Septal Pore Caps in Basidiomycetes, Composition and Ultrastructure

Abstract

Filamentous fungi, including Ascomycota and Basidiomycota, form mycelia that consist of a network of apical growing hyphae. These hyphae are separated into cellular compartments by septa that have pores of about 70 to 500 nm in diameter. The cytoplasm within the mycelium is thus continuous (coenocytic). The septum in the higher Basidiomycota (i.e. Agaricomycotina) is flared towards the pore, forming a barrel-shaped structure that is called the dolipore. These dolipore septa are generally associated with septal pore caps (SPCs). The ultrastructure of the SPC has been relatively well studied by the use of electron microscopy, but the composition and the function of these organelles is unknown. Several functions have been suggested based on the morphological studies. SPCs may be involved in dolipore plugging to prevent lysis of the mycelium after hyphal damage either by producing pore-plug material or by acting as a repository of the pore-plug material. SPCs may also function as a sieve, guide organelles to the dolipore, or prevent organelles to block the entrance of the dolipore. The aim of this Thesis was to identify components of the SPC and to assess whether the SPC ultrastructure reflects the recently revised phylogeny of the Agaricomycotina. It is shown that two orders in the Agaricomycotina, i.e. the Hymenochaetales and Cantharellales, contain two SPC morphologies, namely the imperforate and the perforate SPC-type. Thus, SPCs within the orders of the Agaricomycotina are not monomorphic per se. In addition, two methods were developed to isolate and enrich SPCs from Rhizoctonia solani. This basidiomycetous fungus was chosen because of its well studied, large SPCs that have a diameter of about 1600 to 2000 nm and pores of about 600 to 800 nm. With the first method, fungal septa were isolated by laser microdissection and laser pressure catapulting. A wheat germ agglutinin labeling was used to detect the isolated SPCs by scanning electron microscopy. The second isolation method was based on the combined use of French press, isopycnic centrifugation using a discontinuous sucrose gradient, followed by a treatment with Triton X-100. With this isolation method a SPC protein was identified for the first time, which is named SPC18. The encoding gene SPC18 was isolated. The gene encodes a protein, which contains both a signal peptide to direct it to the ER, and an N-glycosylation motif. Immuno-localization confirmed that the glycoprotein SPC18 is located in the SPCs, but it was also shown to reside in pore-plug material. Taken together, I propose that SPCs of R. solani consist of a proteinaceous core, which is covered with ER-membranes. SPC18 is one of the proteins residing in the SPC core. It may be transported via filaments to the dolipore entrance forming a plug that may contribute to hyphal homeostasis in cases of stress or hyphal damage in basidiomycetous fungi.