Methodology

Development of a gene silencing DNA vector derived from a broad host range geminivirus

Edward M Golenberg¹ , D Noah Sather^1,2 , Leandria C Hancock^1,3 , Kenneth J Buckley⁴ , Natalie M Villafranco⁴ and David M Bisaro⁴

¹  Department of Biological Sciences, Wayne State University, Detroit, MI 48202, USA

²  Seattle Biomedical Research Institute, 307 Westlake Ave. N., Seattle, WA 98109, USA

³  Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, 3901 Rainbow Boulevard, MS1053, G017 Lied Building, Kansas City, KS 66160, USA

⁴  Department of Molecular Genetics and Plant Biotechnology Center, The Ohio State University, Columbus, OH 43210, USA

author email corresponding author email

Plant Methods 2009, 5:9doi:10.1186/1746-4811-5-9

Published:	2 July 2009

Abstract

Background

Gene silencing is proving to be a powerful tool for genetic, developmental, and physiological analyses. The use of viral induced gene silencing (VIGS) offers advantages to transgenic approaches as it can be potentially applied to non-model systems for which transgenic techniques are not readily available. However, many VIGS vectors are derived from Gemini viruses that have limited host ranges. We present a new, unipartite vector that is derived from a curtovirus that has a broad host range and will be amenable to use in many non-model systems.

Results

The construction of a gene silencing vector derived from the geminivirus Beet curly top virus (BCTV), named pWSRi, is reported. Two versions of the vector have been developed to allow application by biolistic techniques or by agro-infiltration. We demonstrate its ability to silence nuclear genes including ribulose bisphosphate carboxylase small subunit (rbcS), transketolase, the sulfur allele of magnesium chelatase (ChlI), and two homeotic transcription factors in spinach or tomato by generating gene-specific knock-down phenotypes. Onset of phenotypes occurred 3 to 12 weeks post-inoculation, depending on the target gene, in organs that developed after the application. The vector lacks movement genes and we found no evidence for significant spread from the site of inoculation. However, viral amplification in inoculated tissue was detected and is necessary for systemic silencing, suggesting that signals generated from active viral replicons are efficiently transported within the plant.

Conclusion

The unique properties of the pWSRi vector, the ability to silence genes in meristem tissue, the separation of virus and silencing phenotypes, and the broad natural host range of BCTV, suggest that it will have wide utility.