Regular Articles The Coprinopsis cinerea septin Cc.Cdc3 is involved in stipe cell elongation Tatsuhiro Shioya a , Hiroe Nakamura a , Noriyoshi Ishii a , Naoki Takahashi a , Yuichi Sakamoto b , Noriaki Ozaki a , Masayuki Kobayashi a , Keiju Okano a , Takashi Kamada c , Hajime Muraguchi a,⇑ a Department of Biotechnology, Faculty of Bioresource Sciences, Akita Prefectural University, Akita 010-0195, Japan b Iwate Biotechnology Research Center, Kitakami, Iwate 024-0003, Japan c Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan article info Article history: Received 4 May 2013 Accepted 13 August 2013 Available online 20 August 2013 Keywords: Coprinopsis cinerea Fruiting Stipe Cell elongation Septin Cdc3 abstract We have identified and characterized a Coprinopsis cinerea mutant defective in stipe elongation during fruiting body development. In the wild-type, stipe cells elongate at the maturation stage of fruiting, resulting in very slender cells. In the mutant, the stipe cells fail to elongate, but become rather globular at the maturation stage. We found that the mutant phenotype is rescued by a gene encoding a homolog of Saccharomyces cerevisiae CDC3 septin, Cc.Cdc3. The C. cinerea genome includes 6 septin genes, 5 of which, including Cc.cdc3, are highly transcribed during stipe elongation in the wild type. In the mutant, the level of Cc.cdc3 transcription in the stipe cells remains the same as that in the mycelium, and the level of Cc.cdc10 transcription is approximately 100 times lower than that in the wild-type stipe cells. No increase in transcription of Cc.cdc3 in the mutant may be due to the fact that the Cc.cdc3 gene has a 4-base pair insertion in its promoter and/or that the promoter region is methylated in the mutant. Overexpressed EGFP-Cc.Cdc3 fusion protein rescues the stipe elongation in the transformants, localizes to the cell cortex and assembles into abundant thin filaments in the elongating stipe cells. In contrast, in vegetative hyphae, EGFP-Cc.Cdc3 is localized to the hyphal tips of the apical cells of hyphae. Cellular defects in the mutant, combined with the localization of EGFP-Cc.Cdc3, suggest that septin filaments in the cell cor- tex provide the localized rigidity to the plasma membrane and allow cells to elongate cylindrically. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction The basidiomycete Coprinopsis cinerea forms a highly differenti- ated structure, the fruiting body (Muraguchi and Kamada; 1998; Kües, 2000). Fig. 1 is a schematic diagram illustrating fruiting in C. cinerea. Fruiting body formation begins with the aggregation of hyphae, producing hyphal knots of approximately 0.2 mm or less in diameter. In the hyphal knots, cells divide rapidly and differen- tiate into a compact core composed of highly branched short cells and a layer of veil cells covering the core (van der Valk and Mar- chant, 1978). Following differentiation of the primordial shaft, the rudimentary pileus (cap) differentiates at the upper region of the primordial shaft, forming a tiny fruiting body primordium (Muraguchi and Kamada, 1998). The stipe tissue differentiates at the central region encompassed by the rudimentary gill. The pri- mordium gradually enlarges and matures under proper light con- ditions, such as a 12 h light/12 h dark cycle (Kamada et al., 1978; Terashima et al., 2005). The maturation stage is triggered by light (0 h in Fig. 1), and stipe elongation starts around the end of the light period in the final day (Kamada, 1994). During stipe elonga- tion, stipe cells elongate without cell division and with nuclear division, becoming multinucleate (Gooday, 1985; Stephenson and Gooday, 1984). The pileus expands to disperse basidiospores near the end of stipe elongation. Pileus expansion and spore dispersion are associated with fruiting body autolysis (Muraguchi et al., 2008), which characterizes the ephemeral life of the Coprinopsis genus. In fruiting, two types of cell expansion occur: a slow process, which is often encountered in primordia, and a more rapid one in- volved specifically in stipe elongation (Reijnders and Moore, 1985). Stipe elongation is almost entirely due to elongation of the stipe cells, which elongate from approximately 0.1 mm to 1 mm during the final phase of fruiting, providing a good opportunity to study cell expansion that is characterized by diffuse extension growth (Kamada and Takemaru, 1977; Kamada, 1994; Gooday, 1985). Dif- fuse extension growth, unlike tip growth in vegetative hyphae, oc- curs throughout the cell surface; the helical or transverse arrangement of chitin microfibrils or glucosaminoglycan chains in the cell wall contributes to the process (Gooday, 1979; Kamada et al., 1991; Mol et al., 1990). Such microfibril arrangement was observed in an initial stage of fruiting body development, i.e., in 1087-1845/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.fgb.2013.08.007 ⇑ Corresponding author. Address: Department of Biotechnology, Faculty of Bioresource Sciences, Akita Prefectural University, Shimoshinjo-nakano, Akita 010-0195, Japan. Fax: +81 18 872 1676. E-mail address: muraguchi@akita-pu.ac.jp (H. Muraguchi). Fungal Genetics and Biology 58–59 (2013) 80–90 Contents lists available at ScienceDirect Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi