Grapevine virus A-mediated gene silencing in Nicotiana benthamiana and Vitis vinifera

https://doi.org/10.1016/j.jviromet.2008.10.010Get rights and content

Abstract

Virus-induced gene silencing (VIGS) is an attractive approach for studying gene function. Although the number of virus vectors available for use in VIGS experiments has increased in recent years, most of these vectors are applied in annual or herbaceous plants. The aim of this work was to develop a VIGS vector based on the Grapevine virus A (GVA), which is a member of the genus Vitivirus, family Flexiviridae. The GVA vector was used to silence the endogenous phytoene desaturase (PDS) gene in Nicotiana benthamiana plants. In addition, an Agrobacterium-mediated method for inoculating micropropagated Vitis vinifera cv. Prime plantlets via their roots was developed. Using this method, it was possible to silence the endogenous PDS gene in V. vinifera plantlets. The GVA-derived VIGS vector may constitute an important tool for improving functional genomics in V. 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.

References (32)

  • E. Bernstein et al.

    Role for a bidentate ribonuclease in the initiation step of RNA interference

    Nature

    (2001)
  • G. Brigneti et al.

    Virus-induced gene silencing in Solanum species

    Plant J.

    (2004)
  • T.M. Burch-Smith et al.

    Applications and advantages of virus-induced gene silencing for gene function studies in plants

    Plant J.

    (2004)
  • R.A. Burton et al.

    Virus-induced gene silencing of a plant cellulose synthase gene

    Plant Cell

    (2000)
  • S. Chang et al.

    A simple and efficient method for isolating RNA from pine trees

    Plant Mol. Biol. Rep.

    (1993)
  • S. Chevalier et al.

    Use of an immunocapture-polymerase chain reaction procedure for the detection of grapevine virus A in Kober stem grooving infected grapevines

    J. Phytopathol.

    (1995)
  • Cited by (0)

    View full text