The low molecular weight fraction of tomato plants inoculated with potato spindle tuber viroid (PSTVd) contains a population of short PSTVd-specific RNAs of either polarity. The main constituents were RNAs of 22 and 23 nt representing different domains of the viroid genome. The occurrence of such distinct RNA species indicated that the nuclear replicating PSTVd RNA induces post-transcriptional gene silencing. The short RNAs were slightly more abundant at 30 days post-inoculation than at later stages and were present in plants infected with a mild, severe or lethal isolate of PSTVD: There was no apparent correlation between the quantity of small PSTVd-specific RNAs and the degree of virulence of the viroid isolate.

Regulation of post-transcriptional gene expression by microRNAs (miRNA) has so far been validated for only a few mRNA targets. Based on the large number of miRNA genes and the possibility that one miRNA might influence gene expression of several targets simultaneously, the quantity of ribo-regulated genes is expected to be much higher. Here, we describe the web tool MicroInspector that will analyse a user-defined RNA sequence, which is typically an mRNA or a part of an mRNA, for the occurrence of binding sites for known and registered miRNAs. The program allows variation of temperature, the setting of energy values as well as the selection of different miRNA databases to identify miRNA-binding sites of different strength. MicroInspector could spot the correct sites for miRNA-interaction in known target mRNAs. Using other mRNAs, for which such an interaction has not yet been described, we discovered frequently potential miRNA binding sites of similar quality, which can now be analysed experimentally. The MicroInspector program is easy to use and does not require specific computer skills. The service can be accessed via the MicroInspector web server at .

Expression or introduction of double-stranded (ds)RNA in eukaryotic cells can trigger sequence-specific gene silencing of transgenes, endogenes, and viruses. Transgenic plants producing dsRNAs with homology to viral sequences are likely to exhibit pathogen-derived resistance to the virus. Cucumber mosaic virus (CMV), a very widespread virus with over 1,000 host species, has the natural ability to suppress silencing in order to establish infection. Here, we report the generation of transgenic tobacco lines, where a DNA transgene containing an inverted repeat of CMV cDNA had been introduced. Expression of this DNA construct delivered an RNA transcript that is able to form an intramolecular double strand. Transgenic plants were challenged with CMV. Three categories of plants could be discriminated: susceptible plants, which typically reacted with milder symptoms than the wild-type control; a "recovery" phenotype, in which newly emerging leaves were free of symptoms; and plants that showed complete resistance. Northern analysis showed that the expression of CMV dsRNA caused, in some transgenic lines, the generation of short RNAs characteristic of posttranscriptional gene silencing. Those lines were CMV resistant. The correlation between the detection of short RNAs and virus resistance provides a molecular marker that makes it possible to predict success in attempts to engineer virus resistance by dsRNA.

No abstract

For the identification of RNA-binding proteins that specifically interact with potato spindle tuber viroid (PSTVd), we subjected a tomato cDNA expression library prepared from viroid-infected leaves to an RNA ligand screening procedure. We repeatedly identified cDNA clones that expressed a protein of 602 amino acids. The protein contains a bromodomain and was termed viroid RNA-binding protein 1 (VIRP1). The specificity of interaction of VIRP1 with viroid RNA was studied by different methodologies, which included Northwestern blotting, plaque lift, and electrophoretic mobility shift assays. VIRP1 interacted strongly and specifically with monomeric and oligomeric PSTVd positive-strand RNA transcripts. Other RNAs, for example, U1 RNA, did not bind to VIRP1. Further, we could immunoprecipitate complexes from infected tomato leaves that contained VIRP1 and viroid RNA in vivo. Analysis of the protein sequence revealed that VIRP1 is a member of a newly identified family of transcriptional regulators associated with chromatin remodeling. VIRP1 is the first member of this family of proteins, for which a specific RNA-binding activity is shown. A possible role of VIRP1 in viroid replication and in RNA mediated chromatin remodeling is discussed.Viroids are the smallest known pathogens of higher plants. They consist of an infectious, single-stranded, covalently closed circular RNA molecule of ca. 250 to 400 nucleotides (nt). Viroids replicate autonomously, causing symptoms in specific host-viroid combinations of developmental disorders (9). Viroids are divided into two structural and functional distinct families. Potato spindle tuber viroid (PSTVd) is the type member of the Pospiviroidae and replicates in the nucleus. Avocado sunblotch viroid, the type member of the Avsunviroidae, replicates in the chloroplast of plant cells (14). Replication of both groups of viroids proceeds via RNA intermediates of complementary polarity. There is no evidence for viroid-encoded polypeptides. Thus, all genetic functions of the viroid replicon, such as cell entry and movement, replication, host specificity, and pathogenicity must be encoded in the RNA sequence and the resulting secondary structure, which is believed to provide specific signals for protein factors of the host. The predicted secondary structure of most viroids is a rod-like structure with five structural domains and an unusually high content of G and C bases (26). Despite the simple genome of viroids and the extensive knowledge of their structure and domains, little is known about host factors that interact with these structural elements.PSTVd replicates in the nucleus and accumulates eventually in the nucleolus as a circular single-stranded molecule, which is defined as positive polarity (44). Inhibition of PSTVd replication by alpha-amanitine indicates that viroid RNA strands of positive and negative polarity are most probably synthesized by the DNA-dependent RNA polymerase II (Pol II) (38,45). This enzyme can also accept PSTVd RNA as a template and "transcribe" it in vitro...