Nucleotide sequence, genetic organization and expression strategy of the double-stranded RNA associated with the '447' cytoplasmic male sterility trait in Vicia faba.

Abstract

The entire nucleotide sequence of the double-stranded (ds) RNA associated with the unconventional '447' cytoplasmic male sterility (CMS) trait in Vicia faba was determined from overlapping cDNA clones and by RT-PCR. Confirming previous observations, it was found that the negative-strand was continuous and 17,635 nt long, while the positive-strand featured an interruption, probably a nick, that could potentially define two subgenomic RNAs of 2735 nt and 14,900 nt, with the smaller RNA being located on the 5' side. The entire positive-strand could encode a single in-frame ORF starting at the first AUG at position 42-44 and ending with a TGA at 17,517-17,519. This long potential polypeptide with a predicted molecular mass of 654,109 is the largest described to date in the plant kingdom and contains conserved amino acid sequence motifs typical of viral helicases and RNA-dependent RNA polymerases (RDRP). Only limited sequence homology was detected with the ORF B encoded by the hypovirulence-associated dsRNA of chestnut blight fungus, a dsRNA replicon similarly contained in host-derived membranous vesicles and considered to share a common ancestry with potyviruses. By contrast, the helicase and RDRP domains were in the same respective arrangement and shared extensive sequence homologies with those identified in the polyprotein encoded by the dsRNA isolated from Japonica rice, another dsRNA replicon featuring a specific nick in the positive-strand. Although no proteolytic self-cleavage activity has yet been demonstrated, it appears likely that this long ORF is a polyprotein that undergoes proteolytic maturation, with one of the polypeptides derived by self-cleavage being the determinant of the CMS trait.