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BREAKTHROUGH TECHNOLOGIES

A Ligation-Independent Cloning Tobacco Rattle Virus Vector for High-Throughput Virus-Induced Gene Silencing Identifies Roles for NbMADS4-1 and -2 in Floral Development^1,[W],[OA]

Peking-Yale Joint Center of Plant Molecular Genetics and Agrobiotechnology, College of Life Sciences, Peking University, Beijing 100871, China (Y.D.); and Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520–8103 (Y.D., T.M.B.-S., Y.L., P.M., S.P.D.-K.)

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.

² Present address: Division of Oncology, Washington University, St. Louis, MO 63110.

³ Present address: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720.

⁴ Present address: Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, People's Republic of China.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Savithramma P. Dinesh-Kumar (savithramma.dinesh-kumar@yale.edu).

^[W] The online version of this article contains Web-only data.

^[OA] Open Access articles can be viewed online without a subscription.

www.plantphysiol.org/cgi/doi/10.1104/pp.107.107391

^* Corresponding author; e-mail savithramma.dinesh-kumar{at}yale.edu.

Received August 14, 2007; accepted September 25, 2007; published October 11, 2007.