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Plant Cell, Vol. 10, 2087-2102, December 1998, Copyright © 1998, American Society of Plant Physiologists

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

Winfried Schiebela, Thierry Pélissiera, Leonhard Riedelb, Sabine Thalmeira, Rosemarie Schiebela, Dirk Kempea, Friedrich Lottspeichc, Heinz L. Sängera, and Michael Wasseneggera
a Max-Planck-Institut für Biochemie, Abteilung Viroidforschung, D-82152 Martinsried, Germany
b Botanisches Institut, Biologie I der RWTH Aachen, Worringer Weg 1, Germany
c Max-Planck-Institut für Biochemie, Abteilung Proteinchemie, D-82152 Martinsried, Germany

Correspondence to: Michael Wassenegger, wasseneg{at}biochem.mpg.de (E-mail), 49-89-8578-2937 (fax).

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.




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Copyright © 1998 by the American Society of Plant Biologists