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

Jeffrey S Larsen,Wayne R Curtis

doi:10.1186/1472-6750-12-21

Abstract

BackgroundPlant 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.ResultsReplicating 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.ConclusionsFor 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.

Highlights

Plant cell suspensions and hairy root cultures represent scalable protein expression platforms
In this study we evaluated the three RNA viral vectors described above for their potential to achieve rapid, high-level Agrobacterium-mediated transient protein expression in N. benthamiana cell suspensions and hairy roots
A replicating Potato virus X (PVX) vector yielded 6.6-fold more GUS when non-essential viral genes were omitted In the present study three different PVX constructs were characterized: a PVX full-length vector containing GUS as a gene insert (PVX-GUS) and derivatives of that vector containing deletions spanning most of the coat protein (PVXΔCP-GUS) or both the coat protein and triple gene block (PVXΔCPΔTGB-GUS)

Summary

Introduction

Plant cell suspensions and hairy root cultures represent scalable protein expression platforms. Plant cell suspensions and hairy roots combine the advantages inherent to plants with the environmental control, scalability, short production cycles, and containment of tissues cultured in bioreactors. Cell suspensions are readily scalable volumetrically in bioreactors by principles already developed for microbial and mammalian cell cultures [7]. Plant cell suspensions have been scaled up to 75,000 L for the production of valuable secondary metabolites [8]. They are amenable to pilot-scale culture in low capital cost bioreactors [9]

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