Title

A novel co-delivery system consisting of a Tomato bushy stunt virus and a defective interfering RNA for studying gene silencing

Abstract

Virus induced gene silencing (VIGS) and suppression are RNA-specific defense and counter-defense circuits in plant–virus interactions. These phenomena have been investigated extensively with an Agrobacterium-mediated transient expression system. In this study, a virus-based transient expression system was developed to study these phenomena. A Tomato bushy stunt virus (TBSV) viral vector with an inactivated P19 suppressor gene, referred to as pHST2-14, was chosen to express the P1 of Tobacco etch virus (TEV). TEV P1 is a component of a well-characterized VIGS suppressor, TEV P1/HC-Pro protein. A TBSV defective interfering RNA (DI) that contains the 3′ proximal portion of a green fluorescence protein (GFP) gene, DI-P, was used as a silencing inducer of the homologous GFP gene on GFP transgenic Nicotiana benthamiana (NbGFP) plants. The TEV P1 gene was inserted into pHST2-14 to generate TBSV-P1. Transcripts of TBSV-P1 were then mixed with DI-P transcripts and inoculated onto NbGFP plants. DI-P consistently accumulated in NbGFP plants that were inoculated with TBSV-P1 and DI-P, and efficiently induced silencing of GFP transgene. These results demonstrate that a TBSV-based co-delivery system can provide a new alternative tool to investigate gene silencing and its influence by a TBSV-expressed foreign protein. It also can be used to elucidate functions of endogenous genes in plants.

Department(s)

Environmental Plant Science and Natural Resources

Document Type

Article

DOI

https://doi.org/10.1016/s0166-0934(03)00149-6

Keywords

tomato bushy stunt virus, defective interfering RNAs, virus induced gene silencing, DI-induced gene silencing

Publication Date

2003

Journal Title

Journal of virological methods

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