Gene silencing is an important but little understood regulatory mechanism in plants. Here

Synergistic viral diseases of higher plants are caused by the interaction of two independent viruses in the same host and are characterized by dramatic increases in symptoms and in accumulation of one of the coinfecting viruses. In potato vírus X (PVX)/potyviral synergism, increased pathogenicity and accumulation of PVX are mediated by the expression of potyviral5' proximal sequences encoding Pl, the helper component proteinase (HC-Pro), and a fraction of P3. Here, we report that the same potyviral sequence (termed Pl/HC-Pro) enhances the pathogenicity and accumulation of two other heterologous viruses: cucumber mosaic virus and tobacco mosaic virus. In the case of PVX-potyviral synergism, we show that the expression of the HC-Pro gene product, but not the RNA sequence itself, is sufficient t o induce the increase in PVX pathogenicity and that both P1 and P3 coding sequences are dispensable for this aspect of the synergistic interaction. In protoplasts, expression of the potyviral Pl/HC-Pro region prolongs the accumulation of PVX (-) strand RNA and transactivates expression of a reporter gene from a PVX subgenomic promoter. Unlike the synergistic enhancement of PVX pathogenicity, which requires only expression of HC-Pro, the enhancement of PVX (-) strand RNA accumulation in protoplasts is significantly greater when the entire Pl/HC-Pro sequence is expressed.These results indicate that the potyviral Pl/HC-Pro region affects a step in disease development that is common to a broad range of virus infections and suggest a mechanism involving transactivation of viral replication.

No abstract

Posttranscriptional gene silencing (PTGS) is an ancient eukaryotic regulatory mechanism in which a particular RNA sequence is targeted and destroyed. The helper component-proteinase (HC-Pro) of plant potyviruses suppresses PTGS in plants. Using a yeast two-hybrid system, we identified a calmodulin-related protein (termed rgs-CaM) that interacts with HC-Pro. Here we report that rgs-CaM, like HC-Pro itself, suppresses gene silencing. Our work is the first report identifying a cellular suppressor of PTGS.

Post-transcriptional gene silencing (PTGS) is a sequence-specific RNA degradation mechanism that is widespread in eukaryotic organisms. It is often associated with methylation of the transcribed region of the silenced gene and with accumulation of small RNAs (21 to 25 nucleotides) homologous to the silenced gene. In plants, PTGS can be triggered locally and then spread throughout the organism via a mobile signal that can cross a graft junction. Previously, we showed that the helper component-proteinase (HC-Pro) of plant potyviruses suppresses PTGS. Here, we report that plants in which PTGS has been suppressed by HC-Pro fail to accumulate the small RNAs associated with silencing. However, the transgene locus of these plants remains methylated. Grafting experiments indicate that HC-Pro prevents the plant from responding to the mobile silencing signal but does not eliminate its ability to produce or send the signal. These results demonstrate that HC-Pro functions downstream of transgene methylation and the mobile signal at a step preceding accumulation of the small RNAs. INTRODUCTIONPost-transcriptional gene silencing (PTGS) is a sequencespecific RNA degradation mechanism first discovered in transgenic plants (Napoli et al., 1990;Smith et al., 1990;van der Krol et al., 1990). Related processes have been found in diverse eukaryotic organisms including Neurospora , in which it is called quelling, and a variety of animal systems, in which it is referred to as RNA interference or RNAi Fire, 1999;Grant, 1999;Kooter et al., 1999;Ding, 2000;Matzke et al., 2001). Sequence-specific RNA degradation is triggered by double stranded RNA (dsRNA) in a variety of organisms Waterhouse et al., 1998;Sharp, 1999;Bass, 2000;Matzke et al., 2001). In plants, PTGS can be induced by RNA viruses, many of which replicate via dsRNA intermediates. Finally, in both plants and Caenorhabditis elegans , the process can be triggered locally and then spread to distant parts of the organism (Palauqui et al., 1997;Voinnet and Baulcombe, 1997;Fire et al., 1998;Jorgensen et al., 1998;Palauqui and Vaucheret, 1998;Voinnet et al., 1998). The relatedness of these sequence-specific RNA degradation processes in different organisms is evidenced by their requirement for a conserved set of gene products Matzke et al., 2001), including a protein with homology to translation factor eIF2C (Tabara et al., 1999;Catalanotto et al., 2000;Fagard et al., 2000), an RNA-dependent RNA polymerase (RdRp) (Cogoni and Macino, 1999a;Dalmay et al., 2000;Mourrain et al., 2000;Smardon et al., 2000), and proteins with homology to DNA helicases and RNase D (Cogoni and Macino, 1999b;Ketting et al., 1999). However, at this point, neither the roles of these various gene products nor the mechanisms for induction, maintenance, and spread of sequence-specific RNA degradation are clearly understood.Several molecular features characterize the sequencespecific RNA degradation processes found in diverse organisms. Studies in both plants and Drosophila have shown that silencing is accompanied b...

No abstract

No abstract

Dicer-like (DCL) enzymes play a pivotal role in RNA silencing in plants, processing the long double-stranded RNA (dsRNA) that triggers silencing into the primary short interfering RNAs (siRNAs) that mediate it. The siRNA population can be augmented and silencing amplified via transitivity, an RNA-dependent RNA polymerase (RDR)-dependent pathway that uses the target RNA as substrate to generate secondary siRNAs. Here we report that Arabidopsis DCL2–but not DCL4-is required for transitivity in cell-autonomous, post-transcriptional silencing of transgenes. An insertion mutation in DCL2 blocked sense transgene-induced silencing and eliminated accumulation of the associated RDR-dependent siRNAs. In hairpin transgene-induced silencing, the dcl2 mutation likewise eliminated accumulation of secondary siRNAs and blocked transitive silencing, but did not block silencing mediated by primary siRNAs. Strikingly, in all cases, the dcl2 mutation eliminated accumulation of all secondary siRNAs, including those generated by other DCL enzymes. In contrast, mutations in DCL4 promoted a dramatic shift to transitive silencing in the case of the hairpin transgene and enhanced silencing induced by the sense transgene. Suppression of hairpin and sense transgene silencing by the P1/HC-Pro and P38 viral suppressors was associated with elimination of secondary siRNA accumulation, but the suppressors did not block processing of the stem of the hairpin transcript into primary siRNAs. Thus, these viral suppressors resemble the dcl2 mutation in their effects on siRNA biogenesis. We conclude that DCL2 plays an essential, as opposed to redundant, role in transitive silencing of transgenes and may play a more important role in silencing of viruses than currently thought.