When viruses or transgenes are introduced into plants, they trigger a host response that is generally referred to as post-transcriptional gene silencing (PTGS) or cosuppression. The mechanism of PTGS is believed to be similar to that of RNA interference (RNAi) in animals. The process is initiated by double-stranded RNA (dsRNA) molecules, which may be generated by replicative intermediates of viral RNAs or by aberrant transgene-coded RNAs (these RNA molecules may become double-stranded when copied by an RNA-dependent RNA polymerase, or RdRP). The dsRNAs are then digested into 21-25 nt small interfering RNAs or siRNAs. The siRNAs subsequently assemble into a nuclease complex called RISC, guiding the complex to bind and destroy homologous transcripts.

PTGS is believed to be an anti-viral response. Viral RNAs not only trigger PTGS, but they also serve as targets. Cleavage of viral RNA results in reduced virus titers in local and distant leaves (referred to as local and systemic silencing, respectively) and a plant "recovery" phenotype. In response, numerous plant viruses have evolved proteins to suppress PTGS (1). However, the mechanisms by which these proteins act have not been determined. In a paper published in the June 2002 issue of EMBO J., Silhavy and colleagues show that one of these suppressor proteins, the tombavirus p19, abolishes gene silencing by binding to 21-25 nt siRNAs (2). For the first time, these results establish a mechanism by which the virus-coded proteins act.

The Findings

The authors first showed that the tombavirus p19 protein could suppress PTGS in Nicotiana benthamiana plants. In one set of experiments, green fluorescent protein (GFP)-transgenic plants were coinfiltrated with Agrobacterium tumefaciens bacteria that carried GFP or p19-expressing tumor-inducing (Ti) plasmids. GFP-transgenic plants have green fluorescence leaves. Infiltration of these plants with GFP resulted in PTGS or cosuppression, marked by a change in leaf fluorescence color from green to red (the red color is due to autofluorescence of chlorophyll). While the mechanism of PTGS in this instance is not clear, it does seem to involve GFP siRNAs. These siRNAs could be generated from transcripts of the GFP expression-construct, transgene, or both (3).

Coinfiltration with both GFP and p19, interestingly, resulted in retention of the green fluorescence in plant leaves. These results suggested that the p19 protein was inhibiting PTGS. In support of this hypothesis, Northern blot analysis showed that GFP/p19 coinfiltration (i.e. suppressed PTGS) resulted in increased GFP mRNA levels and decreased GFP siRNA levels in plant leaves compared to GFP infiltration alone (i.e. PTGS).

The authors then showed that the p19 protein binds specifically to siRNAs. siRNAs are typically 21-25 nt in length and contain 2 nt 3´-overhangs. Using gel shift assays, the authors showed that p19 is able to bind to synthetic 21 nt RNA oligonucleotides with 2 nt 3´-overhangs and to 21-25 nt dsRNAs isolated from tombavirus-infected leaves. However, the protein did not bind to in vitro transcribed long (~100 nt) double- or single-stranded RNA molecules. Moreover, it bound weakly to 21 nt RNA molecules with blunt ends or those with 4 nt 3´-overhangs.

Discussion

The results by Silhavy and colleagues suggest that the tombavirus p19 protein suppresses local PTGS by binding to 21-25 nt siRNAs. Inhibition of local silencing may be achieved by preventing siRNAs from interacting with either the RdRP enzyme or the RISC complex.

In addition to local silencing, the authors showed that p19 could prevent systemic silencing. In systemic silencing, infiltration of plants with GFP allows PTGS to spread to distance leaves. It has been determined that spread of PTGS does not involve movement of the Agrobacterium tumefaciens bacteria or the Ti plasmid. Rather, it has been proposed that this "mobile silencing element" may either be long dsRNAs or siRNAs (4, 5). The results by Silhavy and colleagues suggest that siRNAs, and not the longer dsRNAs, act as this "mobile silencing element", since 1) p19 can inhibit systemic silencing and 2) p19 can only bind to siRNAs.

In addition to suppressing PTGS in plants, p19 may also be used as a tool to inhibit RNAi in other systems. This is suggested by the finding that p19 can bind to siRNAs in vitro without requiring host proteins or other factors. The utility of this protein in RNAi experiments, however, remains to be determined.

References

1. Voinnet O, Pinto YM, and Baulcombe DC. (1999). Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. Proc. Natl. Acad. Sci. USA 96(24):14147-14152.
2. Silhavy D, Molnar A, Lucioli A, Szittya G, Hornyik C, Tavazza M, and Burgyan J. (2002). A viral protein suppresses RNA silencing and binds silencing-generated, 21- to 25-nucleotide double-stranded RNAs. EMBO J. 21(12):3070-3080.
3. Dr. Daniel Silhavy, personal communications.
4. Voinnet O and Baulcomb DC. (1997). Systemic signaling in gene silencing. Nature 389:553.
5. Voinnet O, Lederer C, and Baulcomb DC. (2000). A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana. Cell 103:157-167.
   

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