A putative DEAD-box RNA-helicase is required for normal zoospore development in the late blight pathogen Phytophthora infestans Claire A. Walker a , Maico Köppe a , Laura J. Grenville-Briggs a , Anna O. Avrova b , Neil R. Horner a , Alastair D. McKinnon c , Stephen C. Whisson b , Paul R.J. Birch b , Pieter van West a, * a Aberdeen Oomycete Group, College of Life Science and Medicine, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland, UK b Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, Scotland, UK c Histology and Electron Microscopy Facility, College of Life Science and Medicine, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland, UK article info Article history: Received 22 November 2007 Accepted 12 March 2008 Available online 20 March 2008 Keywords: Phytophthora infestans Oomycete RNA-helicase Zoospore Pathogen Development abstract The asexual multinucleated sporangia of Phytophthora infestans can germinate directly through a germ tube or indirectly by releasing zoospores. The molecular mechanisms controlling sporangial cytokinesis or sporangial cleavage, and zoospore release are largely unknown. Sporangial cleavage is initiated by a cold shock that eventually compartmentalizes single nuclei within each zoospore. Comparison of EST rep- resentation in different cDNA libraries revealed a putative ATP-dependent DEAD-box RNA-helicase gene in P. infestans, Pi-RNH1, which has a 140-fold increased expression level in young zoospores compared to uncleaved sporangia. RNA interference was employed to determine the role of Pi-RNH1 in zoospore devel- opment. Silencing efficiencies of up to 99% were achieved in some transiently-silenced lines. These Pi- RNH1-silenced lines produced large aberrant zoospores that had undergone partial cleavage and often had multiple flagella on their surface. Transmission electron microscopy revealed that cytoplasmic ves- icles fused in the silenced lines, resulting in the formation of large vesicles. The Pi-RNH1-silenced zoo- spores were also sensitive to osmotic pressure and often ruptured upon release from the sporangia. These findings indicate that Pi-RNH1 has a major function in zoospore development and its potential role in cytokinesis is discussed. Ó 2008 Elsevier Inc. All rights reserved. 1. Introduction Oomycetes cause destructive diseases of plants, insects, crusta- ceans, fish and vertebrate animals. Among the oomycetes, Phytoph- thora spp. are arguably the most economically significant pathogens of dicotyledenous plants. The most notorious is Phy- tophthora infestans (Mont.) de Bary, which causes late blight on potatoes and blight on tomatoes, resulting in global losses exceed- ing US$ 5 billion per year (Duncan, 1999). Moreover, the genus in- cludes devastating wide host-range pathogens that threaten natural vegetation, such as P. ramorum that has decimated oak for- ests in California (Appiah et al., 2004). Although they posses a fila- mentous growth habit, they are distantly related to fungi and instead are more closely related to brown algae in the group of Stramenopiles. Oomycetes have thus evolved distinct genetic and biochemical mechanisms for infection (Kamoun, 2003). Infection by Phytophthora spp. typically initiates when sporan- gia release motile, biflagellate zoospores. Zoospores are essential for the disease cycles of many oomycete pathogens and are often the first point-of-contact with the host. Currently, little is known about the molecular biology of zoospore development or the regu- lation of its various stages. On short exposure to low temperature (cold shock) multinucleated sporangia rapidly differentiate by cytoplasmic cleavage to form several zoospores, which are released from the sporangial apex and exhibit an a-helical swimming pat- tern. Understanding the mechanisms underlying the rapidity by which this process occurs is a major goal of our research. Zoospores serve as infectious agents and can swim for hours in the presence of an endogenous food reserve (Carlile, 1986). They remain motile until encystment and can display several tactic behaviors (Deacon and Donaldson, 1993; Hill et al., 1998; Griffith et al., 1988; van West et al., 2002). It is thought that two separate processes, zoospore taxis and zoospore immobilization, define the targeting of host tissue by zoospores. Only directional swimming of zoospores towards chemical, nutrient, ionic or electrical gradi- ents constitutes a genuine tactical response (van West et al., 2002; Appiah et al., 2005). On reaching a host, zoospores encyst. Encystment is a very fast process involving flagella detachment and primary cell wall forma- tion (Griffith et al., 1988). Cysts form a germ tube, which usually differentiates into an appressorium (Grenville-Briggs et al., 2008). The appressorium forms a penetration peg that penetrates the epi- dermal cell layer. Subsequently, during P. infestans leaf infection, 1087-1845/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2008.03.004 * Corresponding author. Fax: +44 1224 555844. E-mail address: p.vanwest@abdn.ac.uk (P. van West). Fungal Genetics and Biology 45 (2008) 954–962 Contents lists available at ScienceDirect Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi