Agriculture Reference
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triggering systemic silencing. Importantly, this study also established that a single
species of synthetic siRNA duplex was nearly as competent in triggering systemic
silencing as was the full-length dsRNA, whereas there was no effect if the individual
siRNA strands were bombarded on their own (Klahre
et al.
, 2002).
Collectively, these results indicate that virtually any type of nucleic acid has
the potential to trigger systemic silencing, provided it bares sequence homology
with the mRNA of the target transgene. Importantly, the second set of experiments
(Klahre
et al.
, 2002) shows that RNA molecules are directly capable of inducing this
phenomenon, excluding the possible complications that are linked to transcription
of the delivered DNA, or the delivered DNA itself. Therefore, it is likely that RNA
is the trigger molecule of systemic silencing, which is consistent with the observa-
tion that the presence of a promoter significantly enhanced silencing initiation by
DNA-based constructs. Moreover, the fact that the two key RNA components of
the silencing pathway (i.e. dsRNA and the siRNAs that derive from this molecule;
see Sections 3.3.1.2 and 3.3.1.3) were potent and direct triggers of systemic silenc-
ing suggests that the other RNA molecules tested in those experiments (sense and
antisense RNAs) were probably ultimately converted into ds- and siRNAs, presum-
ably through the action of a cellular RdRp akin to SDE1. It remains unclear how
promoterless DNA plasmid constructs or PCR-amplified DNA fragments triggered
systemic RNA silencing (Brigneti
et al.
, 1998; Palauqui & Balzergue, 1999), but
spurious transcription from endogenous promoters upon their integration into the
genome could be involved.
3.3.4 Propagation of systemic RNA silencing
3.3.4.1 Long-distance movement of RNA silencing
Long-distance transport occurs in the phloem.
Long-distance movement of
molecules in plants can occur through the xylem and the phloem conduits, both of
which are restored upon graft junctions. However, only the phloem distributes the
photoassimilates throughout the plant, following a specific pattern whereby mature,
photosynthetically autonomous organs export - but do not import - carbohydrates.
These tissues are said to be 'source' of phloem. Conversely, import of carbohydrates
occurs in the new growth (meristems, primordia, young leaves), which has not yet
reached photosynthetic autonomy and is therefore a 'sink' of phloem. The interme-
diate situation is the sink-to-source transition, which typically occurs in leaves in a
basipetal direction down the leaf axis towards the petiole.
Detailed analysis of the pattern and dynamics of silencing activated upon
Agrobacterium
leaf infiltration of the GFP transgenic
N. benthamiana
revealed
that at 20 days post-infiltration of lower leaves, silencing was strongest in sys-
temic, young developing leaves (sink leaves) and was very pronounced in the shoot
tips, although the meristematic regions were still green fluorescent (Voinnet
et al.
,
1998). There was also silencing in upper leaves that were already expanding at the
time of infiltration (sink-source transition leaves), but it was fainter and less exten-
sive than in the new growth. In contrast, the leaves immediately above and below
