Isolation of an RNA-Directed RNA Polymerase–Specific cDNA Clone from Tomato

Abstract

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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