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THE PLANT CELL, Vol 8, Issue 12 2277-2294, Copyright © 1996 by American Society of Plant Biologists
RNA-Mediated Virus Resistance: Role of Repeated Transgenes and Delineation of Targeted Regions
T. Sijen, J. Wellink, J. B. Hiriart and A. van Kammen
Department of Molecular Biology, Wageningen Agricultural University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands
Resistance to cowpea mosaic virus (CPMV) in transgenic Nicotiana
benthamiana plants is RNA mediated. In resistant CPMV movement protein (MP)
gene-transformed lines, transgene steady state mRNA levels were low,
whereas nuclear transcription rates were high, implying that a
post-transcriptional gene-silencing mechanism is at the base of the
resistance. The silencing mechanism can also affect potato virus X (PVX)
RNAs when they contain CPMV MP gene sequences. In particular, sequences
situated in the 3[prime] part of the transcribed region of the MP transgene
direct elimination of recombinant PVX genomes. Remarkably, successive
portions of this 3[prime] part, which can be as small as 60 nucleotides,
all tag PVX genomes for degradation. These observations suggest that the
entire 3[prime] part of the MP transgene mRNA is the initial target of the
silencing mechanism. The arrangement of transgenes in the plant genome
plays an important role in establishing resistance because the frequency of
resistant lines increased from 20 to 60% when transformed with a transgene
containing a direct repeat of MP sequences rather than a single MP
transgene. Interestingly, we detected strong methylation in all of the
plants containing directly repeated MP sequences. In sensitive lines, only
the promoter region was found to be heavily methylated, whereas in
resistant lines, only the transcribed region was strongly methylated.
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