Nuclear-Localized Fluorescent Proteins Enable Visualization of Nuclear Behavior in the Basidiomycete Schizophyllum commune Early Mating Interactions.

Spinning disc confocal microscopical research was conducted on living mating hyphae of the tetrapolar basidiomycete Schizophyllum commune. Haploid strains with either the same or different A and B mating-type genes and expressing differently labelled histone 2B were confronted. In the haploid hyphae histone 2B mCherry and histone 2B EGFP were visualized as red and green nuclei, respectively. In hyphae with the same A but different B genes, the red and green nuclei were observed next to each other. This indicated that nuclear migration between strains, regulated by the B mating type, had taken place. The compatible mating with different A and B genes produced a high number of mixed EFGP/mCherry, yellow nuclei. The mixed nuclei resulted from nearby divisions of nuclei encoding different histones and mating-type genes. During this process, the histones with the different labels were incorporated in the same nuclei, along with the heterodimerized transcription factors encoded by the different A mating-type genes and present around the nuclei. This led to the activation of the A-regulated pathway and indicated that different A genes are important to the cell cycle activation of a compatible mating. Consequently, a yellow nuclear pair stuck together, divided synchronously and proceeded in the migration hyphae towards the colony periphery, where the dikaryotization was promoted by branch formation from the migration hyphae.

Kinesin Motors in the Filamentous Basidiomycetes in Light of the Schizophyllum commune Genome.

Kinesins are essential motor molecules of the microtubule cytoskeleton. All eukaryotic organisms have several genes encoding kinesin proteins, which are necessary for various cell biological functions. During the vegetative growth of filamentous basidiomycetes, the apical cells of long leading hyphae have microtubules extending toward the tip. The reciprocal exchange and migration of nuclei between haploid hyphae at mating is also dependent on cytoskeletal structures, including the microtubules and their motor molecules. In dikaryotic hyphae, resulting from a compatible mating, the nuclear location, synchronous nuclear division, and extensive nuclear separation at telophase are microtubule-dependent processes that involve unidentified molecular motors. The genome of Schizophyllum commune is analyzed as an example of a species belonging to the Basidiomycota subclass, Agaricomycetes. In this subclass, the investigation of cell biology is restricted to a few species. Instead, the whole genome sequences of several species are now available. The analyses of the mating type genes and the genes necessary for fruiting body formation or wood degrading enzymes in several genomes of Agaricomycetes have shown that they are controlled by comparable systems. This supports the idea that the genes regulating the cell biological process in a model fungus, such as the genes encoding kinesin motor molecules, are also functional in other filamentous Agaricomycetes.

The central role of septa in the basidiomycete Schizophyllum commune hyphal morphogenesis.

The purpose of the present research was to observe in the filamentous basidiomycete Schizophyllum commune, the connection between the nuclear division and polymerization of the contractile actin ring with subsequent formation of septa in living hyphae. The filamentous actin was visualized using Lifeact-mCherry and the nuclei with EGFP tagged histone 2B (H2B). Time-lapse fluorescence microscopy confirmed that in monokaryotic and dikaryotic hyphae, the first signs of the contractile actin ring occur at the site of the nuclear division, in one to two minutes after division. At this stage, the telophase nuclei have moved tens of micrometers from the division site. The actin ring is replaced by the septum in six minutes. The apical cells treated with filamentous actin disrupting drug latrunculin A, had swollen tips but the cells were longer than in control samples due to the absence of the actin rings. The nuclear pairing and association with clamp cell development as well as the clamp cell fusion with the subapical cell was disrupted in latrunculin-treated dikaryotic hyphae, indicating that actin filaments are involved in these processes, also regulated by the A and B mating-type genes. This suggests that the actin cytoskeleton may indirectly be a target for mating-type genes.

The making of a mushroom: Mitosis, nuclear migration and the actin network.

