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THE PLANT CELL, Vol 9, Issue 8 1411-1423, Copyright © 1997 by American Society of Plant Biologists
Characterization of Post-Transcriptionally Suppressed Transgene Expression That Confers Resistance to Tobacco Etch Virus Infection in Tobacco
M. M. Tanzer, W. F. Thompson, M. D. Law, E. A. Wernsman and S. Uknes
Department of Botany, North Carolina State University, Raleigh, North Carolina 27695-7612
Tobacco lines expressing transgenes that encode tobacco etch virus (TEV)
coat protein (CP) mRNA with or without nonsense codons give rise to
TEV-resistant tissues that have reduced levels of TEV CP mRNA while
maintaining high levels of transgene transcriptional activity. Two
phenotypes for virus resistance in the lines containing the transgene have
been described: immune (no virus infection) and recovery (initial systemic
symptoms followed by gradual recovery over several weeks). Here, we show
that at early times in development, immune lines are susceptible to TEV
infection and accumulate full-length CP mRNA. Therefore, immune lines also
exhibit meiotic resetting, as is seen in the recovery lines, providing
molecular evidence for a common mechanism of gene silencing and virus
resistance in both cases. We also investigated the characteristics of two
sets of low molecular weight RNAs that appear only in silenced tissue. One
set has nearly intact 5[prime] ends, lacks poly(A) tails, and is associated
with polyribosomes; the second set contains the 3[prime] end of the mRNA.
Treating silenced leaf tissue with cycloheximide resulted in decreased
levels of full-length mRNA and an increase in the levels of the low
molecular weight RNAs, supporting a cytoplasmic decay mechanism that does
not require ongoing translation. Surprisingly, mRNA from the transgene
containing nonsense codons was associated with more ribosomes than
expected, possibly resulting from translation from a start codon downstream
of the introduced translational stop codons. We present a hypothesis for
transgene/viral RNA degradation in which RNA degradation occurs in the
cytoplasm while in association with polyribosomes.
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