Structure and function of the septal pore cap of Schizophyllum commune

Abstract

Filamentous fungi form networks (mycelia) of long, tubular structures (hyphae) that extend at their tip (apex) and branch subapically (behind the tip). Hyphae are compartmentalized by cross walls (septa) that contain a central pore which is covered by a septal pore cap (SPC) in fungi belonging to the Basidiomycetes. In this thesis I studied the composition and function of the SPC of S. commune. SPCs were purified and their composition, proteins and corresponding genes were identified. Analysis of genes of the SPC was combined with improvement of methods for transformation and gene inactivation. Finally, I developed a method to study septa in the mycelium under stress. SPCs of S. commune were purified by separating homogenized mycelium containing 1 % Triton-X-100 on two discontinuous sucrose gradients, followed by filtration over a membrane. The SPC is composed of a proteinaceous core surrounded by membranous material. We show that this core determines the shape of the SPC and consists of two abundant proteins; SPC14 and SPC33. The SPC14 gene encodes a protein of 86 amino acids without known signal or signature sequences. Expressed sequence tag (EST) analysis indicates that SPC33 encodes a 239 and 340 amino acid variant that share a predicted signal anchor, a putative RXR ER localization signal, and a transmembrane region. SPC14 and SPC33 are only found in basidiomycetes with a SPC. Presence of SPC14 and SPC33 in the SPC was confirmed by immuno-localization. Previously, S. commune was transformed to phleomycin resistance using the ble gene of Streptoalloteychus hindustanus. I show that selective concentrations of phleomycin are mutagenic to resistant strains. Therefore, a nourseothricin resistance cassette was constructed with the nat1 gene of Streptomyces noursei. Transformation efficiency with nourseothricin was ten-fold lower, but could be restored by adding phleomycin which was accompanied by an increase of single copy integrations. The nourseothricin and phleomycin cassette were used to construct a vector for rapid screening of homologous integrations and was successfully applied for gene deletions in our lab. However, out of 800 transformants none showed inactivation of the SPC14 or SPC33 gene. Therefore, they might be essential, which is supported by the fact that RNA interference of the SPC14 gene was unstable. Reduced SPC14 mRNA levels, seemingly correlating with reduced growth in monokaryons and abnormal fruiting in dikaryons, was lost upon transfer to fresh medium. Using laser dissection it was assessed whether septa in a mycelium are open or closed. I showed that the apical septum is always open, the second septum is sometimes closed and the third septum is in most cases closed. Upon exposure to antibiotic, heat stress, or hypertonic conditions the first septum closes. The observed plugging mechanism seems to be an emergency response since arrest of growth and vacuolization of cytoplasm precede septal closure. Heat-induced closure was reversible, which indicates that septa are dynamic structures. Apical compartments were found to be able to reinitiate growth after separation from the mycelium. The fact that the third septum is mostly closed challenges the general assumption that mycelia are continuous systems.