Basidiomycetes feature a prolonged dikaryotic life stage. A dispute over open versus closed mitosis could be solved using in vivo fluorescence videomicroscopy of histone 2B::EGFP and Lifeact labeled Schizophyllum commune. It revealed nuclei to condense to approximately one fifth in diameter during mitotic prophase. In addition, the specifics of clamp cell formation typical of many basidiomycetes included an actin network at the future site of nuclear division, which allowed for cessation of nuclear movement and re-localization of one nucleus towards the emerging clamp cell, while the other divided along the hyphal axis. Subsequent fusion of the clamp cell to form the clamp connection restored the close association of the two nuclei in a very fast process after clamp fusion. Septation was preceded by actin patches and vesicles involved in formation of the actin ring.

Novel findings on the role of signal exchange in arbuscular and ectomycorrhizal symbioses.

The availability of genome sequences from both arbuscular and ectomycorrhizal fungi and their hosts has, together with elegant biochemical and molecular biological analyses, provided new information on signal exchange between the partners in mycorrhizal associations. The progress in understanding cellular processes has been more rapid in arbuscular than ectomycorrhizal symbiosis due to its similarities of early processes with Rhizobium-legume symbiosis. In ectomycorrhiza, the role of auxin and ethylene produced by both fungus and host plant is becoming understood at the molecular level, although the actual ligands and receptors leading to ectomycorrhizal symbiosis have not yet been discovered. For both systems, the functions of small effector proteins secreted from the respective fungus and taken up into the plant cell may be pivotal in understanding the attenuation of host defense. We review the subject by comparing cross-talk between fungal and plant partners during formation and establishment of arbuscular and ectomycorrhizal symbioses.

Ras and Rho small G proteins: insights from the Schizophyllum commune genome sequence and comparisons to other fungi.

Unlike in animal cells and yeasts, the Ras and Rho small G proteins and their regulators have not received extensive research attention in the case of the filamentous fungi. In an effort to begin to rectify this deficiency, the genome sequence of the basidiomycete mushroom Schizophyllum commune was searched for all known components of the Ras and Rho signalling pathways. The results of this study should provide an impetus for further detailed investigations into their role in polarized hyphal growth, sexual reproduction and fruiting body development. These processes have long been the targets for genetic and cell biological research in this fungus.

Transcriptome and functional analysis of mating in the basidiomycete Schizophyllum commune.

In this study, we undertook a functional characterization and transcriptome analysis that enabled a comprehensive study of the mating type loci of the mushroom Schizophyllum commune. Induced expression of both the bar2 receptor and the bap2(2) pheromone gene within 6 to 12 h after mates' contact was demonstrated by quantitative real-time PCR. Similar temporal expression patterns were confirmed for the allelic bbr1 receptor and bbp1 pheromone-encoding genes by Northern hybridization. Interestingly, the fusion of clamp connections to the subterminal cell was delayed in mating interactions in which one of the compatible partners expressed the bar2 receptor with a truncated C terminus. This developmental delay allowed the visualization of a green fluorescent protein (Gfp)-labeled truncated receptor at the cell periphery, consistent with a localization in the plasma membrane of unfused pseudoclamps. This finding does not support hypotheses envisioning a receptor localization to the nuclear membrane facilitating recognition between the two different nuclei present in each dikaryotic cell. Rather, Gfp fluorescence observed in such pseudoclamps indicated a role of receptor-pheromone interaction in clamp fusion. Transcriptome changes associated with mating interactions were analyzed in order to identify a role for pheromone-receptor interactions. We detected a total of 89 genes that were transcriptionally regulated in a mating type locus A-dependent manner, employing a cutoff of 5-fold changes in transcript abundance. Upregulation in cell cycle-related genes and downregulation of genes involved in metabolism were seen with this set of experiments. In contrast, mating type locus B-dependent transcriptome changes were observed in 208 genes, with a specific impact on genes related to cell wall and membrane metabolism, stress response, and the redox status of the cell.

Genome sequence of the model mushroom Schizophyllum commune.

