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

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...

In the postgenomic era, large-scale functional genomic approaches are necessary for converting sequence information into functional information. A para-genetic approach, called virus-induced gene silencing (VIGS), offers a rapid means of gaining insight into gene function in plants. VIGS system could be used to suppress endogenous gene expression by infecting plants with a recombinant virus vector (VIGS vector) carrying host-derived sequence. Here, we describe the use of tobacco rattle virus (TRV)-based VIGS technique to study gene function in Nicotiana benthamiana and tomato.

The plant innate immune response is mediated by resistance (R) genes and involves hypersensitive response (HR) cell death. During resistance responses, the host undergoes net changes in the transcriptome. To understand these changes, we generated a whole genome transcript profile for RCY1-mediated resistance to cucumber mosaic virus strain Y (CMV-Y) in Arabidopsis. Using a very stringent selection criterion, we identified 444 putative factors belonging to nine different functional classes that show significant transcript regulation during Arabidopsis-CMV-Y interaction. Genes with unknown function formed the largest class. Other functional classes represented in the resistome include kinases and phosphatases, protein degradation machinery/proteases, transcriptional regulators, and others. Interestingly, several of the unknown function genes possess well characterized domains and secondly many genes encode small peptides with less than 100 amino acids. Analysis of 1.1 kb promoter regions of the 444 genes revealed that 9 out of the 12 known cis-binding elements are significantly associated with pathogen responsive cluster. Location and distribution of five prominent binding elements for select group of disease resistance related and unknown function genes is presented. The analysis also revealed 80 defense-responsive genes that might participate in R gene-mediated defense against both viral and bacterial pathogens. In addition, chromosome distribution of genes that respond to bacterial and viral pathogens suggests that they are located in small gene clusters and may be transcriptionally co-regulated. Exploring the precise function of the new genes identified in this analysis will offer new insights into plant defense.