguish one or more clades of the Agaricomycotina and provide additional signal in phylogenetic analyses.

Septal pore apparatus and nuclear division of Auriscalpium vulgare

Ultrastructure of the septal pore apparatus and nuclear division of Auriscalpium vulgare (Russulales) was examined with freeze substitution and is presented for inclusion in the AFTOL Structural and Biochemical Database (http://aftol.umn.edu). Previously unreported septal characters for the Russulales (Agaricomycotina) were observed: Septa of the hymenophore had bell-shaped perforated septal pore caps that may extend along the septum and a zone of organelle exclusion surrounded the septal pore apparatus. Metaphase I of meiosis and metaphase of mitosis were similar. Globular spindle pole bodies with electron-opaque inclusions were set within polar fenestrae of the nuclear envelope. The nuclear envelope was mostly intact with occasional gaps. Fragments of endoplasmic reticulum were present near the spindle pole bodies but did not form a polar cap. Structural characters may distinguish one or more clades of the Agaricomycotina and provide additional signal in phylogenetic analyses.

Laser microdissection of fungal septa as visualised by scanning electron microscopy

Laser microdissection has been proven a successful technique to isolate single cells or groups of cells from animal and plant tissue. Here, we demonstrate that laser microdissection is suitable to isolate subcellular parts of fungal hyphae. Dolipore septa of Rhizoctonia solani containing septal pore caps were cut by laser microdissection from sections of mycelium and collected by laser pressure catapulting. Subsequently, microdissected septa were visualised using a wheat germ agglutinin labelling of cell walls, septa and septal pore caps and scanning electron microscopy. The use of laser microdissection on fungal cells opens new ways to study subcellular fungal structures and the biochemical composition of hyphal cells.

Marchandiobasidium aurantiacum gen. sp. nov., the teleomorph of Marchandiomyces aurantiacus (Basidiomycota, Ceratobasidiales)

The name Marchandiobasidium aurantiacum gen. sp. nov. is introduced for the teleomorph of Marchandiomyces aurantiacus. Dolipore septa and septal pore caps of the closely related Marchandiomyces corallinus are typical of the Ceratobasidiales, and the basidiomatal characters of Marchandiobasidium aurantiacum are reminiscent of those of the monotypic genus Waitea. Morphological, ultrastructural and molecular data suggest that Marchandiobasidium should not be included in Waitea, but should be treated as a distinct genus.

The taxonomic position of Asterodon, Asterostroma and Coltricia inferred from the septal pore cap ultrastructure

The ultrastructure of the septal pore cap (SPC) of Asterodon, Asterostroma and Coltricia were examined to establish the taxonomic position of these genera. Asterostroma has dolipores with perforate SPCs and is classified in the Lachnocladiaceae. In contrast, Asterodon and Coltricia have dolipores with imperforate SPCs and belong to the Hymenochaetaceae. Other selected species of genera belonging to the Hymenochaetaceae like Hydnochaete, Coltriciella, Inonotus, Onnia, and Cyclomyces also contained imperforate SPCs. Coltriciella, Inonotus and Cyclomyces moreover presented a lamella of endoplasmic reticulum above the imperforate SPC after chemical fixation. Such a lamella could rarely be observed in Coltricia only after high-pressure freezing and freeze substitution. Cryofixed fungal cells of Cyclomyces and Coltricia showed differences in the architecture of the matrix of the SPC. Coltricia showed a more layered matrix structure than the SPC of Cyclomyces. In addition, transmission- and scanning electron microscopy revealed an indent in the centre of the imperforate SPC of Cyclomyces, indicating a reduced thickness, and resulting into a tented profile in cross- sections.

Field-emission scanning electron microscopy of the internal cellular organization of fungi.

Internal viewing of the cellular organization of hyphae by scanning electron microscopy is an alternative to observing sectioned fungal material with a transmission electron microscope. To study cytoplasmic organelles in the hyphal cells of fungi by SEM, colonies were chemically fixed with glutaraldehyde and osmium tetroxide and then immersed in dimethyl sulfoxide. Following this procedure, the colonies were frozen and fractured on a liquid nitrogen-precooled metal block. Next, the fractured samples were macerated in diluted osmium tetroxide to remove the cytoplasmic matrix and subsequently dehydrated by freeze substitution in methanol. After critical point drying, mounting, and sputter coating, fractured cells of several basidiomycetes were imaged with field-emission SEM. This procedure produced clear images of elongated and spherical mitochondria, the nucleus, intravacuolar structures, tubular- and plate-like endoplasmic reticulum, and different types of septal pore caps. This method is a powerful approach for studying the intracellular ultrastructure of fungi by SEM.

