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the infiltrated leaves (source leaves) remained fully green fluorescent. At 30 days
post-infiltration, the stem and roots below the infiltrated leaves also showed GFP
silencing, thus indicating that the movement of the silencing signal was bidirectional
in the plant. Overall, this spatial distribution was consistent with movement occur-
ring through the phloem. The development of silencing in leaves was also similar
to the unloading of a phloem-transported dye through Class I, II and III veins of N.
benthamiana leaves (Roberts et al. , 1997; Voinnet et al. , 1998). Further support for
phloem transport of the signal came from experiments in which the GFP plants were
infiltrated in just one single leaf. These experiments differed from those described
above in which the infiltration involved several leaves on both sides of the plant.
At 1 month post-infiltration, GFP silencing in the stem was restricted to the side of
the originally infiltrated leaf. Shoots that had emerged from the silenced portion of
the stem were silenced, while those emerging from the non-silenced half were not,
a spatial distribution again similar to the movement of phloem-translocated dyes in
N. benthamiana (Roberts et al. , 1997; Voinnet et al. , 1998). Spontaneous Nia co-
suppression in N. tabaccum plants of Class II also occasionally started on one single
leaf situated at the bottom of the plant. In this case, the first leaves to be affected
by chlorosis were also all positioned on the same side of the plant (Palauqui et al. ,
1996).
The fact that systemic spread of RNA silencing is strongly influenced by source-
sink relationships between silenced and non-silenced organs explains why the ef-
ficiency of graft transmission of silencing of a chitinase transgene in tobacco was
highly dependent upon the grafting method used (Crete et al. , 2001). Thus, recipro-
cal grafts involving fully developed sections of plants or exchanges of mature tissue
plugs were unsuccessful for silencing transmission, even if the former method has
been routinely used to indicate the spread of systemic acquired resistance (see, for
instance, Crete et al. , 2001). It appears, from this and other studies, that the top-
grafting method in which a young vegetative shoot scion acts as a strong sink for
the transport of silencing signals from source leaves constitutes the most efficient
approach for transmission of systemic silencing, at least in Solanacaeous species.
The speed of translocation of the silencing signal from leaf to vasculature was
assessed in the GFP transgenic system by removal of the infiltrated leaf 1, 2, 3, 4
or 5 days after infiltration of the A. tumefaciens strain (Voinnet et al. , 1998). There
was systemic loss of GFP fluorescence in 10% of the plants if the infiltrated leaf
was removed 2 days post-infiltration. A progressively higher proportion of plants
exhibited systemic silencing when the infiltrated leaf was removed 3 days post-
infiltration or later. Similar values were obtained with bombardment experiments
carried out in the Class-II Nia tobacco plants (Palauqui & Balzergue, 1999). These
observations indicate that 2-3 days are sufficient for the signal to accumulate and
translocate into the phloem long-distance transport stream.
Molecular requirements for the long-distance transport and systemic effects of RNA
silencing. Triple-grafting experiments in which an intermediate section of non-
transgenic tobacco was inserted between silenced Nia rootstocks, and non-silenced
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