Plant viral suppressors of RNA silencing
Introduction
RNA silencing is an adaptive defense mechanism that is triggered by double stranded RNA (dsRNA). Three initially unrelated lines of research led to the recognition of RNA silencing as an important means of defense against viruses and other nucleic acid invaders. The discovery that plant viruses encode suppressors of RNA silencing is a key element in the story. The first line of research led to the discovery of transgene-induced RNA silencing. Attempts to over-express endogenous genes by introducing additional copies resulted instead in turning off the endogenous gene as well as the transgene (Napoli et al., 1990, Smith et al., 1990, van der Krol et al., 1990). The next piece of the puzzle came from studies of pathogen-derived resistance in which RNA silencing directed against a viral transgene provided resistance to any virus carrying the targeted sequence (Baulcombe, 1996, Dougherty and Parks, 1995, English et al., 1996, Goodwin et al., 1996, Lindbo et al., 1993, Smith et al., 1994). Thus, viruses could be targets of RNA silencing. The third clue came from studies of synergistic viral diseases caused by certain pairs of co-infecting viruses. A viral protein called helper component proteinase (HC-Pro) was shown to mediate one class of viral synergistic disease (Shi et al., 1997, Vance et al., 1995). Expression of HC-Pro in transgenic plants allowed a broad range of heterologous viruses to accumulate beyond the normal level, suggesting that HC-Pro blocked a general plant defense mechanism (Pruss et al., 1997). Remarkably, the mechanism blocked by HC-Pro was found to be RNA silencing (Anandalakshmi et al., 1998, Brigneti et al., 1998, Kasschau and Carrington, 1998). Since the initial demonstration that HC-Pro blocks RNA silencing, many other plant viral suppressors of silencing have been identified (see Table 1).
Section snippets
RNA silencing
The term RNA silencing refers to a set of related pathways found in a broad range of eukaryotic organisms. Genetic and biochemical experiments have established a general mechanistic model for these related pathways and identified factors that are required for RNA silencing in a variety of organisms (Fig. 1). The process is initially triggered by dsRNA, which can be introduced experimentally or arise from endogenous transposons, replicating RNA viruses, or the transcription of transgenes. The
Functional assays used to identify suppressors of RNA silencing
Three major approaches have been widely used to identify plant viral suppressors of RNA silencing: (1) transient expression assays, (2) the reversal of silencing assay, and (3) stable expression assays. These assays are described below and shown in cartoon form in Fig. 2, Fig. 4.
Mechanism of suppression
The assays discussed above have proven extremely useful as rapid and sensitive methods to identify suppressors of silencing. They have also enabled rudimentary characterization of suppressor mechanism, but conflicting results from different assays have made it difficult to draw firm conclusions for many suppressors. Interestingly, the currently known suppressors share no obvious similarities at either the nucleic acid or the protein level, perhaps reflecting differences at the mechanistic level
Cucumber mosaic virus (CMV) 2b
The CMV 2b protein was one of the first identified suppressors of RNA silencing and also one of the best studied from a mechanistic standpoint. The initial indication that CMV 2b suppressed silencing came from the reversal of silencing assay in which 2b expressed from PVX could prevent the initiation of silencing but could not reverse silencing that was already established (Brigneti et al., 1998). That early result raised the possibility that 2b might block systemic silencing. Subsequently,
Do all plant viruses have suppressors of silencing?
The discovery that plants have a generalized antiviral defense mechanism triggered by dsRNA has revolutionized our thinking about plant–virus interactions. In the euphoric aftermath of the initial identification of plant viral suppressors of silencing, a popular expectation was that most or all plant viruses would encode a suppressor of RNA silencing. Although many different viral suppressors have been identified, the fact that HC-Pro helps so many different viruses suggests that a lot of
Suppressors of silencing as tools
The finding that certain viral proteins suppress RNA silencing has provided a new tool for technologies utilizing genetically modified plants and is, therefore, of practical significance. Many biotechnological applications are impaired by RNA silencing, and suppressors of silencing can be used to attain consistent, high-level expression of transgenes in plants (Mallory et al., 2002a, Voinnet et al., 2003). With silencing under control, transgenic plants can be engineered to produce a range of
Acknowledgements
VBV gratefully acknowledges support from the USDA Competitive Grants Program, NIH, and Dow AgroSciences LLC.
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