Note Localization of protein secretion in fungal colonies using a novel culturing technique; the ring-plate system Ana M. Levin, Ronald P. de Vries, Han A.B. Wösten ⁎ Microbiology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands Received 29 August 2006; received in revised form 9 January 2007; accepted 10 January 2007 Available online 18 January 2007 Abstract A novel culturing technique, called the ring-plate system, is described. Fungal colonies are grown on a polycarbonate membrane placed on a plate with 6 ring-shaped wells that are filled with liquid medium. This culturing technique enables simultaneous monitoring of environmental conditions and secretion in different parts of the fungal colony. © 2007 Elsevier B.V. All rights reserved. Keywords: Ring plate system; Fungal colonies; Secretion; Aspergillus niger; Vegetative mycelium; Fungi; Xylanase Filamentous fungi colonize moist substrates by means of hyphae that grow at their tips and that branch subapically. The resulting mycelium secretes a wide variety and large amounts of proteins that degrade the substrate into molecules that can be taken up to serve as nutrients. Their enormous secretory capacity makes fungi interesting cell factories for production of homologous and heterologous proteins (Punt et al., 2002). Within the mycelium not all hyphae grow and are active in protein secretion. It was shown that these processes are confined to two zones in colonies of Aspergillus niger and Phanerochaete chrysosporium (Wösten et al., 1991; Moukha et al., 1993). Glucoamylase (GlaA) is secreted in the outer growth zone of A. niger colonies, whereas lignin peroxidase (LiPH8) is released in the central growth zone of colonies of P. chrysosporium. In previous studies protein secretion was localized by growing fungal colonies in between two polycarbonate membranes that had been placed on a protein binding polyvinylidenedifluoride (PVDF) membrane that itself was positioned on a nutrient agar medium (Wösten et al., 1991; Moukha et al., 1993). In this experimental set-up secreted proteins are immobilized by the PVDF membrane shortly after their secretion. Immuno-labeling on the PVDF membrane allows the localization of specific proteins but the protein profile released at each part of the colony cannot be easily assessed. Moreover, determining local medium conditions such as pH and the concentration of nutrients is hampered by lateral diffusion and the presence of agar in the medium. Determining these local conditions, as well as local gene expression, is crucial to understand processes underlying secretion in a fungal colony. We here describe a new culturing method, called the ring plate system, which allows us to determine the secretome at each part of the colony and to correlate it with environmental conditions. As an example, secretion was studied in colonies of A. niger . The ring plate consists of a polycarbonate disc (9 cm in diameter, 1.2 cm thick) with 6 ring-shaped wells (Fig. 1A). The inner two rings are collectively called ring 1 because of their small volume and the outer ring is thus called ring 5. The wells are separated by 0.1 cm and are 0.5 cm deep and 0.5 cm in width. After sterilization of the ring plate at 123 °C under pressurized steam, wells were filled with minimal medium (MM) (de Vries et al., 2004) using 25 mM xylose as carbon source. A porous polycarbonate membrane (diameter 76 mm, thickness 5–10 μm, 6×10 8 pores cm - 2 , pore size 0.1 μm; GE Osmonics, USA) was placed on top of the ring plate and 1.5 μl of spore suspension (10 8 spores μl - 1 ) of A. niger strain N402 (Bos et al., 1988) was inoculated in the center. Cultures were grown at 30 °C under constant light in a box with water- saturated air. Experiments were replicated as biological triplicates and representative results have been presented below. Journal of Microbiological Methods 69 (2007) 399 – 401 www.elsevier.com/locate/jmicmeth ⁎ Corresponding author. Tel.: +31 30 2533448; fax: +31 30 2513655. E-mail address: h.a.b.wosten@bio.uu.nl (H.A.B. Wösten). 0167-7012/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2007.01.003