Septal Pore Caps in Basidiomycetes Composition and Ultrastructure Septal Pore Caps in Basidiomycetes Composition and Ultrastructure Septumporie-kappen in Basidiomyceten Samenstelling en Ultrastructuur (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof.dr. J.C. Stoof, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op maandag 17 december 2007 des middags te 16.15 uur door Kenneth Gregory Anthony van Driel geboren op 31 oktober 1975 te Terneuzen Promotoren: Prof. dr. A.J. Verkleij Prof. dr. H.A.B. Wösten Co-promotoren: Dr. T. Boekhout Dr. W.H. Müller voor mijn ouders Cover design by Danny Nooren. Scanning electron micrographs of septal pore caps of Rhizoctonia solani made by Wally Müller. Printed at Ponsen & Looijen b.v., Wageningen, The Netherlands. ISBN 978-90-6464-191-6 CONTENTS Chapter 1 General Introduction 9 Chapter 2 Septal Pore Complex Morphology in the Agaricomycotina 27 (Basidiomycota) with Emphasis on the Cantharellales and Hymenochaetales Chapter 3 Laser Microdissection of Fungal Septa as Visualized by 63 Scanning Electron Microscopy Chapter 4 Enrichment of Perforate Septal Pore Caps from the 79 Basidiomycetous Fungus Rhizoctonia solani by Combined Use of French Press, Isopycnic Centrifugation, and Triton X-100 Chapter 5 SPC18, a Novel Septal Pore Cap Protein of Rhizoctonia 95 solani Residing in Septal Pore Caps and Pore-plugs Chapter 6 Summary and General Discussion 113 Samenvatting 123 Nawoord 129 List of Publications 131 Curriculum vitae 133 Chapter 1 General Introduction Kenneth G.A. van Driel*, Arend F. van Peer*, Wally H. Müller, Teun Boekhout & Han A.B. Wösten *Both authors equally contributed to this work Chapter 1 Filamentous Fungi Filamentous fungi grow by means of hyphae that extend at their apices while branching subapically. This mode of growth together with hyphal fusion (anastomosis) results in an interconnected network of hyphae called a mycelium. Hyphae of the lower fungi, i.e. the Glomeromycota, Zygomycota, and Chytridiomycota (Schüßler et al., 2001; Bauer et al., 2006), are sparsely, if at all, septated (Barr, 2001; Benny et al., 2001). In contrast, the hyphae of the filamentousAscomycota and Basidiomycota are regularly septated. Their septa contain pores of about 50 to 500 nm, which are covered with a continuous plasma membrane and allow streaming of cytoplasm including large organelles like mitochondria (Bracker & Butler, 1963, 1964; Gull, 1978; Van Driel et al., 2007). The continuity of the cytoplasm discriminates cells of filamentous fungi from those of plants and animals. In these latter two kingdoms there are also intercellular cytoplasmic connections but they are much smaller. Gap junctions in animals have pores of about 1.6 to 3.0 nm in diameter and allow streaming of inorganic ions and small water-soluble organic molecules (Veenstra, 1996; Perkins et al., 1997). Plasmodesmata in plants are small microchannels of 1.5 to 2.0 nm, restricting also in this case the intercellular flow to small molecules (reviewed by Ghoshroy et al., 1997). It should be noted that the channels in plasmodesmata are dynamic and can be closed or increased in diameter to 5 to 9 nm. In this chapter the ultrastructure, the composition and the function of fungal septa and their associated organelles (i.e. the Woronin bodies and the septal pore caps) are described. The mechanisms underlying the formation of the septa and the associated organelles are also briefly discussed. This is followed by a description of Rhizoctonia solani (Basidiomycota) as a model system to study septal pore caps in Basidiomycota. At the end of this chapter the aim of this Thesis is described followed by a brief summary of each of the chapters. The Septum and Woronin Bodies in Filamentous Ascomycota The ascomycetous septum consists of a cross-wall with a central pore (Figure 1A). The septum is tapered towards the pore and is usually referred to as “simple” septum. The diameter of the septal pore varies between 50 to 500 nm allowing passage of mitochondria, nuclei and other organelles (Shatkin & Tatum, 1959; Moore & McAlear, 1962; Gull, 1978). Although the septa are extensions of the lateral cell wall, their chemical compositions differ (Gull, 1978). The septum plate is build up by chitin microfibrils and β-glucans, but α-glucan, which is a component of the lateral wall, is not found (Griffin, 1994). Older septa may be covered with an amorphous protein layer (Gull, 1978). The ascomycetous septum is associated with Woronin bodies (Figure 1A) that are also found at the hyphal tip (Momany et al., 2002). Woronin first observed Woronin bodies in 1864 by light microscopy (Woronin, 1864; Buller, 1933). They have a spherical 10 General introduction Figure 1 – Transmission electron micrographs of several septum types that are found in the fungal kingdom. A) Septal pore of Aspergillus nidulans (Ascomycota). The septum is tapered towards the pore, and is also referred as “simple” septum. Woronin bodies are associated at the pore (image is adopted from Momany et al., 2002). B) Septal pore of Sporidiobolus ruineniae (Pucciniomycotina). Septum is tapered towards the pore, but without Woronin bodies (Boekhout et al., 1992). C) Dolipore septum of Itersonilia perplexans (Agaricomycotina) without septal pore caps (SPC). Membrane bands are located between the swollen pore that is covered by endoplasmic reticulum (Boekhout, 1991). D) Dolipore septum (DP) of Rhizoctonia solani (Agaricomycotina) associated with perforate SPCs. A nucleus (Nu) is blocked passage by the pores in the septal pore caps (Müller et al., unpublished). Bars represent 250 nm in A, 500nm in B and D, and 200 nm in C. or hexagonal shape with a diameter of 150 to 500 nm (Markham & Collinge, 1987). Woronin bodies rapidly plug septal pores when hyphae are damaged to prevent loss of cell content (Trinci & Collinge, 1974). HEX-1 is the main protein of these organelles in Neurospora crassa (Jedd & Chua, 2000). It forms the crystalline core of the Woronin body (Yuan et al., 2003), which is surrounded by a single membrane (Markham & Collinge, 1987). Phosphorylation of HEX-1 is important for multimerization of the protein and proper formation of the Woronin bodies (Juvvadi et al., 2007). The HEX-1 protein has a C-terminal peroxisome targeting signal (PTS1) and therefore Woronin bodies have been suggested to be peroxisomes (Jedd & Chua, 2000; Tenney et al., 2000). HEX-1 homologues have been found in several other filamentous Ascomycota, like Aspergillus nidulans and Magnaporthe grisea (Jedd & Chua, 2000; Soundarajan et al., 2004) but not in ascomycetous yeasts nor in Basidiomycota. Ultrastructure of the Septum and the Septal Pore Cap in Basidiomycota Within the Basidiomycota, the three major groups Pucciniomycotina (Urediniomycetes; Swann & Taylor, 1995), Ustilaginomycotina (Ustilaginomycetes; Swann & Taylor, 1995), and Agaricomycotina (Hymenomycetes; Swann & Taylor, 1995) are distinguished (Bauer et al., 2006; James, et al., 2006; Hibbett et al., 2007). These groups are amongst others characterized by the ultrastructure of their septum. The Pucciniomycotina contain the rust fungi, which have septa with a pore morphology as found in the filamentous 11 Chapter 1 Ascomycota (Oberwinkler & Bandoni, 1982; Swann et al., 2001; Bauer et al., 2006), though without Woronin bodies (Figure 1B). The Ustilaginomycotina that include the smut fungi also possess a septum with a septal pore that is similar to that found in the hyphae of Ascomycota, but may have a slightly swollen rim around the pore. These septal pores may also be associated with membrane caps or membrane bands (Bauer et al., 1997, 2001). The Agaricomycotina (i.e. Tremellomycetes, Dacrymycetes, and Agaricomycetes) includes jelly fungi and mushroom-forming fungi. They have a barrel-shaped swelling around the pore, the dolipore (Figure 1C, D), which generally is associated with a septal pore cap (SPC) (Figure 1D) (Bracker & Butler, 1963). The SPC is also known as Verschlußband (Girbardt, 1958) or parenthesome (Moore & McAlear, 1962). The dolipore channel is about 70 to 500 nm in diameter (Bracker & Butler, 1964 ; Setliff et al., 1972; Patton & Marchant, 1978), but SPCs that cover the dolipore restrict the passage of large organelles, such as nuclei (Figure 1D). Like in the Ascomycota, the composition of the basidiomycetous septum is different from that of the lateral cell wall. The lateral cell wall consists of chitin, β-1,3/β-1,6- glucan and α-1,3-glucan. The septal plate contains chitin and β-1,3/β-1,6-glucan but no α-1,3-glucan, whereas the septal swelling contains α-1,3-glucan, β-1,3-glucan, and β- 1,6-glucan (Janszen & Wessels, 1970; Müller et al., 1998a, 2000a). The dolipore swelling contains more β-1,6-glucan than the septal plate (Müller et al., 1998a, 2000a). Staining of the polysaccharides according to Thiéry (1967) showed that filaments of the inner electron dense septal layer radiate into the swelling of the dolipore and form a distinct rim visible in median and traverse sections. This rim intertwines as a loose network of stained fibrous material into the non-stained peripheral part of the septal swelling (Bracker & Butler, 1963; Van der Valk & Wessels, 1976). At the dolipore septum, several SPC-types can be distinguished, which can be used as a phylogenetic marker (e.g. McLaughlin et al., 1995; Fell et al., 2001; Hibbett & Thorn, 2001; Wells & Bandoni, 2001; Lutzoni et al., 2004). The vesicular (tubular or saccular) SPC-type is found in members of the Tremellomycetes. This SPC-type consists of a group of vesicles or tubules arranged in a hemisphere surrounding the dolipore, e.g. in Trichosporon sporotrichoides (Figure 2A). The imperforate SPC-type is found in the Dacrymycetes and Agaricomycetes (Wells & Bandoni, 2001) and consists of a slightly flattened closed membranous structure, e.g. in Epulorhiza anaticula (Figure 2B). This SPC-type may have an inward growth with reduced thickness or a minute pore in the centre of the membrane (Müller et al., 2000b).