Technical advance RNA-mediated gene silencing in the cereal fungal pathogen Cochliobolus sativus YUEQIANG LENG 1 , CHENGXIANG WU 2 , ZHAOHUI LIU 1 , TIMOTHY L. FRIESEN 3 , JACK B. RASMUSSEN 1 AND SHAOBIN ZHONG 1, * 1 Department of Plant Pathology, North Dakota State University, Fargo, ND 58108, USA 2 Department of Microbiology, University of Hawaii at Manoa, Honolulu, HI, USA 3 USDA-ARS Cereal Crops Research Unit, Northern Crop Science Laboratory, Fargo, ND 58105, USA SUMMARY A high-throughput RNA-mediated gene silencing system was developed for Cochliobolus sativus (anamorph: Bipolaris soro- kiniana), the causal agent of spot blotch, common root rot and black point in barley and wheat. The green fluorescent protein gene (GFP) and the proteinaceous host-selective toxin gene (ToxA) were first introduced into C. sativus via the polyethylene glycol (PEG)-mediated transformation method. Transformants with a high level of expression of GFP or ToxA were generated. A silencing vector (pSGate1) based on the Gateway cloning system was developed and used to construct RNA interference (RNAi) vectors. Silencing of GFP and ToxA in the transformants was demonstrated by transformation with the RNAi construct expressing hairpin RNA (hpRNA) of the target gene. The polyketide synthase gene (CsPKS1), involved in melanin biosyn- thesis pathways in C. sativus, was also targeted by transforma- tion with the RNAi vector (pSGate1-CsPKS1) encoding hpRNA of the CsPKS1 gene. The transformants with pSGate1-CsPKS1 exhibited an albino phenotype or reduced melanization, suggest- ing effective silencing of the endogenous CsPKS1 in C. sativus. Sectors exhibiting the wild-type phenotype of the fungus appeared in some of the CsPKS1-silenced transformants after subcultures as a result of inactivation or deletions of the RNAi transgene. The gene silencing system established provides a useful tool for functional genomics studies in C. sativus and other filamentous fungi. INTRODUCTION RNA-mediated gene silencing or RNA interference (RNAi) has been found to be a common phenomenon in eukaryotic organisms. In this process, double-stranded RNA (dsRNA), which can be induced by viral RNA, hairpin RNA (hpRNA) or virus- induced gene silencing encoded RNA (Waterhouse and Helliwell, 2003), is degraded into small interfering RNAs (siRNAs). These siRNAs are incorporated into the RNA-induced silencing complex (RISC) to target and degrade the mRNA of genes with the complementary sequence (Liu et al., 2002; McDonald et al., 2005; Nakayashiki and Nguyen, 2008; Waterhouse and Helliwell, 2003). RNAi has been demonstrated to silence genes in a number of fungi (for a review, see Kück and Hoff, 2010). Examples include Cryptococcus neoformans (Liu et al., 2002), Magnaporthe oryzae (Jeon et al., 2007; Kadotani et al., 2003; Nakayashiki et al., 2005), Venturia inaequalis (Fitzgerald et al., 2004), Histoplasma capsulatum (Rappleye et al., 2004), Schizophyllum commune (de Jong et al., 2006), Coprinopsis cinerea (syn. Coprinus cinereus) (Namekawa et al., 2005; Wälti et al., 2006), Mortierella alpina (Takeno et al., 2005), Dictyostelium discoideum (Martens et al., 2002), Aspergillus and Fusarium species (McDonald et al., 2005), Bipolaris oryzae (Moriwaki et al., 2007) and Ophiostoma novo- ulmi (Carneiro et al., 2010). Most of the studies mentioned above used RNAi vectors containing inverted repeats of the target gene or its partial sequence for transformation. Construction of RNAi vectors often involves several cloning steps, and thus is laborious and time-consuming. Nakayashiki et al. (2005) developed a pHANNIBAL-like silencing vector, pSilent-1, for RNA silencing studies in filamentous fungi. The applicability of pSilent-1 was demonstrated in several phytopathogenic ascomycete fungi, including M. oryzae, Colletotrichum lagenarium and B. oryzae (Moriwaki et al., 2007; Nakayashiki et al., 2005). However, pSilent-1 is a restriction enzyme-based cloning vector and there- fore may not be suitable for the construction of large numbers of RNAi vectors for high-throughput gene silencing studies. Recently, a pHellsgate-like RNAi vector (pTroya) based on Invit- rogen’s (Carlsbad, CA, USA) Gateway technology has been devel- oped and shown to be useful in Colletotrichum gloeosporioides (Shafran et al., 2008). This type of RNAi vector should have wide application in many other plant pathogenic fungi. *Correspondence: Email: shaobin.zhong@ndsu.edu MOLECULAR PLANT PATHOLOGY (2011) 12 (3), 289–298 DOI: 10.1111/J.1364-3703.2010.00666.X MOLECULAR PLANT PATHOLOGY © 2010 BSPP AND BLACKWELL PUBLISHING LTD NO CLAIM TO ORIGINAL US GOVERNMENT WORKS 289