Isolation of an RNA-Directed RNA polymerase-specific cDNA clone from tomato

A 3600-bp RNA-directed RNA polymerase (RdRP)-specific cDNA comprising an op en reading frame (ORF) of 1114 amino acids was isolated from tomato. The pu tative protein encoded by this ORF does not share homology with any charact erized proteins. Antibodies that were raised against synthetic peptides who se sequences have been deduced from the ORF were shown to specifically dete ct the 127-kD tomato RdRP protein. The immunoresponse to the antibodies cor related with the enzymatic activity profile of the RdRP after chromatograph y on Q-, poly(A)-, and paly(U)-Sepharose, hydroxyapatite, and Sephadex G-20 0 columns. DNA gel blot analysis revealed a single copy of the RdRP gene in tomato. RdRP homologs from petunia, Arabidopsis, tobacco, and wheat were i dentified by using polymerase chain reaction. A sequence comparison indicat ed that sequences homologous to RdRP are also present in the yeast Schizosa ccharomyces pombe and in the nematode Caenorhabditis elegans. The previousl y 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.