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.</p

Nuclear-Localized Fluorescent Proteins Enable Visualization of Nuclear Behavior in the Basidiomycete <i>Schizophyllum commune</i> 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

Cytoskeletal components in the arbuscular mycorrhizal fungus Glomus mosseae

In Glomus mosseae microtubules and microfilaments were visualized by indirect immunofluorescence microscopy in hyphae quick-frozen, freeze-substituted, and treated with cell-wall-degrading enzymes. Microtubules were distinguished in both the cortical and the central parts of hyphae, while microfilaments were revealed only in the cortical part. In immunoblotting, tubulin and actin were detected in an extract from hyphae elicited by host plants

Dynamics of cytoskeletal proteins in developing pine ectomycorrhiza

Mycorrhizal short roots of Pinus contorta Dougl. ex Loud colonized by Suillus variegatus (Sow. ex Fr.) O. Kuntze or Paxillus involutus (Batsch) Fr. were collected 1->60 days after fungal contact. The proteins of the inoculated roots were extracted, electrophoretically separated, blotted and immunostained for alpha-tubulin and actin. The development of the mycorrhiza was also followed microscopically. The signal of plant alpha-tubulin was stronger than the signal of fungal alpha-tubulin during the first 5 days in S. variegatus mycorrhiza and was then exceeded by fungal alpha-tubulin. This correlated well with the increase of fungal mycelium in the mycorrhiza. A transient drop in both plant and fungal alpha-tubulin signals was observed 20 days after fungal contact, suggesting a change in the metabolism of the mycorrhiza. The signals for plant and fungal actins in the mycorrhiza increased steadily during early infection and then remained at a high level as the mycorrhiza matured. Similar trends were observed in P. contorta-P. involutus mycorrhiza. The data from P. contorta-S. variegatus mycorrhizas suggests that alpha-tubulin is a growth-related protein, subject to changes, while the amount of actin reflects the general metabolic activity of the mycorrhiza. In both mycorrhizal systems clear alpha-tubulin and actin signals were detected 60 days after colonization, which indicates that the mycorrhizas were metabolically active in spite of their withered appearance