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. 2011 Mar 11;7:6.
doi: 10.1186/1746-4811-7-6.

Development of Expression Vectors Based on Pepino Mosaic Virus

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Free PMC article

Development of Expression Vectors Based on Pepino Mosaic Virus

Raquel N Sempere et al. Plant Methods. .
Free PMC article

Abstract

Background: Plant 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.

Results: Agroinfectious 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.

Conclusions: A 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.

Figures

Figure 1
Figure 1
Multiplication of PepMV CP-mutants in N. benthamiana protoplasts. (a) Schematic representation of PepXL6 and its derived CP mutants (PepXL6agg and PepGFPΔCP). RdRp-RNA-dependent RNA polymerase ORF; TG1, TGB2 and TGB3-triple gene block ORFs; CP-coat protein ORF. (b) Northern blot of total RNA prepared from N. benthamiana protoplasts 24 h post inoculation, hybridized with a replicase-specific RNA probe. Ethidium bromide-stained rRNA is shown (bottom panels).
Figure 2
Figure 2
Effect of CP deletion on PepMV multiplication in agroinfiltrated N. benthamiana tissues, with and without CP trans complementation. (a) Leaves agroinfiltrated with pBPepGFPΔCP (left) and pBGFP (rigth) at 6 days post inoculation (dpi). (b) Leaves co-agroinfiltrated with pBPepGFPΔCP and the construct for CP expression, at 6 dpi (left) and 10 dpi (right). The agroinfiltration assays were carried out in the presence of pB19. Photographs at 6 dpi were taken by confocal laser scanning microscopy and the photograph at 10 dpi was taken under illumination with a handheld UV lamp.
Figure 3
Figure 3
PepMV vectors containing a duplicated subgenomic mRNA promoter. (a) In Pep5.128, the GFP gene was positioned downstream of the CP gene, whereas it was positioned upstream of the CP gene in Pep501. The duplicated SGP spanned positions -76 to +36 relative to the CP start codon. Pep352 and Pep640 include successive deletions at the 3' end of the putative CP SGP. SGP-CP subgenomic mRNA promoter; SGPd-duplicated CP subgenomic mRNA promoter. (b) Green fluorescence visible in systemically-infected N. benthamiana leaves from plants agroinfiltrated with pBPep501 at 5 and 7 dpi. (c) Northern blot of leaf tissue hybridized with a CP-specific RNA probe to determine Pep501 stability. Total RNA was prepared from systemically-infected leaves at 5 and 7 dpi. The band corresponding to the GFP-CP sgRNA is marked with star. Ethidium bromide-stained rRNA is shown (bottom panels). (d) N. benthamiana leaves infiltrated with pBPep352 or pBPep501 vectors, showing fluorescence emitted under UV illumination with a handheld UV lamp. Photographs were taken at 5 or 7 dpi.
Figure 4
Figure 4
PepMV vectors with a heterologous CP subgenomic mRNA promoter. (a) Schematic representation of Pep505. SGP-Ps5 corresponds to the PepMV Ps5 (Chilean genotype) CP subgenomic mRNA promoter. (b) Fluorescence in systemically-infected leaves of N. benthamiana plants agroinfiltrated with pBPep505. Plants were photographed under illumination with a handheld UV lamp at 7 dpi. (c) Northern blot of total RNA from leaf tissues hybridized with a CP-specific RNA probe to determine Pep505 stability. Total RNA was prepared from agroinfiltrated (IN) and systemically-infected (SYS) leaves at 7 and 10 dpi. The band corresponding to the CP-GFP sgRNA is marked with star. Ethidium bromide-stained rRNA is shown (bottom panels).
Figure 5
Figure 5
PepMV vector including a GFP:CP fusion separated by the FMDV 2A catalytic peptide sequence. (a) Schematic representation of PepGFP2a. The position of the 2A sequence insertion is indicated. (b) Fluorescence in systemically infected N. benthamiana leaves from plants agroinfiltrated with pBPepGFP2a. (c) Northern blot of leaf tissue hybridized with a CP-specific RNA probe to determine PepGFP2a stability. Total RNA was prepared from agroinfiltrated and systemically-infected leaves showing fluorescence at 7 and 14 dpi, and from plants mechanically inoculated with PepGFP2a at 7 dpi after the second and third passages. The band corresponding to the GFP-CP sgRNA is marked with star. Ethidium bromide-stained rRNA is shown (bottom panels). (d) GFP expression after second and third passage of PepGFP2a. Plants were photographed under white and UV light at 7 and 12 dpi.
Figure 6
Figure 6
Western blot analysis of total proteins extracted from systemically-infected leaves agroinfiltrated with PepGFP2a. Soluble leaf proteins were separated by 15% SDS PAGE, followed by western blotting using an antibody against (a) the CP or (c) GFP. (b) Gel stained with Coomasie brilliant blue.
Figure 7
Figure 7
PepMV based gene silencing vector. (a) Schematic representation of PepPDS2a. (b) Representative leaves from N. benthamiana plants infected with the PepPDS2a vector showing different phenotypes of photobleaching (L1, L2 and L3). (c) Quantification of pds mRNA levels by semi-quantitative RT-PCR in plants inoculated with water (mock), pBPepGFP2a and pBPepPDS2a, as indicated. Lanes 1-3 correspond to mock-inoculated controls using 20, 25 and 30 amplification cycles. Thirty cycles were used for amplifications in lanes 4-7. L1, L2 and L3 correspond to the leaves with different phenotypes of photobleaching shown in the panel b. The relative (%) accumulation of pds mRNA in relation to mock-inoculated leaves is indicated below each lane.

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