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Research Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo, Japan (Received on November 20, 2011; Revised on November 28, 2011; Accepted on November 28, 2011)We developed a reassortant RNA virus vector derivedfrom Cucumber mosaic virus (CMV), which has advant-ages of very wide host range and can efficiently inducegene silencing in a few model plants. Certain CMVisolates, however, show limited host ranges presumablybecause they naturally co-evolved with their own hosts.We used a reassortant comprised of two strains of CMV,Y-CMV and Gn-CMV, to broaden the host range and todevelop a virus vector for virus-induced gene silencing(VIGS). Gn-CMV could infect chili pepper and tomatomore efficiently than Y-CMV. Gn-CMV RNA1, 3 andY-CMV RNA2-A1 vector were newly reconstructed,and the transcript mixture of RNA1 and 3 genomes ofGn-CMV and RNA2 genome of Y-CMV RNA2 con-taining portions of the endogenous phytoene desaturase(PDS) gene (CMV2A1::PDSs) was inoculated onto chilipepper (cv. Chung-yang), tomato (cvs. Bloody butcher,Tigerella, Silvery fir tree, and Czech bush) and Nicotianabenthamiana. All the tested plants infected by the reas-sortant CMV vector showed typical photo-bleachingphenotypes and reduced expression levels of PDSmRNA. These results suggest that the reassortant CMVvector would be a useful tool for the rapid induction ofthe RNA silencing of endogenous genes in chili pepperand tomato plants.Keywords : Cucumber mosaic virus vector, endogenousgene, phytoene desaturase, RNA silencingIn plants, the RNA silencing mechanism is conserved andresults from sequence-specific degradation of target RNAs,which has an essential role in most development stages.RNA silencing depends on small RNAs (21 to 24 nt),which are the key regulators of RNA silencing pathways ineukaryotic organisms (Fire et al., 1998; Pickford and Cogoni,2003; Vance and Voucheret, 2001; Waterhouse et al., 2001).Recently the transcription of double-stranded RNA (dsRNA)was shown to effectively down-regulate a gene (Hamiltonet al., 1998; Waterhouse et al., 1998). When viruse wasmanipulated to carry a region of the host gene sequences,the process can also be targeted against the correspondingmRNA. This is called virus-induced gene silencing (VIGS).After virus replication, replicative form (dsRNA) inter-mediates can trigger the RNA-mediated defense mechanism,and dsRNA intermediates are processed into short inter-fering RNAs (siRNAs). These small RNAs are producedfrom dsRNA by Dicer-like (DCL) enzymes, and act asguides in RNA-induced silencing complexes (RISCs) totarget RNA or DNA sequences (Bernstein et al., 2001;Hamilton and Baulcombe, 1999; Hammond et al., 2000;Zamore et al., 2000). The phenotypic changes in the infect-ed plant reflect the loss of expression of the encoded protein(Lu et al., 2003). Over the past 15 years, several RNA virusvectors have been developed for VIGS. These includeTobacco mosaic virus (Kumagai et al., 1995), Potato virusX (Ruiz et al., 1998), Tobacco rattle virus (Liu et al., 2002;Ratcliff et al., 1999), Barley stripe mosaic virus (Holzverg,2002), Pea early browning virus (Constantin et al., 2004),Bean pod mottle virus (Zhang and Ghabrial, 2006),Cucumber mosaic virus (Nagamatsu, 2007; Otagaki et al.,2006), and Pepper mottle mosaic virus (Lee et al., 2011).VIGS has many advantages over other methods, in which itis rapid and does not require a recalcitrant plant trans-formation (Burch-smith et al., 2004). VIGS has beensuccessfully applied to dicotyledonous species includingArabidopsis (Burch-Smith et al., 2006), potato (Brigneti etal., 2004), tomato (Liu et al., 2002), pepper (Chung et al.,2004) and to several monocots including barley (Holzberget al., 2002; Hein et al., 2005), wheat (Scofield et al., 2005)and rice (Ding et al., 2006; Ding et al., 2007). Cucumber mosaic virus (CMV) is one of the well-characterized positive-sense single stranded RNA viruses

Application of a Reassortant Cucumber mosaic virus Vector for Gene Silencing in Tomato and Chili Pepper Plants