AN ANALYSIS OF SPONTANEOUS DELETION SITES IN SOIL-BORNE WHEAT MOSAIC-VIRUS RNA2

Plants mechanically inoculated with soil-borne wheat mosaic virus (SBW MV, Oklahoma isolate) generated several deleted forms of RNA2, the sma ller genomic RNA (3593 nt). Four naturally deleted forms were cloned b y RT-PCR methods and the sequences around each deletion site were comp ared. SBWMV RNA2 molecules were deleted for 519, 759, 964, or 1030 nt, respectively, each within the coal protein-readthrough domain. No com mon sequences were found flanking the 5'- and 3'-sites of deletion; ho wever, the 5'-site of each deletion lay in the region between genome c oordinates 1417-1465. The Delta 759nt clone isolated from virus-infect ed plants after a single mechanical passage was identical to a clone d escribed previously (J. Chen, S. A. MacFarlane, and T. M. A. Wilson, 1 994, Virology 202, 921-929) which became the dominant, stable form of SBWMV RNA2 after five or more serial mechanical transfers. In this stu dy, 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 c lones, which were found only during the first four passages, had delet ions at one of four alternative sites. After the fifth mechanical pass age 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 (Okla homa) is also reported. (C) 1995 Academic Press, Inc.