Tobacco rattle virus–based virus-induced gene silencing in Nicotiana benthamiana

• Published in: Nature protocols Vol. 9; no. 7; pp. 1549 - 1562
• Main Authors: Senthil-Kumar, Muthappa, Mysore, Kirankumar S
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.07.2014; Nature Publishing Group
• Subjects: 631/1647/1513; 631/208/191/505; 631/449/2491; Agrobacterium; Analysis; Analytical Chemistry; Biological Techniques; Cloning; Computational Biology/Bioinformatics; Flowers & plants; Gene expression; Gene Knockdown Techniques; Gene Silencing; Genetic aspects; Genetic Vectors; Genetically modified organisms; Genomes; Leaves; Life Sciences; Microarrays; MicroRNAs; Nicotiana - genetics; Nicotiana - virology; Nicotiana benthamiana; Organic Chemistry; Physiological aspects; Plant species; Plant Viruses - genetics; Plants; protocol; RNA polymerase; Tobacco; Tobacco (Plant); Tobacco rattle virus
• ISSN: 1754-2189; 1750-2799; 1750-2799
• DOI: 10.1038/nprot.2014.092

This Protocol describes how to downregulate specific plant genes using tobacco rattle virus virus-induced gene silencing (TRV-VIGS). The method can be used in a range of plants, but N. benthamiana is used here as an example. Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) is widely used in various plant species to downregulate the expression of a target plant gene. TRV is a bipartite, positive-strand RNA virus with the TRV1 and TRV2 genomes. To induce post-transcriptional gene silencing (PTGS), the TRV2 genome is genetically modified to carry a fragment of the target gene and delivered into the plant (along with the TRV1 genome) by agroinoculation. TRV1- and TRV2-carrying Agrobacterium strains are then co-inoculated into 3-week-old plant leaves by one of three methods: a needleless syringe, the agrodrench method or by pricking with a toothpick. Target gene silencing occurs in the newly developed noninoculated leaves within 2–3 weeks of TRV inoculation. The TRV-VIGS protocol described here takes only 4 weeks to implement, and it is faster and easier to perform than other gene silencing techniques that are currently available. Although we use Nicotiana benthamiana as an example, the protocol is adaptable to other plant species.