Previous analysis of potato spindle tuber viroid (PSTVd) RNA-infected tobacco plants has suggested that an RNA-DNA interaction could trigger de novo methylation of PSTVd transgene sequences. Using the genomic sequencing technique, the methylation pattern associated with the RNA-directed DNA methylation process has been characterized. Three different PSTVd transgene constructs all showed a similar pattern of methylation. Most of the cytosines at symmetrical as well as non-symmetrical positions appeared to be methylated in both DNA strands of the viroid sequences. Heavy methylation was mostly restricted to the viroid cDNA sequences. Flanking DNA regions immediately adjacent to the viroid cDNA displayed a lower but significant level of cytosine methylation. The observation that the heavy methylation was essentially co-extensive with the length of the PSTVd cDNA sequences provided evidence that a direct RNA-DNA interaction can act as a strong and highly specific signal for de novo DNA methylation. These data also confirmed that de novo methylation was not limited to canonical CpG and CpNpG sites, but can also involve all the cytosine residues located in the genomic region where the RNA-DNA interaction takes place.

A 3600-bp RNA-directed RNA polymerase (RdRP)-specific cDNA comprising an open reading frame (ORF) of 1114 amino acids was isolated from tomato. The putative protein encoded by this ORF does not share homology with any characterized proteins. Antibodies that were raised against synthetic peptides whose sequences have been deduced from the ORF were shown to specifically detect the 127-kD tomato RdRP protein. The immunoresponse to the antibodies correlated with the enzymatic activity profile of the RdRP after chromatography on Q-, poly(A)-, and poly(U)-Sepharose, hydroxyapatite, and Sephadex G-200 columns. DNA gel blot analysis revealed a single copy of the RdRP gene in tomato. RdRP homologs from petunia, Arabidopsis, tobacco, and wheat were identified by using polymerase chain reaction. A sequence comparison indicated that sequences homologous to RdRP are also present in the yeast Schizosaccharomyces pombe and in the nematode Caenorhabditis elegans. The previously described induction of RdRP activity upon viroid infection is shown to be correlated with an increased steady state level of the corresponding mRNA. The possible involvement of this heretofore functionally elusive plant RNA polymerase in homology-dependent gene silencing is discussed.

A 3600-bp RNA-directed RNA polymerase (RdRP)-specific cDNA comprising an open reading frame (ORF) of 1114 amino acids was isolated from tomato. The putative protein encoded by this ORF does not share homology with any characterized proteins. Antibodies that were raised against synthetic peptides whose sequences have been deduced from the ORF were shown to specifically detect the 127-kD tomato RdRP protein. The immunoresponse to the antibodies correlated with the enzymatic activity profile of the RdRP after chromatography on Q-, poly(A)-, and poly(U)-Sepharose, hydroxyapatite, and Sephadex G-200 columns. DNA gel blot analysis revealed a single copy of the RdRP gene in tomato. RdRP homologs from petunia, Arabidopsis, tobacco, and wheat were identified by using polymerase chain reaction. A sequence comparison indicated that sequences homologous to RdRP are also present in the yeast Schizosaccharomyces pombe and in the nematode Caenorhabditis elegans. The previously described induction of RdRP activity upon viroid infection is shown to be correlated with an increased steady state level of the corresponding mRNA. The possible involvement of this heretofore functionally elusive plant RNA polymerase in homology-dependent gene silencing is discussed. INTRODUCTIONRNA-directed RNA polymerase (RdRP) from healthy tomato leaf tissue seems to represent a plant-specific and hence exceptional nucleic acid-synthesizing enzyme because higher plants are the only eukaryotes in which the presence of a cellular RdRP has been unambiguously demonstrated to date (for discussion, see Schiebel et al., 1993aSchiebel et al., , 1993b. RdRP activity has been detected in Chinese cabbage (Astier-Manifacier and Cornuet, 1971), cauliflower (Astier-Manifacier and Cornuet, 1978), tobacco (Duda et al., 1973;Duda, 1979;Takanami and Fraenkel-Conrat, 1982), tomato (Boege and Sänger, 1980), cowpea (Dorssers et al., 1982), and cucumber (Khan et al., 1986), but only the RdRP from tomato leaf tissue has been isolated and characterized with respect to its physicochemical (Schiebel et al., 1993a) and in vitro catalytic (Schiebel et al., 1993b) properties. These cellular RdRPs should not be mistaken for RNA-dependent RNA polymerases (EC 2.7.7.48), which become detectable when bacteria and eukaryotes are infected with RNA viruses. RNA-dependent RNA polymerases mediate viral RNA replication and are therefore much more appropriately called virus RNA replicases.Despite all of these studies, the origin and the actual biological function(s) of plant-encoded RdRP have remained unresolved and are enigmatic because its cognate template(s) and in vivo transcription products remain unknown. Nevertheless, we surmised (Schiebel et al., 1993b) that in the cell, RdRP might be of paramount importance because it transcribes from corresponding RNA sequences small RNA molecules that control the synthesis of nucleic acids and their translation into proteins.Studies on the induction of a highly specific antiviral state in transgenic plants led to a hypothesis that cellular RdRP c...

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Covalently closed circular (+) RNA of the potato spindle tuber viroid (PSTVd) can efficiently dimerize noncovalently upon heating and slow cooling in the presence of monovalent cations or Mg2+. In vitro transcription of subgenomic fragments reveals that the ability to dimerize resides in the "upper strand" of its self-complementary rod-like structure. Nuclease probing of these fragments, namely, molecules spanning either the upper or the lower strand of PSTVd, confirms the existence of the previously proposed hairpins I-III, of which hairpin I might contain noncanonical G.A and A.A base pairs. In addition, the upper and lower (+) strands contain large hairpin loops consisting of stretches rich in either adenosine or uridine. Dimerization of the upper (+) strand results in a nuclease-resistant core encompassing hairpin I and is inhibited by an antisense oligonucleotide spanning the entire hairpin; this palindromic domain thus represents the dimerization site. When upper and lower strands were heated and cooled together, no annealing to a viroid-like duplex of both molecules occurs, only dimerization of the upper strand. Therefore, the dimerization hairpin of viroid RNA represents a unique conformational signal that is homologous to similar regions in the human immunodeficiency virus and other retroviruses.