Plant Viruses as Gene Expression and Silencing Vectors

Abstract

Plant viruses have been modified to express heterologous proteins in plants and to downregulate the expression of genes of host plants through virus‐induced gene silencing (VIGS). Many viruses have been adopted as vectors for either or both purposes, with varying effectiveness and popularity.

Key Concepts

  • Plant‐infecting viruses can be engineered to express heterologous proteins or impart silencing of host genes.
  • Gene expression vectors can be used to produce stand‐alone proteins or proteins/peptides fused to viral proteins.
  • Gene‐specific silencing can be achieved by incorporating partial fragments of target genes in VIGS vectors.
  • For most VIGS vectors, delivery remains a bottleneck in need of further improvements.

Keywords: plant virus vector; gene expression; virus‐induced gene silencing; plant virus genome organization; (+) ssRNA virus

Figure 1. Simplified scheme of an RNA‐silencing (RNA interference) pathway in plants.
Figure 2. Schematic representation of two different gene expression strategies used by (+) ssRNA viruses. The lines represent genomic (g) and subgenomic (sg) RNAs of the viruses, whereas the rectangular boxes on the lines represent individual proteins encoded by these RNAs. Viruses using strategy I for gene expression translate the 5′ proximal protein(s) directly from gRNAs (solid box). The 5′ encoded proteins then direct the synthesis of sgRNAs for the translation of 3′ proximal proteins. By contrast, viruses using strategy II encode all of their protein products in the form of a polyprotein precursor, which is then processed by virus‐encoded proteases to individual mature proteins.
Figure 3. TMV‐based gene expression vector. (a) TMV and other similar viruses like TVCV and crTMV encode four different viral proteins. The 126K and 183K proteins, the latter a ‐terminally extended form of the former, are essential for viral genome replication. The movement protein (MP) and CP are both translated from their own sgRNAs. (b) Commonly used TMV‐based expression vectors. Both versions of TMV expression vectors are chimeras of two closely related viruses, denoted by black and blue lines and boxes, with their connection pints near the end of MP‐coding region. See Shivprasad . () and Marillonnet . () for details. In addition, both contain multiple mutations or artificial introns (denoted as *) within the 126K/183K‐coding region.
Figure 4. CPMV‐based gene expression vectors. (a) Genome organisation of the bipartite CPMV. RNA1‐encoded proteins, consisting of protease cofactor (C‐Pro), helicase (Hel), viral protein genome‐linked (VPg), protease (Pro) and RNA‐dependent RNA polymerase (RdRP), are needed for the replication of RNA genome. RNA2 encodes MP, larger and smaller CP (L‐CP and S‐CP) subunits. Both RNAs translate polyprotein precursors that are subsequently processed into various mature viral proteins. (b) Various types of CPMV‐based vectors. Small foreign peptides can be displayed on the surface of the virions by inserting their coding sequence at certain positions of S‐CP‐coding region. Alternatively, stand‐alone foreign proteins can be produced by inserting their coding sequences between MP and L‐CP‐coding sequences. Note that the protease‐processing sites need to be duplicated (pink boxes) in order to release the expressed foreign proteins. Protein‐of‐interest can also be expressed from CPMV by linking its coding sequence to the ‐terminus of S‐CP, separated by a self‐cleaving peptide (light green box) derived from foot mouth disease virus.
Figure 5. TRV‐based VIGS vector. (a) Organisation of the bipartite TRV genome. The coding strategy of TRV RNA1 is highly similar to TMV except for the 3′ utmost open reading frame which in TRV encodes the 16K suppressor of RNA silencing. Two of the RNA2‐encoded proteins, 29K and 33K, can be deleted without affecting the infectivity of TRV. (b) In the TRV‐based VIGS vector, the bulk of 29K/33K‐coding sequence was replaced by a DNA fragment that contains the recognition sites of multiple restriction enzymes, facilitating the insertion of fragments of host plant genes. Adapted with permission from Liu et al. (2002). © John Wiley & Sons.
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Further Reading

Baulcombe DC (1999) Fast forward genetics based on virus‐induced gene silencing. Current Opinion in Plant Biology 2: 109–113.

Cañizares MC, Nicholson L and Lomonossoff GP (2005) Use of viral vectors for vaccine production in plants. Immunology and Cell Biology 83: 263–270.

Carillo‐Tripp J, Shimada‐Beltran H and Rivera‐Bustamante R (2006) Use of geminiviral vectors for functional genenomics. Current Opinion in Plant Biology 9: 209–215.

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Lomonossoff GP and Hamilton WDO (1999) Cowpea mosaic virus‐based vaccines. Current Topics in Microbiology and Immunology 240: 177–189.

Lomonossoff GP (2005) Antigen delivery systems: use of recombinant plant viruses. In: Mestecky J, Bienenstock J, Lamm ME, Mayer L, McGhee JR and Strober W (eds) Mucosal Immunology, 3rd edn, pp. 1061–1072. Amsterdam: Elsevier.

Pogue GP, Lindbo JA, Garger SJ and Fitzmaurice WP (2002) Making an ally from an enemy: plant virology and the new agriculture. Annual Review of Phytopathology 40: 45–74.

Porta C and Lomonossoff GP (2002) Viruses as vectors for the expression of foreign sequences in plants. Biotechnology and Genetic Engineering Reviews 19: 245–291.

Robertson D (2004) VIGS vectors for gene silencing: many targets, many tools. Annual Review of Plant Biology 55: 495–519.

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Qu, Feng(Apr 2016) Plant Viruses as Gene Expression and Silencing Vectors. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0020709.pub2]