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Open Access Highly Accessed Research article

RNA viral vectors for improved Agrobacterium-mediated transient expression of heterologous proteins in Nicotiana benthamiana cell suspensions and hairy roots

Jeffrey S Larsen and Wayne R Curtis*

Author Affiliations

Department of Chemical Engineering, The Pennsylvania State University, University Park, Pennsylvania, 16802, USA

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BMC Biotechnology 2012, 12:21  doi:10.1186/1472-6750-12-21

Published: 6 May 2012

Abstract

Background

Plant cell suspensions and hairy root cultures represent scalable protein expression platforms. Low protein product titers have thus far limited the application of transient protein expression in these hosts. The objective of this work was to overcome this limitation by harnessing A. tumefaciens to deliver replicating and non-replicating RNA viral vectors in plant tissue co-cultures.

Results

Replicating vectors derived from Potato virus X (PVX) and Tobacco rattle virus (TRV) were modified to contain the reporter gene β-glucuronidase (GUS) with a plant intron to prevent bacterial expression. In cell suspensions, a minimal PVX vector retaining only the viral RNA polymerase gene yielded 6.6-fold more GUS than an analogous full-length PVX vector. Transient co-expression of the minimal PVX vector with P19 of Tomato bushy stunt virus or HC-Pro of Tobacco etch virus to suppress post-transcriptional gene silencing increased GUS expression by 44 and 83%, respectively. A non-replicating vector containing a leader sequence from Cowpea mosaic virus (CPMV-HT) modified for enhanced translation led to 70% higher transient GUS expression than a control treatment. In hairy roots, a TRV vector capable of systemic movement increased GUS accumulation by 150-fold relative to the analogous PVX vector. Histochemical staining for GUS in TRV-infected hairy roots revealed the capacity for achieving even higher productivity per unit biomass.

Conclusions

For the first time, replicating PVX vectors and a non-replicating CPMV-HT vector were successfully applied toward transient heterologous protein expression in cell suspensions. A replicating TRV vector achieved transient GUS expression levels in hairy roots more than an order of magnitude higher than the highest level previously reported with a viral vector delivered by A. tumefaciens.

Keywords:
Plant tissue culture; Gene silencing; Viral vectors; Hairy roots; Plant cell suspensions; Potato virus X; Tobacco rattle virus; Transient protein expression; Agrobacterium tumefaciens