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provided a visual phenotype of the silenced state (Palauqui & Vaucheret, 1995). De-
tailed analysis of the spatial and temporal pattern of the chlorotic phenotype in those
plants showed that development of silencing was a dynamic process (Palauqui et al. ,
1996). It was first apparent within one or several expanding clusters of cells that were
stochastically distributed in mature leaves. These silencing foci eventually reached
avein from which the co-suppressed state was transmitted to the new growth, fol-
lowing a pattern reminiscent of the phloem translocation (Palauqui et al. , 1996).
The existence of a signal molecule for RNA silencing was confirmed in elegant
experiments involving grafting of non-silenced transgenic plants onto the Nia co-
suppressed plants (Palauqui et al. , 1997). Grafting resulted in 100% transmission of
the silenced state from the rootsocks to transgenic scions that expressed an initially
nonsilenced Nia transgene. These experiments provided a clear demonstration that
a silencing signal emitted from the rootstocks was able to travel over long distances
through the vascular system and could trigger de novo silencing of a homologous
transgene in remote tissues of the plant. Interestingly, systemic patterns similar to
those of the Nia silenced plants had been observed with other tobacco-based silenc-
ing systems that involved co-suppression of S -adenosyl-L-methionine synthetase
and chitinase genes (Boerjan et al. , 1994; Kunz et al. , 1996). This suggested that
systemic RNA silencing was not a peculiarity of the Nia transgenic system.
Transgenic N. benthamiana plants expressing the GFP provided a powerful exper-
imental system to confirm that non-cell-autonomous silencing was indeed a broadly
applicable principle in plants (Voinnet & Baulcombe, 1997). Infiltration of a culture
of recombinant Agrobacterium was used to allow a local and transient production of
GFP RNA within a cluster of mesophyll cells in one or two leaves of the GFP trans-
genic plants. This treatment triggered first the loss of GFP fluorescence within the
infiltrated region of the leaf as a result of local PTGS of both the stably integrated and
ectopic GFP transgenes. Phenotypically, silencing was manifested as the appearance
of red fluorescent tissue under ultraviolet (UV) illumination, owing to chlorophyll
autofluorescence. This red (i.e. silenced) tissue was restricted to the Agrobacterium
infiltrated area, whereas the surrounding non-silenced tissue remained green under
UV light. Although silencing had been triggered locally, it moved to remote parts
of the plants, which eventually became uniformly silenced for GFP (Voinnet &
Baulcombe, 1997).
A remarkable aspect of the long-distance signalling elicited in the Nia and GFP
systems was its nucleotide sequence specificity, both in terms of initiation and prop-
agation. Hence, graft transmission of Nia silencing was not observed if the scions
were from transgenic plants expressing a transgene that was divergent in sequence
from the co-suppressed Nia transgene in the rootstocks (Palauqui et al. , 1997). In
addition, systemic silencing of GFP did not take place unless the Agrobacterium -
infiltrated cultures carried a transgene that was identical in sequence to the stably
integrated GFP transgene (Voinnet & Baulcombe, 1997). Therefore, to account for
its nucleotide sequence-specific effects, it was proposed that the systemic signal for
RNA silencing must have a nucleic acid component (Palauqui et al. , 1997; Voinnet
& Baulcombe, 1997).
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