A Ligation-Independent Cloning Tobacco Rattle Virus Vector for High-Throughput Virus-Induced Gene Silencing Identifies Roles forNbMADS4-1and -2in Floral Development

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.The last decade has seen an explosion in the availability of plant gene sequences. The genomes of the model species Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) have both been sequenced, while those of tomato (Solanum lycopersicum) and maize (Zea mays) are currently being sequenced (Mueller et al., 2005; http://www.sgn.cornell.edu/about/tomato_project/; http://www.maizesequence.org/index.html). Large collections of ESTs have also been generated for a variety of species that are widely used for research purposes. Concomitant with the availability of this sequence information, many important aspects of plant growth and development have been analyzed by DNA microarrays, leading to the identification of numerous genes potentially involved in these processes. At this time then, the challenge to most plant biologists is to effectively mine this data to identify and characterize the genes and gene products that are critical to the crucial processes that have been investigated. This calls for techniques that start with a known DNA sequence and allow the determination of biological function. This approach is called reverse genetics and some of the most common methods for performing reverse genetic studies are based on RNA silencing.Although recently discovered, 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. RNA silencing is also indispensable for normal plant growth and development, regulating the expression of central genes in flowering, meristem identity, and other processes (Meins et al., 2005). In plants, RNA silencing plays critical roles in viral ...

A Root-Expressed l-Phenylalanine:4-Hydroxyphenylpyruvate Aminotransferase Is Required for Tropane Alkaloid Biosynthesis in Atropa belladonna

“…For the A. belladonna GenBank results, the percentage of GenBank entries with a match to the A. belladonna unigene set is reported. Ab-ArAT4 and Tropane Alkaloid BiosynthesisVIGS for functional genomics in A. belladonna was investigated using the TRV two-component system, with constructs assembled in the TRV2 vector by ligation-independent cloning (Dong et al, 2007). Initially, the effectiveness of VIGS in A. belladonna was assessed using constructs targeting PHYTOENE DESATURASE (PDS) and ACTIN.…”

“…VIGS experiments were performed using the two-component TRV system with target gene constructs assembled by ligation independent cloning in the TRV2-LIC vector (Ratcliff et al, 2001;Dong et al, 2007). PCR fragments of A. belladonna PDS, ACTIN, PMT, H6H, and ArATs were amplified from cDNA synthesized from RNA extracted from either the secondary roots or sterile seedlings of A. belladonna using primer pairs described in Supplemental Table 4.…”

“…In addition, a rapid, targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method for the simultaneous detection of hyoscyamine, scopolamine, and selected precursors in a single extract was developed, together with a tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) system, to facilitate functional analysis of candidate genes (Ratcliff et al, 2001;Liu et al, 2002;Dong et al, 2007). These resources were used to identify a phylogenetically distinct, root-expressed aromatic amino acid aminotransferase (ArAT), designated Ab-ArAT4, which preferentially catalyzes the transamination of phenylalanine (L-Phe) to phenylpyruvate, the initial step leading to formation of littorine, a key intermediate in hyoscyamine and scopolamine biosynthesis.…”

Virus-Based MicroRNA Silencing in Plants

“…Here, we modified the TRV RNA2-derived vector pYL170 (Dong et al, 2007) by inserting the PEBV CP subgenomic promoter (Wang et al, 1997) and the ccdB (for control of cell death B) gene with ligation-independent cloning (LIC) adaptor sequences immediately downstream of the TRV CP gene to generate the TRV expression vector pTRV2e (Fig. 1).…”

A Visual Reporter System for Virus-Induced Gene Silencing in Tomato Fruit Based on Anthocyanin Accumulation

“…With the exception of hypersus-ceptible Nicotiana benthamiana, the penetration of VIGS phenotypes in most plant species is only partial and shows a patchy tissue distribution. Tobacco rattle virus (TRV), for example, which drives a highly efficient VIGS in all newly developed organs of N. benthamiana (Ruiz et al, 1998;Ratcliff et al, 2001;Dong et al, 2007), works less efficiently in potato (Solanum tuberosum) and tomato (Liu et al, 2002;Brigneti et al, 2004;Ryu et al, 2004), probably because parts of the plant remain poorly silenced or not silenced at all (Rotenberg et al, 2006;Senthil-Kumar et al, 2007). Tomato plants inoculated at the cotyledon stages show silenced symptoms in about half of their fruit.…”