Determination of sequence and structural requirements for pathogenicity of a cucumber mosaic virus satellite RNA...

We describe the use of biologically active cDNA clones to investigate genetic determinants of a satellite RNA that modulates symptoms normally induced by its helper virus, cucumber mosaic virus (CMV). For this purpose, we have investigated a CMV satellite RNA (Y-satRNA) that induces bright yellow symptoms on tobacco and necrosis on tomato. To determine the pathogenicity-modulating domain of Y-satRNA, several insertion and deletion mutants were created by using various restriction sites in the cDNA of Y-satRNA, and RNA transcripts derived from the clones were mixed with CMV and used to inoculate plants. Although the satellite RNA was able to tolerate small insertions (as much as 4 bases at present), small deletions were deleterious, indicating that the sequence requirements for viability of the satellite RNA are relatively inflexible. Biological activity assays of chimeric satellite RNAs between Y-satRNA and a non-necrogenic satellite RNA, T73-satRNA, suggested that only two (or at least one of two) specific bases (positions 318 and 325) in the 3' region direct the necrogenic property of Y-satRNA. Sequences involved in production of yellow symptoms were investigated by constructing chimeras between Y-sat cDNA and cDNA of a satellite RNA designated S19-satRNA. S19-satRNA has considerable homology to Y-satRNA but does not elicit yellow symptoms on tobacco. Chimeric clones were constructed by using a BstXI site that cuts within a stable secondary structure in the region between positions 100 and 200 (region Y). The results of infectivity tests with RNA transcripts suggest that formation of a secondary structure in region Y may be involved in induction of yellow symptoms as well as viability of Y-satRNA.