Research on plant viruses has yielded a variety of tools for use in planta such as functional promoters, transient expression systems, and, most recently, methods for gene silencing. Viral vectors may be utilized for over-expression or suppression of target genes in plants. However, the mechanisms by which these processes occur are not yet fully known. In a review article entitled “Virus-mediated reprogramming of gene expression in plants” published in the June 2001 issue of Current Opinion in Plant Biology (4(3):181-5), Guy della-Cioppa and his associates at Large Scale Biological Corporation (Vacaville, CA) discuss the use of viral vectors to manipulate the expression of target genes.
The authors review approaches for over-expression of genes of interest in plants using viral vectors. Vectors are generated from viruses such as tobacco mosaic virus (TMV) or potato virus X (PVX), and may be used to produce higher levels of desired proteins or metabolites than would be tolerated in plants engineered by conventional transformation practices. Two examples are given. In the first, researchers achieved a tenfold increase in phytoene production in Nicotiana benthamina using a viral vector containing a phytoene synthase gene. In the second case, N. benthamina accumulated a substantial pool of a desired carotenoid, capsanthin, when a capsanthin-capsorubin synthase gene from a Capsicum species was over-expressed using a viral vector.
The authors describe how viral vectors are well suited for use in high-throughput studies, such as gene shuffling or molecular breeding projects. Libraries of mutant or chimeric versions of genes may be cloned into viral vectors and used to generate over-expressing plant lines. Plants with desired characteristics may then be selected. In theory, such an approach could generate enhanced enzyme activities, such as thermally activated cellulases and ligninases that would facilitate pulp and paper production.
Viral vectors can also be used to suppress gene expression in plants. To illustrate the biology behind this process, the authors describe several related gene silencing events: post-transcriptional gene silencing (PTGS), co-suppression, pathogen-derived resistance (PDR), viral-induced gene silencing (VIGS), and the non-plant phenomena RNA interference (RNAi) and quelling. Co-suppression was first observed when researchers generated transgenic plants engineered for strong constitutive expression of a target gene. In some lines, both the transgene and the endogenous gene were unexpectedly silenced, an event correlated with low steady-state mRNA levels of the target gene. Independently, other researchers began to develop transgenic plants resistant to viral pathogens by constitutive in planta expression of viral genes such as those encoding viral coat proteins. The constant expression of these viral elements in the host was thought to interfere with the normal life cycle of the virus during infection, an event called pathogen derived resistance. However, the level of resistance did not correlate with the level of transgene expression. Then, careful observation of PDR transgenic plants resistant to tobacco etch virus (TEV) through the constitutive expression of coat proteins (CP) showed that resistance required only CP-encoding transcripts, not protein, and that high steady-state levels of CP transcripts were avoided by a RNA degradation activity. This general mechanism was named PTGS. It appears to be responsible for many subsets of gene silencing events, including co-suppression, VIGS, and PDR.
PTGS is likely to have evolved in nature to combat attacks by viral pathogens. The production of foreign viral RNA transcripts is a central event in the life cycle of many plant viral pathogens. Thus, development of a mechanism in plants to recognize transcripts that rise above a threshold level and selectively degrade them would confer an evolutionary advantage to plant species under selection pressure from viral pests. The authors list evidence in favor of PTGS existing as a viral defense strategy; for example, PTGS activity is observed in plant tissue that has resisted a viral attack or plants that experience cross-protection from another viral pest. Arabidopsis mutants that are impaired in the PTGS response are hyper-susceptible to viral infection. Double-stranded RNA, which strongly induces PTGS, is also an element of many plant viral life cycles. Finally, the PTGS signal is mobile in a manner similar to the movement of plant viruses throughout their hosts.
In the host-pathogen arms race, there are likely to be countermeasures developed by viruses against the plant PTGS response. The authors theorize a number of different ways in which this could happen, including avoidance of viral detection by the host or translocation through the host body faster than the mobile PTGS signal. Viral expression of suppressors of PTGS machinery have indeed been identified in some virus groups, and an analysis of these suppressors should allow further elucidation of the basic machinery of this phenomenon.
The mechanism of silencing by viral vectors continues to be an area of active investigation. Recent studies have shown viral vectors containing target gene fragments, rather than the entire gene, are effective for silencing. These gene fragments elicit silencing when present in either sense or anti-sense orientation. In theory, chimeric genes should induce the silencing of multiple gene targets, a feature that would be of particular utility for studies of gene families.
It is clear that the biology of various viruses dictates the extent to which vectors derived from them will be useful. For example, viruses that possess PTGS suppressors may be of limited value for derivation of vectors. On the other hand, some viruses cause effective systemic gene silencing in the host. These excellent silencers induce a promotive response in the host rather than manifesting persistent infection symptoms. In these virus-plant systems, a host sequence-specific RNA degradation process is triggered. Thus, such viruses hold particular promise for development of new vectors.
In conclusion, the authors express hope that understanding the mechanisms leading to effective gene silencing will promote the development of additional viral vectors, which will further promote target gene expression manipulation.