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

RNA silencing is a remarkable type of gene regulation based on sequence-specific targeting and degradation of RNA. The term encompasses related pathways found in a broad range of eukaryotic organisms, including fungi, plants, and animals. In plants, it serves as an antiviral defense, and many plant viruses encode suppressors of silencing. The emerging view is that RNA silencing is part of a sophisticated network of interconnected pathways for cellular defense, RNA surveillance, and development and that it may become a powerful tool to manipulate gene expression experimentally.

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.

To address the role of small regulatory RNAs in rice development, we generated a large data set of small RNAs from mature leaves and developing roots, shoots, and inflorescences. Using a spatial clustering algorithm, we identified 36,780 genomic groups of small RNAs. Most consisted of 24-nt RNAs that are expressed in all four tissues and enriched in repeat regions of the genome; 1029 clusters were composed primarily of 21-nt small RNAs and, strikingly, 831 of these contained phased RNAs and were preferentially expressed in developing inflorescences. Thirty-eight of the 24-mer clusters were also phased and preferentially expressed in inflorescences. The phased 21-mer clusters derive from nonprotein coding, nonrepeat regions of the genome and are grouped together into superclusters containing 10-46 clusters. The majority of these 21-mer clusters (705/831) are flanked by a degenerate 22-nt motif that is offset by 12 nt from the main phase of the cluster. Small RNAs complementary to these flanking 22-nt motifs define a new miRNA family, which is conserved in maize and expressed in developing reproductive tissues in both plants. These results suggest that the biogenesis of phased inflorescence RNAs resembles that of tasiRNAs and raise the possibility that these novel small RNAs function in early reproductive development in rice and other monocots.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression in animals and plants. Comparative genomic computational methods have been developed to predict new miRNAs in worms, flies, and humans. Here, we present a novel single genome approach for the detection of miRNAs in Arabidopsis thaliana. This was initiated by producing a candidate miRNA-target data set using an algorithm called findMiRNA, which predicts potential miRNAs within candidate precursor sequences that have corresponding target sites within transcripts. From this data set, we used a characteristic divergence pattern of miRNA precursor families to select 13 potential new miRNAs for experimental verification, and found that corresponding small RNAs could be detected for at least eight of the candidate miRNAs. Expression of some of these miRNAs appears to be under developmental control. Our results are consistent with the idea that targets of miRNAs encompass a wide range of transcripts, including those for F-box factors, ubiquitin conjugases, Leucine-rich repeat proteins, and metabolic enzymes, and that regulation by miRNAs might be widespread in the genome. The entire set of annotated transcripts in the Arabidopsis genome has been run through findMiRNA to yield a data set that will enable identification of potential miRNAs directed against any target gene.