Elsevier

Virus Research

Volume 125, Issue 2, May 2007, Pages 169-175
Virus Research

Efficient gene silencing induction in tomato by a viral satellite DNA vector

https://doi.org/10.1016/j.virusres.2006.12.016Get rights and content

Abstract

Virus-induced gene silencing (VIGS) in tomato (Lycopersicon esculentum Mill.) is currently routinely analysed using Tobacco rattle virus (TRV)-based vector. We recently reported a new vector system modified from DNAβ (DNAmβ) of Tomato yellow leaf curl China virus (TYLCCNV) for VIGS analysis in Solanaceous species including tomato. Here, we describe DNAmβ-induced gene silencing in tomato. We found that DNAmβ-induced gene silencing was initiated from vascular tissues, and later scattered to other tissues. Once initiated in seedlings, the silencing phenotype lasted for the entire life span of the plants, was expressed in a variety of tissues and organs including leaf, shoot, stem, flower and fruit, and could be achieved at any growth stage. It was insensitive to temperature as high as 32 °C and no symptoms were observed in silenced plants. The DNAmβ vector worked efficiently in at least seven tomato cultivars, indicating that this system has great potential as a versatile VIGS system for routine functional analysis of genes in tomato.

Introduction

Virus-induced gene silencing (VIGS) is a technology that employs recombinant viruses to knock down expression of plant endogenous genes involving a homology-based degradation mechanism triggered by double stranded ribonucleic acid (RNA) molecules (Baulcombe, 1999). VIGS has obvious advantages over other known approaches for gene function analysis. It does not require development of stable genetic transformants, and thus is rapid and less laborious. It can be applied for functional analysis of genes for which only partial sequences are available, such as expressed sequence tags (ESTs). It also allows characterization of phenotypes that are lethal in stable lines, and is able to silence either individual or multiple members of a gene family (Burch-Smith et al., 2004, Lu et al., 2003).

Over 10 VIGS vectors have been reported (Burch-Smith et al., 2004, Constantin et al., 2004, Fofana et al., 2004, Tao and Zhou, 2004). Most of them are applicable to Solanaceous species, particularly in Nicotiana benthamiana (Brigneti et al., 2004, Burch-Smith et al., 2004, Faivre-Rampant et al., 2004, Valentine et al., 2004), while Barley stripe mosaic virus vector works in monocotyls barley and wheat (Hein et al., 2005, Holzberg et al., 2002, Lacomme et al., 2003, Scofield et al., 2005).

VIGS in tomato (Lycopersicon esculentum Mill.) has been achieved using Tobacco rattle virus (TRV)-based vectors in different tomato tissues and organs including leaf, root and fruit (Ekengren et al., 2003, Fu et al., 2005, Liu et al., 2002, Ryu et al., 2004, Valentine et al., 2004). It is highly efficient with only mild viral symptom, and shows great persistency and consistent observations between experiments (Burch-Smith et al., 2004, Ekengren et al., 2003). However, this vector system is efficient only when the growth temperature is lower than 22 °C (Burch-Smith et al., 2004, Ekengren et al., 2003).

Germiniviruses have been successfully modified into VIGS vectors (Carrillo-Tripp et al., 2006). Most germiniviral VIGS vectors are derived from the DNA components of bipartite begomoviruses, which include Tomato golden mosaic virus (TGMV) (Kjemtrup et al., 1998, Peele et al., 2001), Cabbage leaf curl virus (CaLCuV) (Turnage et al., 2002) and African cassava mosaic virus (ACMV) (Fofana et al., 2004). High temperature is not permissive for TRV-mediated VIGS, but is effective for ACMV-mediated VIGS (Chellapan et al., 2005). We recently reported a new germinivirus vector system modified from DNAβ associated with a monopartite begomovirus Tomato yellow leaf curl China virus (TYLCCNV) (DNAmβ), which is efficient for VIGS analysis in Solanaceous species including tomato (Tao and Zhou, 2004). In this study, we evaluated the potential of DNAmβ as a VIGS system for routine functional analysis of genes in tomato, by detailing DNAmβ-induced gene silencing in tomato in relation to aspects of developmental dynamics, persistency, cultivar permissiveness, growth stage, tissues and organs for expression of silencing phenotype and sensitivity to high temperature.

Section snippets

Plant materials and growth conditions

Seeds of tomato cultivars, Moneymaker, Pearson, Rio Grande, and four Chinese hybrid cultivars, Zao Feng, Zao Kui, Li Chun and He Zuo 903, were sown in small pots, and grown in plant growth chambers at 22 °C with a 16 h/8 h light/dark regime. Plants older than eight-leaf stage were transferred to greenhouse under the conditions of 22–25 °C with a 16 h/8 h light/dark regime.

Construction of the gene silencing constructs

A 351-bp fragment of magnesium chelatase subunit (ChlI otholog, also called Su) gene (BT012789:155-505) was PCR-amplified from

Development of gene silencing in tomato leaves induced by DNAmβ

The ChlI otholog (ChlI) encodes a component of the magnesium chelatase complex required for chlorophyll production. Silencing of the ChlI gene leads to blocking of chlorophyll production, making the tissue turning yellow initially and later white (Robertson, 2004). The plasmids DNAmβ-ChlI and the control TRV-ChlI both containing a 351-bp fragment of the tomato ChlI gene were delivered by agro-injection or agro-infiltration into tomato plants.

In DNAmβ-ChlI-silenced plants, yellowing phenotype

Discussion

Tomato is an economically important crop, as well as one of the model plants for biological research. Exploitation of VIGS, a robust functional genomic tool in tomato has recently received great attention. However, despite the fact that over 10 VIGS vectors have been reported (Burch-Smith et al., 2004, Constantin et al., 2004, Fofana et al., 2004), only TRV has been successfully used in tomato VIGS analysis before DNAmβ was reported. TRV-induced gene silencing can be applied to different

Acknowledgements

The TRV silencing vector used in this study is kindly provided by Dr. S.P. Dinesh-Kumar (Yale University, USA). This work was financially supported by the Cultivation Fund of the Key Scientific and Technical Innovation Project, Ministry of Education of China (No. 705025), the National Basic Research Program of China (No. 2006CB101903), the Fok Ying Tong Education Foundation (No. 101032), and the China National Natural Science Foundation (No. 30671352).

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    1

    These authors contributed equally to this work.

    2

    Present address: College of Life and Environmental Science, Zhejiang Normal University, Jinhua 321004, PR China.

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