THE PLANT CELL, Vol 1, Issue 12 1165-1173, Copyright © 1989 by American Society of Plant Biologists
Determination of Sequence and Structural Requirements for Pathogenicity of a Cucumber Mosaic Virus Satellite RNA (Y-satRNA)
C. Masuta and Y. Takanami
Life Science Research Laboratory, Japan Tobacco Inc., 6-2, Umegaoka, Midori-ku, Yokohama 227, Japan
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[prime] 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.
