Grapevine virus A-mediated gene silencing in Nicotiana benthamiana and Vitis vinifera
Introduction
Infection of a plant by a virus often triggers a defense response and activates post-transcriptional gene silencing in the plant, a key mechanism for protecting plants against viral invasion. In this virus-induced gene silencing (VIGS) response, the plant defense system specifically targets RNAs derived from the viral genome for degradation (Bernstein et al., 2001, Nykanen et al., 2001). As a consequence, if the virus is harboring a fragment of a plant gene in its genome, the defense system will trigger the destruction of the corresponding plant mRNA, as well. On this basis, in recent years, genomes of plant viruses have been widely utilized to knock down expression of either transgenes or endogenous genes, and have been proven to be important tools for the analysis of gene function in plants (Atkinson et al., 1998, Baulcombe, 1999, Burton et al., 2000, Lu et al., 2003, Burch-Smith et al., 2004). Although the number of developed VIGS vectors has increased in recent years, most of these vectors are meant to be used in annual or herbaceous plants (Kumagai et al., 1995, Ruiz et al., 1998, Ratcliff et al., 2001, Holzberg et al., 2002, Brigneti et al., 2004). However, new virus vectors are required to expand the application of VIGS to a wider range of plants. The aim of this study was to test whether the Grapevine virus A (GVA) could be used as a VIGS vector.
GVA is closely associated with the economically important rugose wood (RW) disease of grapevine, specifically with Kober stem grooving (Garau et al., 1994, Chevalier et al., 1995, Chevalier et al., 1997). This virus, which is spread through infected propagation plant materials and by mealybugs, is a member of the genus Vitivirus, family Flexiviridae (Martelli et al., 2007). It is a filamentous particle about 800 nm long, and is considered to be a phloem-associated virus. The GVA genome (∼7.4 kb) consists of five open reading frames (ORFs; Galiakparov et al., 1999, Saldarelli et al., 2000, Galiakparov et al., 2003c). ORF1, located at the 5′-terminus of the genome, encodes a 194-kDa polypeptide with conserved motifs of replication-related proteins. ORF2 encodes a ∼20-kDa protein whose function is unknown. ORF3 is the movement protein (MP) gene, ORF4 encodes the coat protein (CP) and ORF5 encodes a small protein that exhibits sequence similarities to small RNA binding proteins of various plant viruses (Galiakparov et al., 2003b) and suppresses RNA silencing (Chiba et al., 2006, Zhou et al., 2006).
Recently a GVA-derived expression vector was developed and used to express the beta-glucuronidase (GUS) gene in Nicotiana benthamiana plants (Haviv et al., 2006). Infection of Vitis vinifera plants with the GVA vector has not been developed so far. Infection of this host with a cloned infectious cDNA of GVA is difficult to accomplish using the simple agro-infiltration methods that are used for most plant viruses. The present study describes: (i) the use of the GVA-derived vector for silencing the endogenous phytoene desaturase (PDS) gene in N. benthamiana, (ii) an Agrobacterium-mediated method developed for inoculating in vitro-propagated V. vinifera plantlets with cloned infectious GVA cDNAs, through their roots, and (iii) the use of the method developed for applying the GVA-derived vector for silencing the endogenous PDS gene in V. vinifera plantlets.
Section snippets
Plant material
N. benthamiana and V. vinifera cv. Prime were used in this study. N. benthamiana plants were grown in pots under greenhouse conditions: 25 °C; a 16-h light cycle and 60% humidity. V. vinifera plantlets, which were obtained from plant material treated by cryopreservation to ensure virus elimination, were micropropagated and maintained at a temperature of 26 °C under a 16-h photoperiod, as described by Wang et al. (2003).
GVA constructs
Fig. 1 presents schematic maps of the GVA-derived constructs used in this
VIGS in N. benthamiana
The vector GVA-118, described previously by Haviv et al. (2006), was designed to express the foreign gene through the MP-sgRNA promoter. A 500 nts product of the PDS gene of N. benthamiana (NbPDS) was PCR-amplified and cloned between the Not I and Apa I cleavage sites of the GVA-118. The resulting GVA-NbPDS cDNA was then inserted into the pCAMBIA2301 to generate pGVA-nbPDS-349, as described in Materials and Methods (Fig. 1). Plants of N. benthamiana were inoculated with Agrobacterium carrying
Conclusions
VIGS is considered to be a powerful tool for studying the functions of host genes (Burch-Smith et al., 2004). Until recently, the only VIGS vectors available were suitable mainly for use in annual plants. To the best of our knowledge, the GVA-derived VIGS vector is the first to be used for silencing the endogenous PDS genes in both N. benthamiana and V. vinifera plants. Unlike in most of the known VIGS vectors, silencing of the PDS gene in N. benthamiana via the GVA-derived VIGS vector is
Acknowledgements
This research was supported by grants IS-3784-05 and CB-9020-05 from BARD, the United States–Israel Binational Agricultural Research and Development Fund, and by grant number 565/05 from the Israeli Science Foundation. This report was approved for publication as Agricultural Research Organization, Journal Series No. 508/08.
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