Much remains to be learned about the biology of mushroom-forming fungi, which are an important source of food, secondary metabolites and industrial enzymes. The wood-degrading fungus Schizophyllum commune is both a genetically tractable model for studying mushroom development and a likely source of enzymes capable of efficient degradation of lignocellulosic biomass. Comparative analyses of its 38.5-megabase genome, which encodes 13,210 predicted genes, reveal the species's unique wood-degrading machinery. One-third of the 471 genes predicted to encode transcription factors are differentially expressed during sexual development of S. commune. Whereas inactivation of one of these, fst4, prevented mushroom formation, inactivation of another, fst3, resulted in more, albeit smaller, mushrooms than in the wild-type fungus. Antisense transcripts may also have a role in the formation of fruiting bodies. Better insight into the mechanisms underlying mushroom formation should affect commercial production of mushrooms and their industrial use for producing enzymes and pharmaceuticals.

Basidiomycete mating type genes and pheromone signaling.

The genome sequences of the basidiomycete Agaricomycetes species Coprinopsis cinerea, Laccaria bicolor, Schizophyllum commune, Phanerochaete chrysosporium, and Postia placenta, as well as of Cryptococcus neoformans and Ustilago maydis, are now publicly available. Out of these fungi, C. cinerea, S. commune, and U. maydis, together with the budding yeast Saccharomyces cerevisiae, have been investigated for years genetically and molecularly for signaling in sexual reproduction. The comparison of the structure and organization of mating type genes in fungal genomes reveals an amazing conservation of genes regulating the sexual reproduction throughout the fungal kingdom. In agaricomycetes, two mating type loci, A, coding for homeodomain type transcription factors, and B, encoding a pheromone/receptor system, regulate the four typical mating interactions of tetrapolar species. Evidence for both A and B mating type genes can also be identified in basidiomycetes with bipolar systems, where only two mating interactions are seen. In some of these fungi, the B locus has lost its self/nonself discrimination ability and thus its specificity while retaining the other regulatory functions in development. In silico analyses now also permit the identification of putative components of the pheromone-dependent signaling pathways. Induction of these signaling cascades leads to development of dikaryotic mycelia, fruiting body formation, and meiotic spore production. In pheromone-dependent signaling, the role of heterotrimeric G proteins, components of a mitogen-activated protein kinase (MAPK) cascade, and cyclic AMP-dependent pathways can now be defined. Additionally, the pheromone-dependent signaling through monomeric, small GTPases potentially involved in creating the polarized cytoskeleton for reciprocal nuclear exchange and migration during mating is predicted.

Dichotomization of mycorrhizal and NPA-treated short roots in Pinus sylvestris.

Conifers like Scots pine (Pinus sylvestris) have a complicated root system consisting of morphologically and anatomically different root types, of which the short roots have a very limited ability to elongate. Short roots have an important role in nature since they are able to establish ectomycorrhizal symbiosis, in which the growth of fungal mycelium between the epidermal cells and in the intercellular space between cortical cells leads to formation of dichotomous short roots, which may, through further splitting of the meristem, form coralloid root structures. Dichotomous short roots have been suggested to result from changes in either auxin or ethylene concentrations due to the fungal growth inside the root. NPA, the inhibitor of polar auxin transport, enhances the dichotomization of P. sylvestris short root tips similarly to the fungal growth in the root, thus confirming that auxin plays a role in short root morphogenesis. Ethylene is also known to have an important role in the regulation of root morphogenesis. In future the research dealing with the root system and ectomycorrhiza development in P. sylvestris must take into account that both auxin and ethylene are involved and that there is no contradiction in obtaining the same phenotype with both hormones. The expression analysis of PIN proteins, auxin efflux carriers, could give valuable information about the role of auxin transport in regulating the root growth and morphogenesis of coniferous root system and mycorrhiza.

Interaction with mycorrhiza helper bacterium Streptomyces sp. AcH 505 modifies organisation of actin cytoskeleton in the ectomycorrhizal fungus Amanita muscaria (fly agaric).

