Agriculture Reference
In-Depth Information
interpretation of these observations is that the signal moves from cell to cell via
the symplasm through the plasmodesmata.
A particularly striking aspect of silencing movement in tissues that are competent
for signal perception and mRNA degradation is the extent and uniformity of the
silenced phenotype. This aspect is even more apparent in bombardment experiments
in which complete systemic silencing in the new growth can be elicited with a
limited quantity of trigger nucleic acid within a few cells of a single, mature leaf
(Voinnet et al. , 1998; Palauqui & Balzergue, 1999; Klahre et al. , 2002). Moreover,
transmission of silencing becomes rapidly independent of the initially bombarded
leaves, as shown by the leaf detachment experiments described in Section 3.4.1.1.
Taken together, these observations indicate that movement of silencing involves a
relay-amplification process, whereby secondary signal molecules are synthesized
as silencing moves away from its sites of initiation. This property is most clearly
evidenced upon phloem unloading of the signal, as silencing initially restricted
around the veins progressively invades the entire leaf lamina.
As explained previously, RdRps have been implicated in the amplification of
silencing effector molecules, both in plants and in C. elegans (Dalmay et al. , 2000;
Mourrain et al. , 2000; Sijen et al. , 2001). An early indication of the potential in-
volvement of RdRps in the extensive nature of silencing movement in plants came
from biolistic experiments carried out in the GFP transgenic N. benthamiana plants
(Voinnet et al. , 1998). In those experiments, the DNA constructs used to initiate sys-
temic silencing were PCR-amplified fragments with sequence of the 5 part of the
GFP cDNA. It was shown that silencing developing in systemic leaves was targeted
not only against the region of the GFP transcript corresponding to the bombarded
DNA, but also against regions located outside of the sequence used to trigger RNA
silencing. This 'transitive silencing' is diagnostic of the activity of RdRps (Sijen
et al. , 2001; Vaistij et al. , 2002) and this prompted the evaluation of the contribution
of SDE1 - the Arabidopsis RdRp required for initiation of sense transgene silencing
(Dalmay et al. , 2000; Mourrain et al. , 2000) - to the extent of silencing cell-to-cell
movement.
To recreate in Arabidopsis the phloem unloading and subsequent cell-to-cell
spread of silencing observed in N. benthaminana leaves, a system was set up
that involved Arabidopsis plants carrying a constitutively expressed GFP transgene
(Himber et al. , 2003). A second set of plants was from an isogenic GFP line carry-
ing, in addition to the GFP transgene, a null mutation in the gene for SDE1. Both
types of plants were supertransformed with a second construct in which a pan-
handled transgene with GFP sequences was cloned under the control of the phloem
companion cell-specific AtSUC2 promoter. Thus, silencing of the GFP transgene
was triggered specifically in the vasculature of those plants, allowing a precise as-
sessment of its movement into the neighbouring mesophyll cells in both wt and
sde1 backgrounds (see Plate 3.2A, following page 146). All of the transformants
with wt copies of SDE1 exhibited a uniformly red (i.e. GFP silenced; Plate 3.2B)
phenotype, whereas 80% of the sde1 mutant plants showed a silencing phenotype
that was centred around the vascular network, affecting a near constant number of
Search WWH ::




Custom Search