An Analysis of Spontaneous Deletion Sites in Soil-Borne Wheat Mosaic Virus RNA2

Abstract

Plants mechanically inoculated with soil-borne wheat mosaic virus (SBWMV, Oklahoma isolate) generated several deleted forms of RNA2, the smaller genomic RNA (3593 nt). Four naturally deleted forms were cloned by RT-PCR methods and the sequences around each deletion site were compared SBWMV RNA2 molecules were deleted for 519, 759, 964, or 1030 nt, respectively, each within the coat protein-readthrough domain. No common sequences were found flanking the 5′- and 3′-sites of deletion; however, the 5′-site of each deletion lay in the region between genome coordinates 1417-1465. The Δ759nt clone isolated from virus-infected plants after a single mechanical passage was identical to a clone described previously (J. Chen, S. A. MacFarlane, and T. M. A. Wilson, 1994, Virology 202, 921-929) which became the dominant, stable form of SBWMV RNA2 after five or more serial mechanical transfers. In this study, a total of 33 independent, RT-PCR clones with an apparent deletion of 759 nt, isolated after one to seven mechanical passages, were also sequenced and their precise deletion sites were compared. Twenty-six clones contained the original "stable," 759-nt deletion, whereas 7 clones, which were found only during the first four passages, had deletions at one of four alternative sites. After the fifth mechanical passage the stable 759-nt deletion dominated. The results also show that, in general, the smaller deletions are not intermediates in the larger deletion process. The complete nucleotide sequence of SBWMV RNA2 (Oklahoma) is also reported.