Viral and Cellular Factors Involved in Phloem Transport of Plant Viruses

In transgenic and nontransgenic plants, viruses are both initiators and targets of a defense mechanism that is similar to posttranscriptional gene silencing (PTGS). Recently, it was found that potyviruses and cucumoviruses encode pathogenicity determinants that suppress this defense mechanism. Here, we test diverse virus types for the ability to suppress PTGS. Nicotiana benthamiana exhibiting PTGS of a green fluorescent protein transgene were infected with a range of unrelated viruses and various potato virus X vectors producing viral pathogenicity factors. Upon infection, suppression of PTGS was assessed in planta through reactivation of green fluorescence and confirmed by molecular analysis. These experiments led to the identification of three suppressors of PTGS and showed that suppression of PTGS is widely used as a counter-defense strategy by DNA and RNA viruses. However, the spatial pattern and degree of suppression varied extensively between viruses. At one extreme, there are viruses that suppress in all tissues of all infected leaves, whereas others are able to suppress only in the veins of new emerging leaves. This variation existed even between closely related members of the potexvirus group. Collectively, these results suggest that virus-encoded suppressors of gene silencing have distinct modes of action, are targeted against distinct components of the host gene-silencing machinery, and that there is dynamic evolution of the host and viral components associated with the gene-silencing mechanism.

One of the functions of RNA silencing in plants is antiviral defense. A hallmark of RNA silencing is spreading of the silenced state through the plant. Little is known about the nature of the systemic silencing signal and the proteins required for its production, transport, and reception in plant tissues. Here, we show that the RNA-dependent RNA polymerase RDR6 in Nicotiana benthamiana is involved in defense against potato virus X at the level of systemic spreading and in exclusion of the virus from the apical growing point. It has no effect on primary replication and cell-to-cell movement of the virus and does not contribute significantly to the formation of virus-derived small interfering (si) RNA in a fully established potato virus X infection. In grafting experiments, the RDR6 homolog was required for the ability of a cell to respond to, but not to produce or translocate, the systemic silencing signal. Taking these findings together, we suggest a model of virus defense in which RDR6 uses incoming silencing signal to generate double-stranded RNA precursors of secondary siRNA. According to this idea, the secondary siRNAs mediate RNA silencing as an immediate response that slows down the systemic spreading of the virus into the growing point and newly emerging leaves.

Posttranscriptional gene silencing (PTGS) processes double-stranded (ds) RNAs into 21-25 nucleotide (nt) RNA fragments that direct ribonucleases to target cognate mRNAs. In higher plants, PTGS also generates mobile signals conferring sequence-specific silencing in distant organs. Since PTGS acts as an antiviral system in plants, successful virus infection requires evasion or suppression of gene silencing. Here we report that the 19 kDa protein (p19) of tombusviruses is a potent silencing suppressor that prevents the spread of mobile silencing signal. In vitro, p19 binds PTGS-generated, 21-25 nt dsRNAs and 21-nt synthetic dsRNAs with 2-nt 3' overhanging end(s), while it barely interacts with single-stranded (ss) RNAs, long dsRNAs or blunt-ended 21-nt dsRNAs. We propose that p19 mediates silencing suppression by sequestering the PTGS-generated 21-25 nt dsRNAs, thus depleting the specificity determinants of PTGS effector complexes. Moreover, the observation that p19-expressing transgenic plants show altered leaf morphology might indicate that the p19-targeted PTGS pathway is also important in the regulation of plant development.