Development of expression vectors based on pepino mosaic virus

Abstract

BackgroundPlant viruses are useful expression vectors because they can mount systemic infections allowing large amounts of recombinant protein to be produced rapidly in differentiated plant tissues. Pepino mosaic virus (PepMV) (genus Potexvirus, family Flexiviridae), a widespread plant virus, is a promising candidate expression vector for plants because of its high level of accumulation in its hosts and the absence of severe infection symptoms. We report here the construction of a stable and efficient expression vector for plants based on PepMV.ResultsAgroinfectious clones were produced from two different PepMV genotypes (European and Chilean), and these were able to initiate typical PepMV infections. We explored several strategies for vector development including coat protein (CP) replacement, duplication of the CP subgenomic promoter (SGP) and the creation of a fusion protein using the foot-and-mouth disease virus (FMDV) 2A catalytic peptide. We found that CP replacement vectors were unable to move systemically and that vectors with duplicated SGPs (even heterologous SGPs) suffered from significant transgene instability. The fusion protein incorporating the FMDV 2A catalytic peptide gave by far the best results, maintaining stability through serial passages and allowing the accumulation of GFP to 0.2-0.4 g per kg of leaf tissue. The possible use of PepMV as a virus-induced gene silencing vector to study gene function was also demonstrated. Protocols for the use of this vector are described.ConclusionsA stable PepMV vector was generated by expressing the transgene as a CP fusion using the sequence encoding the foot-and-mouth disease virus (FMDV) 2A catalytic peptide to separate them. We have generated a novel tool for the expression of recombinant proteins in plants and for the functional analysis of virus and plant genes. Our experiments have also highlighted virus requirements for replication in single cells as well as intercellular and long-distance movement.

A Narcissus mosaic viral vector system for protein expression and flavonoid production

“…The capability to express GUS protein (68 KDa) in a NMV system suggests that large transcription factors, such as bHLHs that are involved in the regulation of flavonoid biosynthesis pathways, could also be expressed with this system. The pattern of GFP spread (locally and systemically) found in this study suggests that NMV shows behaviors similar to those characterized for other Potex viruses [16,54,55]. This long period of stable protein expression, in combination with the ease of systemic infection, suggests that the NMV system may provide an efficient platform for over-expression, purification and sub-cellular localization of proteins of interest.…”

“…A range of virus-based plant transient expression systems has been developed for protein expression [13,16,35-39]. Plant viral vector systems have several advantages over other systems, such as rapid and systemic infections, and also avoid the risks associated with the use of animal pathogens for production of recombinant proteins or vaccines [11,40-43].…”

“…Therefore, a viral expression system, combined with the appropriate host plant, could provide an efficient tool for studying protein functions and producing valuable secondary metabolites in planta without having to generate stable transgenic plants. While plant viral vectors have been widely used to down-regulate the expression of endogenous genes [4-9], they have been also developed to over-express heterologous proteins [3,10-16]. However, there can be limitations associated with the different viral systems used.…”

New phytoviral vector for superexpression of target proteins in plants

“…Èç ëèòåðàòóðíûõ äàííûõ èçâåñòíî, ÷òî ïîâòîðÿ-þùèåñÿ ïîñëåäîâàòåëüíîñòè ãåíîìîâ âèðóñíûõ âåê-òîðîâ ìîãóò âûùåïëÿòüñÿ âñëåäñòâèå ðåïëèêàòèâ-íîé ðåêîìáèíàöèè [10]. Äàííûé ôàêò ìîaeåò áûòü ïðè÷èíîé îòñóòñòâèÿ çíà÷èòåëüíîãî óâåëè÷åíèÿ ýô-ôåêòèâíîñòè ýêñïðåññèè ÁÎ AltMV-MU â òðàíñ-Ðèñ.…”

Translating 2A Research into Practice

“…The first types of genetically modified organisms created using 2A to co-express multiple proteins were plants, initially as a research tool, but also to improve drought-resistance (Kwon et al, 2004); disease-resistance (François et al, 2004;Geu-Flores et al, 2009) and nutritional qualities (Randall et al, 2004). Plant virus vectors based on potato virus X (PVX), cowpea mosaic virus (CPMV), pepino mosaic virus (PepMV), and bean pod mottle virus (BPMV) have been engineered with F2A and used to produce functional recombinant proteins including vaccines and antibodies (Smolenska et al, 1998;Gopinath et al, 2000;Marconi et al, 2006;Zhang et al, 2010;Sempere et al, 2011).…”