title: 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.
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.
authors.affiliation: Peking-Yale Joint Center of Plant Molecular Genetics and Agrobiotechnology, College of Life Sciences, Peking University, Beijing 100871, China.
authors.forename: Yiyu
authors.initials: Y
authors.lastname: Dong
authors.forename: Tessa M
authors.initials: TM
authors.lastname: Burch-Smith
authors.forename: Yule
authors.initials: Y
authors.lastname: Liu
authors.forename: Padmavathi
authors.initials: P
authors.lastname: Mamillapalli
authors.forename: Savithramma P
authors.initials: SP
authors.lastname: Dinesh-Kumar
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D020224:Expressed Sequence Tags; D035264:Flowers / Q000254:growth & development*; D020868:Gene Silencing; D005801:Genes, Homeobox; D005822:Genetic Vectors; D018551:Solanum lycopersicum / Q000235:genetics; D026161:MADS Domain Proteins / Q000502:physiology*; D010641:Phenotype; D010942:Plant Viruses / Q000235:genetics*; D014026:Nicotiana / Q000235:genetics* / Q000254:growth & development / Q000821:virology
chemical_list: D026161:MADS Domain Proteins