REVIEW Gorou Horiguchi RNA silencing in plants: a shortcut to functional analysis Received October 31, 2003; accepted December 8, 2003 Abstract RNA silencing is a rapidly expanding research field, not only because it is a fundamental biological issue but also because its application in the control of gene expression is highly promising. Post- transcriptional gene silencing in plants is a form of RNA silencing by which target RNA is degraded in a sequence-specific manner. Findings regarding the cen- tral role that double-stranded RNA plays in triggering RNA silencing have prompted the development of many modified methods for RNA silencing. These methods, in combination with the development of genomic resources, have provided rapid and efficient means by which to investigate gene function in a wide range of plant species. This review addresses the technical aspects of RNA silencing in plants by introducing the principles of several methods of RNA silencing, as well as the advantages and disadvantages of each method. Key words dsRNA  inverted repeat  reverse genetics  RNA silencing  siRNA Introduction RNA silencing is a sequence-specific RNA degradation system that is conserved in a wide range of organisms, and termed post-transcriptional gene silencing (PTGS), RNA interference (RNAi), and quelling, in plants, animals, and fungi, respectively. Genetic and biochem- ical studies of different organisms have identified homologous genes involved in RNA silencing, such as those encoding RNA-dependent RNA polymerase (RdRP), RNase III-like endonuclease (Dicer), and PAZ/Piwi-domain protein (ARGONAUTE), suggesting that RNA silencing has an ancient origin (Cogoni and Macino, 1999; Catalanotto et al., 2000; Dalmay et al., 2000, 2001; Fagard et al., 2000; Hammond et al., 2000; Mourrain et al., 2000; Smardon et al., 2000; Bernstein et al., 2001; Knight and Bass, 2001; Martinez et al., 2002; summarized by Hutva´ gner and Zamore, 2002). These findings led to the establishment of a general model for RNA silencing consisting of three steps (Fig. 1). First, RNA silencing is initiated by the formation of double-stranded RNA (dsRNA). Second, dsRNA is processed by Dicer into 21- to 26-nucleotide (nt) dsRNA fragments called short interfering RNAs (siRNAs). Finally, the RNA-induced silencing complex (RISC) incorporates an siRNA and cleaves a target RNA that has a region complementary to the siRNA sequence (Hamilton and Baulcombe, 1999; Matzke et al., 2001a; Hannon, 2002; Hutva´ gner and Zamore, 2002). Investigations of the molecular mechanisms under- lying PTGS in plants have contributed to the identifica- tion of siRNA and genes involved in the RNA silencing pathway found commonly in eukaryotes and specifically in plants (Hamilton and Baulcombe, 1999; Dalmay et al., 2000, 2001; Fagard et al., 2000; Mourrain et al., 2000). These efforts have also provided evidence that in plants RNA silencing plays a role in defense against mobile genetic elements, such as viruses and transposons (Vance and Vaucheret, 2001; Waterhouse et al., 2001b; Baul- combe, 2002). In addition to the role of RNA silencing as a defense system, recent extensive identification of microRNAs (miRNAs) from various organisms, includ- ing plants, has added a novel role for RNA silencing, in that it is important for developmental control of gene expression (McConnell et al., 2001; Park et al., 2002; Schauer et al., 2002; Finnegan et al., 2003; Palatnik et al., 2003; Tang et al., 2003). For example, jaw-1D mutant of Arabidopsis thaliana exhibits abnormal leaf development due to the overexpressison of an miRNA (Fig. 2; Palatnik et al., 2003). A deeper understanding of the mechanisms of RNA silencing has stimulated its use as a reverse genetics tool, Gorou Horiguchi National Institute for Basic Biology/Center for Integrative Bioscience Myodaiji-cho, Okazaki 444-8585, Japan Tel: 181-564-55-7513 Fax: 181-564-55-7512 E-mail: ghori@nibb.ac.jp U.S. Copyright Clearance Center Code Statement: 0301–4681/2004/7202/3–65 $ 15.00/0 Differentiation (2004) 72:65–73 r International Society of Differentiation 2004