The actin cytoskeleton (AC) of fungal hyphae is a major determinant of hyphal shape and morphogenesis, implicated in controlling tip structure and secretory vesicle delivery. Hyphal growth of the ectomycorrhizal fungus Amanita muscaria and symbiosis formation with spruce are promoted by the mycorrhiza helper bacterium Streptomyces sp. AcH 505 (AcH 505). To investigate structural requirements of growth promotion, the effect of AcH 505 on A. muscaria hyphal morphology, AC and actin gene expression were studied. Hyphal diameter and mycelial density decreased during dual culture (DC), and indirect immunofluorescence microscopy revealed that the dense and polarised actin cap in hyphal tips of axenic A. muscaria changes to a loosened and dispersed structure in DC. Supplementation of growth medium with cell-free bacterial supernatant confirmed that reduction in hyphal diameter and AC changes occurred at the same stage of growth. Transcript levels of both actin genes isolated from A. muscaria remained unaltered, indicating that AC changes are regulated by reorganisation of the existing actin pool. In conclusion, the AC reorganisation appears to result in altered hyphal morphology and faster apical extension. The thus improved spreading of hyphae and increased probability to encounter plant roots highlights a mechanism behind the mycorrhiza helper effect.

Cloning of Pinus sylvestris SCARECROW gene and its expression pattern in the pine root system, mycorrhiza and NPA-treated short roots.

The SCARECROW (SCR) gene is central to root radial patterning. Its expression has not been investigated in conifers with morphologically different root types. Additional interest in SCR functions in the Pinus sylvestris root system comes from the effect of ectomycorrhiza formation on the short root apical structure. Here, the P. sylvestris SCR gene (PsySCR) was cloned and its expression investigated by northern blot and in situ hybridization of primary, lateral and short roots and mycorrhiza. Short root dichotomization was induced by auxin transport inhibitor (N-1-naphthylphthalamic acid (NPA)). PsySCR has conserved GRAS family protein motifs at the C-terminus and a variable N-terminus. PsySCR expression occurred in young root tissue and mycorrhiza. In root sections the PsySCR signal runs through the tip in initials for stele and root cap column and becomes upwards-restricted to endodermis in all root types. The PsySCR expression pattern suggests for the first time a regulatory role for SCR in maintaining the endodermal characteristics and radial patterning of roots with open meristem organization. The specific PsySCR localization is also an excellent marker for investigation of the dichotomization process in short roots.

Ras GTPase-activating protein gap1 of the homobasidiomycete Schizophyllum commune regulates hyphal growth orientation and sexual development.

The white rot fungus Schizophyllum commune is used for the analysis of mating and sexual development in homobasidiomycete fungi. In this study, we isolated the gene gap1 encoding a GTPase-activating protein for Ras. Disruption of gap1 should therefore lead to strains accumulating Ras in its activated, GTP-bound state and to constitutive Ras signaling. Haploid Deltagap1 monokaryons of different mating types did not show alterations in mating behavior in the four different mating interactions possible in fungi expressing a tetrapolar mating type system. Instead, the growth rate in Deltagap1 monokaryons was reduced by ca. 25% and ca. 50% in homozygous Deltagap1/Deltagap1 dikaryons. Monokaryons, as well as homozygous dikaryons, carrying the disrupted gap1 alleles exhibited a disorientated growth pattern. Dikaryons showed a strong phenotype during clamp formation since hook cells failed to fuse with the peg beside them. Instead, the dikaryotic character of the hyphae was rescued by fusion of the hooks with nearby developing branches. Deltagap1/Deltagap1 dikaryons formed increased numbers of fruitbody primordia, whereas the amount of fruitbodies was not raised. Mature fruitbodies formed no or abnormal gills. No production of spores could be observed. The results suggest Ras involvement in growth, clamp formation, and fruitbody development.

Ectopic expression of a constitutively active Cdc42 small GTPase alters the morphology of haploid and dikaryotic hyphae in the filamentous homobasidiomycete Schizophyllum commune.