Automated Electron Tomography of the Septal Pore Cap in Rhizoctonia solani

Dolipore septa and septal pore caps (SPCs) in filamentous basidiomycetes may play an important role in maintaining the integrity of hyphal cells. We have investigated the ultrastructure of the dolipore septum and the SPC in Rhizoctonia solani hyphal cells after high-pressure freezing, freeze substitution, and Spurr embedding. We visualized the SPC with associated cell ultrastructures in three dimensions by automated electron tomography of thick-sectioned cells, followed by 3D tomographic reconstructions. Using these methods we were able to document the passage of mitochondria through the SPC, small tubular membranous structures at the entrance of the septal pore channel, filamentous structures connecting the inner side of the SPC with pore-plugging material, thin filaments anchoring the pore-plugging material with the plasma membrane, small vesicles attached to the plugging material, and tubular endoplasmic reticulum continuous with the base of the SPC. We hypothesize that the SPC, the filamentous structures, the plugging material, and the endoplasmic reticulum act in a coordinated fashion to maintain cellular integrity, intercellular communication, and the transport of solutes and cell organelles in the filamentous fungus R. solani.

Field emission gun-scanning electron microscopy of septal pore caps of selected species in the Rhizoctonia s.l. complex

Techniques of freeze-fracturing and cytoplasmic maceration were combined to reveal the septal pore cap in some members of the Rhizoctonia s.l. complex by the use of field emission gun-scanning electron microscopy. Perforate septal pore caps were observed in Rhizoctonia solani, Aquathanatephorus pendulus, Ceratobasidium cornigerum, and Waitea circinata, showing a successive decrease in cap size. W. circinata revealed an irregular distribution of holes in the cap. Imperforate septal pore caps were observed in Epulorhiza anaticula and Sebacina vermifera. The cap of E. anaticula was connected to tubular endoplasmic reticulum, while the cap of S. vermifera was connected to platelike endoplasmic reticulum. Scanning microscopy is a useful method to complement our knowledge of the septal pore cap as it shows the septal pore cap in situ in relation with other organelles. Septal pore caps present within the Rhizoctonia s.l. complex can be subdivided into: (i) a perforate septal pore cap with either regularly or irregularly distributed holes; (ii) an imperforate septal pore cap connected with tubular endoplasmic reticulum or with platelike endoplasmic reticulum. Our results sustain the current classification of the investigated species in four orders.

Field Emission Gun-Scanning Electron Microscopy of Septal Pore Caps of Selected Species in the Rhizoctonia s.l. Complex

Field Emission Gun-Scanning Electron Microscopy of Septal Pore Caps of Selected Species in the Rhizoctonia s.l. Complex

Scanning electron microscopy of the septal pore cap of the basidiomycete Schizophyllum commune

As part of a comparative study of the structure and function of pore structures in heterobasidiomycetous yeasts, dikaryotic hyphae of Schizophyllum commune were subjected to chemical fixation, freeze fracturing, maceration, and freeze substitution, and were subsequently prepared for scanning electron microscopy. The interior of the hyphal cell was visualized and revealed the perforated septal pore cap or parenthesome, mitochondria, vacuoles, and tubular endoplasmic reticulum. The septal pore cap showed connections with tubular endoplasmic reticulum. This tubular endoplasmic reticulum covered the dolipore septal surface. The results presented here complement and extend the ultrastructural image of the septal pore cap obtained from transmission electron micrographs.Key words: septal pore cap, Schizophyllum commune, freeze fracture, maceration, scanning electron microscopy.

Endoplasmic reticulum: a dynamic patchwork of specialized subregions.

Endoplasmic reticulum: a dynamic patchwork of specialized subregions.

Septal ultrastructure of some fungal endophytes from boreal orchid mycorrhizas

The septal ultrastructure of nine fungal endophytes from mycorrhizas of boreal orchids was examined to corroborate identifications made previously on the basis of cultural and microscopic characteristics. Leptodontidium orchidicola has a simple septum perforated by a single central pore flanked by Woronin bodies. Epulorhiza anaticula, E. repens , and a Sebacina sp. all have dolipore septa with flattened septal pore caps some of which have a minute single central perforation. Ceratobasidium cornigerum, C. obscurum, Moniliopsis anomala, Thanatephorus pennatus and Sistotrema sp. have dolipore septa with dome-shaped, perforated septal pore caps.

Septal and lateral pores in the fungal endophytes of two pasture grasses

Septal spores of two fungal endophytes of Lolium and Festuca are more elaborate, in presporulation hyphae, than the simple pore of many ascomycetous fungi. The pore of the Gliocladium-like endophyte consists of a dome-shaped, membraneous cap formed of vesicle-like structures that encloses the aperture. Woronin bodies in the pore vicinity appear to bear fine fibrillar processes extending radially from their surfaces, often making contact with the pore cap. In septal pores of the Phialophora-like endophyte the hemispherical boundary of the pore lumen bears an irregular electron-opaque deposit, and fibrillar processes from Woronin bodies have not been observed. Lateral pores occur in both endophytes in areas of massed contiguous hyphae. Reduction of two hyphal walls to a narrow strip occurs at the pore site. Development of a pore between the hyphae probably results from degradative activities of a fibrillar band extending between two Woronin bodies, one in each hypha, and penetrating the dividing wall. In lateral pore formation, involvement of Woronin bodies with an associated fibrillar band at the future pore site may support their enzymatic nature. Similarly, in septal pores of the Gliocladium-like endophyte, fibrillar extensions from Woronin bodies to the septal pore cap may indicate enzymatic control of the pluglike contents of the pore. Key words: fungi, septal pores, lateral pores, ultrastructure, endophytes, grasses.