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by localized virus replication was primed not only against the viral genome but also
against the corresponding endogenous transcripts, therefore generating a visual sys-
temic silencing phenotype. Systemic silencing was apparent only when replication
competent PVX was used as inoculum (Voinnet
et al.
, 2000). Because PVX has
an RNA genome that is replicated via RNA intermediates, this result is consistent
with the idea that the observed signal, or at least part of it, has an RNA compo-
nent.
Further support for an antiviral role of RNA silencing came from the finding of
viral-encoded proteins that are specifically directed against systemic, as opposed to
intracellular RNA, silencing. For instance, systemic silencing from PVX could be
achieved only if the ORF for the 25-kDa movement protein (P25) was deleted in the
viral genome, whereas silencing within the inoculated region remained unaffected
by the presence or absence of P25 (Voinnet
et al.
, 2000). Likewise, the 2b protein
of cucumber mosaic virus was found to have a poor effect on intracellular silenc-
ing triggered by a transgene. However, triple-grafting experiments demonstrated
unambiguously that 2b was a very potent inhibitor of the physical long-distance
trafficking of silencing (Guo & Ding, 2002). A detailed analysis of the contribution
of silencing suppression to systemic viral infection was provided in a recent study
of the P19 protein of cymbidium ringspot virus (CymRSV) (Havelda
et al.
, 2003).
Previous work had already established that the P19-defective CymRSV accumulates
to wt levels in single cells, suggesting that P19 targets a non-cell autonomous step
of RNA silencing (Silhavy
et al.
, 2002). Using a combination of
in situ
hybridiza-
tion and immunohistochemistry, the authors have now convincingly demonstrated
that the lack of P19 does not alter the phloem-dependent movement or the replica-
tion of CymRSV in and around the vascular bundles of systemic leaves (Havelda
et al.
, 2003). However, it prevents further viral invasion of the leaf lamina, which,
although virus free, exhibits nucleotide sequence-specific resistance to secondary
infection with either CymRSV or an unrelated recombinant virus carrying sequence
identity to CymRSV (Szittya
et al.
, 2002). Therefore, P19 most likely prevents the
onset or movement of a mobile virus induced silencing signal. Upon phloem un-
loading of the pathogen, the signal primes the destruction of viral RNAs ahead of
the infection front. As observed for P19-deficient CymRSV, deletion of P25 caused
PVX to be confined within the vasculature of systemically infected leaves. Thus,
the movement-promoting function of P25 may also be explained in terms of pre-
venting the synthesis or spread of a virus-induced silencing signal ahead of the
virus. However, P25 has also been shown to facilitate cell-to-cell trafficking of PVX
by increasing plasmodesmata size-exclusion limit in infected cells (Angell
et al.
,
1996). Therefore, PVX movement might depend on both physical gating of the viral
genome and inhibition of an antiviral signal.
3.3.8.2 A role in non-cell autonomous regulation of gene expression?
Since plant miRNAs resemble siRNAs, both biochemically and functionally (Bartel
& Bartel, 2003), the possibility of their movement appears to be a legitimate ques-
tion in light of the data summarized in this chapter. Limited silencing movement