Cloning of the Cdc42 gene from Schizophyllum commune enabled investigation of the role of ScCdc42 in the regulation of vegetative growth and sexual reproduction in this fungus, which has a well-characterized hyphal cell structure, cytoskeleton, and mating system. Ectopic expression of the constitutively active Sccdc42(G12V) or Sccdc42(Q61L) alleles from native or inducible ScCel1 promoters in haploid hyphae had dramatic effects on hyphal morphology, cytoskeletal structure, and Cdc42 localization. For transformants with constitutively active Sccdc42, polar tip growth of apical cells in the leading hyphae was normal but polar tip growth in side branches was altered, implying different regulation of polarity establishment in the two groups of apical cells. Branch emergence at exceptional sites and isotropic growth of cells near the septum indicated that ScCdc42 regulates branch site selection and subsequent hyphal development. Poor dikaryotization along with irregular clamp connections in mates expressing Sccdc42(G12V) or Sccdc42(Q61L) suggested that Cdc42 also contributes to efficient mating in S. commune.

Hypaphorine, an indole-3-acetic acid antagonist delivered by the ectomycorrhizal fungus Pisolithus tinctorius, induces reorganisation of actin and the microtubule cytoskeleton in Eucalyptus globulus ssp bicostata root hairs.

Hypaphorine, an indole alkaloid from the ectomycorrhizal fungus Pisolithus tinctorius Coker & Couch., counteracts indole-3-acetic acid (IAA) activity and controls the rate of root hair elongation in Eucalyptus globulus ssp. bicostata. The present investigation shows that hypaphorine changes cytoskeletal organisation in elongating root hairs of the host. The actin cytoskeleton was investigated by two different fixation and labelling procedures, which gave similar results. In control root hairs, actin organisation was characterised by (i) an actin cap at the very tip region, (ii) a subapical region with reduced labelling and containing fine actin filaments, and (iii) axial bundles of actin filaments running from the subapical part to the base of the root hair. In the hypaphorine-treated root hairs no actin cap was distinguished. The fine actin filaments occurring in the subapical region were replaced by a few thick actin filament bundles that extended from the subapical region toward the root hair tip. In the hypaphorine-treated hairs the total number of actin filament bundles along most of the root hair length was significantly reduced, presumably due to aggregation of pre-existing actin filaments. The first signs of alteration to the cytoskeleton could be detected as soon as 15 min after hypaphorine treatment. In hypaphorine-treated, but not in control root hairs, a patch of aggregated microtubules regularly occurred at a distance of approximately 10 microm from the tip, possibly as a consequence of changes induced by hypaphorine in the actin cytoskeleton. The hypaphorine-induced aggregations in the actin and microtubule cytoskeletons could stabilise the structure of cytoskeletal elements, which in turn could hinder the vesicle delivery at the tip necessary for elongation. Such cytoskeletal alterations may be a consequence of the antagonism between IAA and hypaphorine. The latter view was supported by restoration of the actin cytoskeleton in hypaphorine-treated root hairs by IAA application.

A peroxidase gene family and gene trees in Heterobasidion and related genera.

Four putative peroxidase-encoding gene fragments, named mnp1a, mnp1b, mnp2 and mnp3, were amplified with degenerative primers from the white-rot basidiomycete genus Heterobasidion. The fragments were cloned and sequenced. Similar fragments were produced and analyzed from the related genera Amylostereum, Bondarzewia and Echinodontium. Each amplified fragment contains three identically positioned introns. According to the predicted amino acid sequence, these fragments are most similar to two Mn peroxidase-encoding genes (MPGI and mnp2) and gene pgv of Trametes versicolor. Conserved residues thought to be essential for peroxidase function were identified. All four MnP gene loci of Heterobasidion were detected only in H. parviporum. Variation occurred in the predicted amino-acid sequences (131-132 amino acids) of all four fragments originating from the 47 Heterobasidion isolates tested. Amino acid variation in fragments of mnp2 and mnp3 separated European Heterobasidion parviporum ("S-type") and H. abietinum ("F-type"), known to have identical rDNA sequences. Asian and western North American isolates from fir, spruce and other hosts had the peroxidase amino acid sequences of European H. parviporum. American and European H. annosum ("P-type") isolates had different amino acid sequences and might be cryptic species.

T-DNA transfer and integration in the ectomycorrhizal fungus Suillus bovinus using hygromycin B as a selectable marker.

