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ARS Home » Northeast Area » Washington, D.C. » National Arboretum » Floral and Nursery Plants Research » Research » Publications at this Location » Publication #321366

Title: Utilization of a tobacco rattle virus vector to clone an Nicotiana benthamiana cDNA library for VIGS

item SEO, EUN-YOUNG - Chungnam National University
item KIM, HYUN-SEUNG - Chungnam National University
item KIM, JUNG-KYU - Chungnam National University
item GOTOH, TAKAFUMI - Kyushu University
item Hammond, John
item LIM, HYOUN-SUB - Chungnam National University

Submitted to: Journal of Faculty of Agriculture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/19/2015
Publication Date: 10/13/2015
Citation: Seo, E., Kim, H., Kim, J., Gotoh, T., Hammond, J., Lim, H. 2015. Utilization of a tobacco rattle virus vector to clone an Nicotiana benthamiana cDNA library for VIGS. Journal of Faculty of Agriculture. 60:331-337.

Interpretive Summary: Genome sequencing can reveal the entire genome of important crop plants, but does not reveal the function of genes not previously characterized in another organism. Reverse genetics by means of Virus-Induced Gene Silencing (VIGS) is a technique for rapid phenotypic assessment by reducing the levels of expression of individual genes, thus potentially allowing rapid identification of gene function. A Tobacco rattle virus VIGS vector was utilized to examine visually distinct phenotypes in the model plant Nicotiana benthamiana. Three VIGS cDNA clones inducing leaf formation and necrosis were identified; two were shown to be derived from previously uncharacterized genes, whereas the third had been identified from the related Nicotiana tabacum as a gene that is expressed after cold treatment. The induction of necrosis is consistent with the prior identification as an oxidative stress response gene, which is known to be encoded in the chloroplast genome. These results demonstrate that a high-throughput system for reverse genetics has been developed and can be applied to gene function discovery; this is of potential value to plant scientists working with many crops, including crops for which the full genome is not yet available.

Technical Abstract: Virus-induced gene silencing (VIGS) is an efficient and rapid method to identify plant gene functions. One of the most widely used VIGS vectors is Tobacco rattle virus (TRV) which has been used successfully for RNA interference (RNAi) in N. benthamiana and tomato. We previously modified a TRV VIGS vector to contain the Gateway system for high throughput cloning, and utilized this system to express a library of N. benthamiana cDNA. Random c.300 bp N. benthamiana cDNA fragments were generated by ultrasonication and inserted into the TRV VIGS vector by Gateway cloning. N. benthamiana were agroinfiltrated with randomly selected TRV cDNA constructs in Agrobacterium tumefaciens GV 2260. Distinct visible phenotypes were identified in three sets of the inoculated N. benthamiana plants. The three distinguished phenotypes showed leaf malformation and necrosis. The three expressed gene inserts were homologous to EST fragments identified as CK290013.1, CK296346.1, and AM8112161.1, and presumably these genes are related to TRV pathogenesis in N. benthamiana. Identification of the selected genes by VIGS will aid further analysis to determine the relationship between VIGS phenotype and gene function.