Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus. One of the plant glutathione S-transferase (GST) genes, NbGSTU4, responds as an upregulated gene in Nicotiana benthamiana post BaMV infection. In order to identify the role of NbGSTU4 in BaMV infection, the expression of NbGSTU4 was knocked down using a virus-induced gene silencing technique or was transiently expressed in N. benthamiana in BaMV inoculation. The results show a significant decrease in BaMV RNA accumulation when the expression level of NbGSTU4 is reduced; whereas the viral RNA accumulation increases when NbGSTU4 is transiently expressed. Furthermore, this study identified that the involvement of NbGSTU4 in viral RNA accumulation occurs by its participation in the viral early replication step. The findings show that the NbGSTU4 protein expressed from Escherichia coli can interact with the 3′ untranslated region (UTR) of the BaMV RNA in vitro in the presence of glutathione (GSH). The addition of GSH in the in vitro replication assay shows an enhancement of minus-strand but not plus-strand RNA synthesis. The results suggest that the plant GST protein plays a role in binding viral RNA and delivering GSH to the replication complex to create a reduced condition for BaMV minus-strand RNA synthesis. Keywords: Bamboo mosaic virus (BaMV), glutathione (GSH), glutathione S-transferase (GST), in vitro RNA replication, redox, viral RNA replication, virus-induced gene silencing (VIGS) Introduction Glutathione S-transferases (GSTs) are grouped into a large family of both eukaryotes and prokaryotes and catalyze a variety of reactions (Sheehan et al., 2001; Dixon et al., 2002; Allocati et al., 2009). Conventionally, GSTs catalyze the transfer of a reduced tripeptide glutathione (GSH; glutamyl-cysteinyl-glycine) to various substrates containing a reactive electrophilic center, to form a polar S-glutathionylated product for reducing oxidative stress (Dixon et al., 2002). Plant GSTs were first recognized in 1970, and have been proven to play a role in the detoxification occurring in herbicide injuries (Frear & Swanson 1970). These plant GSTs are classified into eight distinct groups: phi, tau, theta, zeta, lambda, DHAR, TCHQD and microsomal (Edwards & Dixon, 2005). As more plant genomes are sequenced, the list of GSTs continues to grow. In Arabidopsis, 48 GST-like genes were grouped into 28 tau, 13 phi, 3 theta, 2 zeta and 2 lambda GSTs (Dixon et al., 2002); 42 maize GST-like genes were grouped into 12 phi, 28 tau and 2 zeta GSTs; and 25 soybean GST-like genes were grouped into 20 tau, 1 zeta and 4 phi GSTs (McGonigle et al., 2000). The members of the phi and tau family are specific to plants and are the most abundant. They are induced by various treatments that induce general oxidative stress, such as osmotic stress, temperature stress and chemical toxins (Mauch & Dudler, 1993; Marrs, 1996). Furthermore, the role of tau GSTs has been characterized in the tolerance to cold temperature and the oxidative stress that occurs when they are overexpressed in tobacco seedlings (Roxas et al., 1997). Similar responses were also observed in yeast (Kampranis et al., 2000; Kilili et al., 2004). These observations suggest that the tau GSTs can play a significant role in protecting plants against oxidative stress. Moreover, three tau GSTs (NbGSTU1, NbGSTU2 and NbGSTU3) and one phi GST (NbGSTF1) from Nicotiana benthamiana plants were examined for their roles in fungal infections (Dean et al., 2005). The expression levels of NbGSTU1 and NbGSTU3 were found to be upregulated post-infection, although those of NbGSTU2 and NbGSTF1 were unaffected. Further analysis revealed that only the NbGSTU1-knockdown plant showed more lesions compared to the others when inoculated with Colletotrichum orbiculare. These findings suggest that, although a relatively large number of different GSTs are present in plants, only a few are involved in disease development (Dean et al., 2005). Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus. An RNA genome of c. 6.4 kb with a 5′-cap and a 3′-poly(A) tail contains five open-reading frames (Lin et al., 1992, 1994). ORF1 encodes a 155 kDa replicase for viral RNA replication (Li et al., 2001; Huang et al., 2004). ORF2 to ORF4 encode movement proteins that participate in intra- and intercellular movement of the virus (Lin et al., 2004, 2006). ORF5 encodes the capsid protein for virus encapsidation. Potexvirus coat protein is also involved in virus movement (Cruz et al., 1998). The 3′ untranslated region (UTR) of BaMV has been structurally mapped (Cheng & Tsai, 1999); it contains cis-acting elements for the initiation of minus-strand RNA synthesis (Cheng & Tsai, 1999; Huang et al., 2001; Cheng et al., 2002), viral RNA long-distance movement (Chen et al., 2003), and the polyadenylation of plus-strand RNA (Chen et al., 2005). Furthermore, the host factor chloroplast phosphoglycerate kinase has been reported to interact with the 3′ UTR, and is essential for efficient BaMV accumulation in plants (Lin et al., 2007). A few differentially expressed genes from N. benthamiana plants infected with BaMV were recently identified in viral infection cycles (Cheng et al., 2010). One of these cDNA fragments had similarities to a GST from N. tabacum (Cheng et al., 2010). This study is involved in a cloning of the full-length new tau group GTS gene NbGSTU4 from the N. benthamiana plant. NbGSTU4 not only binds to the 3′ UTR of the BaMV RNA, but also enhances the viral RNA replication in vitro. This study details the role of the NbGSTU4 in BaMV infection cycle.