Abstract
Virus-induced gene silencing (VIGS) is a widely used, powerful technique for reverse genetics. VIGS vectors derived from the Tobacco rattle virus (TRV) are among the most popular for VIGS. We have developed a TRV RNA2 vector that allows the insertion of gene silencing fragments by ligation-independent cloning (LIC). This new vector has several advantages over previous vectors, particularly for applications involving the analysis of large numbers of sequences, since TRV-LIC vectors containing the desired insert are obtained with 100% efficiency. Importantly, this vector allows the high-throughput cloning of silencing fragments without the use of costly enzymes required for recombination, as is the case with GATEWAY-based vectors. We generated a collection of silencing vectors based on 400 tomato (Solanum lycopersicum) expressed sequence tags in this TRV-LIC background. We have used this vector to identify roles for SlMADS1 and its Nicotiana benthamiana homologs, NbMADS4-1 and -2 in flowering. We find that NbMADS4-1 and -2 act nonredundantly in floral development and silencing of either gene results in loss of organ identity. This TRV-LIC vector should be a valuable resource to the plant community.
Highlights
Virus-induced gene silencing (VIGS) is a widely used, powerful technique for reverse genetics
It was originally described for use in tobacco (Nicotiana tabacum) and its relative Nicotiana benthamiana (Kumagai et al, 1995; Ruiz et al, 1998), but VIGS has been adopted for gene analysis in many dicotyledonous species including Arabidopsis (Burch-Smith et al, 2006; Wang et al, 2006), potato (Solanum tuberosum; Brigneti et al, 2004), tomato (Liu et al, 2002a), and pepper (Capsicum annum; Chung et al, 2004)
We report here the construction of a Tobacco rattle virus (TRV)-ligation-independent cloning (LIC) vector that facilitates the high-throughput cloning of silencing fragments
Summary
Virus-induced gene silencing (VIGS) is a widely used, powerful technique for reverse genetics. We generated a collection of silencing vectors based on 400 tomato (Solanum lycopersicum) expressed sequence tags in this TRV-LIC background. We have used this vector to identify roles for SlMADS1 and its Nicotiana benthamiana homologs, NbMADS4-1 and -2 in flowering. We find that NbMADS4-1 and -2 act nonredundantly in floral development and silencing of either gene results in loss of organ identity This TRV-LIC vector should be a valuable resource to the plant community. RNA silencing is a wellcharacterized, endogenous system for monitoring RNA inside a cell and eliminating foreign molecules or inhibiting mRNA translation (for review, see Brodersen and Voinnet, 2006) It is a homology-based process that uses small RNA fragments to identify targets for destruction or inhibition. VIGS has been developed for use in monocots and it has been described in barley (Hordeum vulgare; Holzberg et al, 2002; Hein et al, 2005), wheat (Triticum aestivum; Scofield et al, 2005), and rice (Ding et al, 2006, 2007)
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