The T-DNA of Agrobacterium tumefaciens can be transferred to plants, yeasts, fungi and human cells. Using this system, dikaryotic mycelium of the ectomycorrhizal fungus Suillus bovinus was transformed with recombinant hygromycin B phosphotransferase (hph)and enhanced green fluorescent protein (EGFP) genes fused with a heterologous fungal promoter and CaMV35S terminator. Transformation resulted in hygromycin B-resistant clones, which were mitotically stable. Putative transformants were analysed for the presence of hph and EGFP genes by PCR and Southern analysis. The latter analysis proved both multiple- and single-copy integrations of the genes in the S. bovinus genome. A. tumeficiens transformation should make possible the development of tagged mutagenesis and targeted gene disruption technology for S. bovinus.

Maturation of barley cysteine endopeptidase expressed in Trichoderma reesei is distorted by incomplete processing.

Maturation of barley cysteine endopeptidase B (EPB) in Trichoderma reesei was studied with metabolic in hibitors, Western blotting, and immuno microscopy. The inactive 42-kDa recombinant EPB proprotein, first detected in apical cells, was sequentially processed in a time-dependent manner to a secreted polypeptide of 38.5 kDa, and thereafter, to polypeptides of 37.5, 35.5, and 32 kDa exhibiting enzyme activity both in the hyphae and culture medium. The sizes of the different forms of recombinant EPB were in accordance with molecular masses calculated from the deduced amino acid sequence, assuming cleavage at four putative Kex2p sites present in the 42-kDa proprotein. Both the liquid and the zymogram in-gel activity assays indicated that the 32-kDa enzyme produced in T. reesei in vivo was 2 kDa larger and four times less active than the endogenous EPB. Brefeldin A treatment prevented the last Kex2p processing step of EPB from a 35.5- to a 32-kDa protein. This coincided with a significant increase in the immuno-gold label for EPB and in modified Golgi-like bodies, which suggests that the processing step probably took place in medial Golgi. A 30.5-kDa EPB polypeptide was observed when glycosylation was inhibited by tunicamycin (TM) or when deglycosylation was carried out enzymatically. Deglycosylation increased the enzyme activity twofold, which was also indicated by an increased fluorescence by TM treatment in the zymogram in-gel activity assay. Simultaneous incubation with TM and monensin produced a peptide of 31.5 kDa. Therefore, monensin may inhibit the final processing step of an unglycosylated EPB by an unknown protease in the fungus. In any case, the final recombinant EPB product in Trichoderma differs from the mature endogenous 30-kDa enzyme produced in barley.

Effect of cytochalasin A on apical growth, actin cytoskeleton organization and enzyme secretion in Aspergillus nidulans.

The role of actin in apical growth and enzyme secretion in the filamentous fungus Aspergillus nidulans was studied by treating the hyphae with cytochalasin A (CA), which inhibits actin polymerization. Indirect immunofluorescence microscopy revealed actin at the tips of main hyphae and branches, and at the site of developing septa. CA inhibited the growth of the fungus and changed the growth pattern of hyphal tips from cylindrical tubes to spherical beads. The regions with swellings showed no actin fluorescence, and neither was actin seen in association with septa. After 4 h exposure, hyphae were able to resume the normal tip growth pattern in the presence of CA for a short period of time and new cylindrical hyphae, with actin fluorescence at the apex, emerged from the swollen tips. Later, the tips of the hyphae swelled again, which led to a beaded appearance. We also studied the effect of CA on the secretion of alpha- and beta-galactosidase. alpha-Galactosidase is secreted into the culture medium, whereas beta-galactosidase remains in the mycelium, with part of its activity bound to the cell wall. When A. nidulans mycelium was incubated in the presence of CA, a reduction in the secretion of alpha-galactosidase into the culture medium and a decrease in the alpha- and beta-galactosidase activities bound to the cell wall was detected. However, the CA dose used for the hyphae did not modify the secretion of the enzymes from protoplasts. Results described here provide evidence that a polymerized actin cytoskeleton is required for normal apical growth, hyphal tip shape and polarized enzyme secretion in A. nidulans. Cytochalasin-induced disruptions of the actin cytoskeleton could result in the alterations of apical growth and inhibition of enzyme secretion observed by blocking secretory vesicle transport to the apex.