Method: low-cost delivery of the cotton leaf crumple virus-induced gene silencing system

John Tuttle,Candace H Haigler,Dominique Robertson

doi:10.1186/1746-4811-8-27

John Tuttle, Candace H Haigler + Show 1 more

Open Access

https://doi.org/10.1186/1746-4811-8-27

Copy DOI

Journal: Plant methods	Publication Date: Jan 1, 2012
Citations: 51	License type: cc-by

Affiliation: North Carolina State University

Abstract

BackgroundWe previously developed a virus-induced gene silencing (VIGS) vector for cotton from the bipartite geminivirusCotton leaf crumple virus (CLCrV). The original CLCrV VIGS vector was designed for biolistic delivery by a gene gun. This prerequisite limited the use of the system to labs with access to biolistic equipment. Here we describe the adaptation of this system for delivery by Agrobacterium (Agrobacterium tumefaciens). We also describe the construction of two low-cost particle inflow guns.ResultsThe biolistic CLCrV vector was transferred into two Agrobacterium binary plasmids. Agroinoculation of the binary plasmids into cotton resulted in silencing and GFP expression comparable to the biolistic vector. Two homemade low-cost gene guns were used to successfully inoculate cotton (G. hirsutum) and N. benthamiana with either the CLCrV VIGS vector or the Tomato golden mosaic virus (TGMV) VIGS vector respectively.ConclusionsThese innovations extend the versatility of CLCrV-based VIGS for analyzing gene function in cotton. The two low-cost gene guns make VIGS experiments affordable for both research and teaching labs by providing a working alternative to expensive commercial gene guns.

Highlights

We previously developed a virus-induced gene silencing (VIGS) vector for cotton from the bipartite geminivirusCotton leaf crumple virus (CLCrV)
When the virus infects the plant, it triggers post-transcriptional gene silencing (PTGS) against the viral genome as well as the included host sequence. This leads to the degradation of RNAs with homology to the viral genome and produces a knockdown phenotype for the targeted gene(s). Because it can rapidly silence genes without the need for stable transformation, VIGS has become an attractive alternative to other reverse genetics strategies, which are timeconsuming and especially difficult in plant species like cotton that are recalcitrant to transformation/regeneration
Two additional A-DNA plasmids, one for silencing Chelatase subunit I (ChlI) and one expressing GFP, were made by swapping XbaI/SacI fragments consisting of the 3' region of the AL1 gene, AL2, AL3, the multiple cloning site, and one of the two duplicated common regions with the same region from the biolistic vector (Figure 1). These plasmids will be referred to as CLCrVA:ChlI and CLCrVA:GFP, respectively

Summary

Introduction

We previously developed a virus-induced gene silencing (VIGS) vector for cotton from the bipartite geminivirusCotton leaf crumple virus (CLCrV). The original CLCrV VIGS vector was designed for biolistic delivery by a gene gun. When the virus infects the plant, it triggers PTGS against the viral genome as well as the included host sequence This leads to the degradation of RNAs with homology to the viral genome and produces a knockdown phenotype for the targeted gene(s). Because it can rapidly silence genes without the need for stable transformation, VIGS has become an attractive alternative to other reverse genetics strategies, which are timeconsuming and especially difficult in plant species like cotton that are recalcitrant to transformation/regeneration. Vectors have region between the replication origins is released in planta to form a functional viral episome [7,8]

Methods

Results

Conclusion