Agriculture Reference
In-Depth Information
protein interacts specifically
in vitro
with PSTVd RNA and immunoprecipitation
from PSTVd-infected tissues confirmed that this complex does exist
in vivo
. Using
the three-hybrid system, interaction domains between VirP1 and PSTVd RNA were
mapped to an asymmetric internal loop called the
RY motif
(Gozmanova
et al.
, 2003;
Maniataki
et al.
, 2003). This motif is contained within an approximately 80 nt long,
bulged hairpin at the right-end side of the PSTVd RNA.
Sequence alterations in PSTVd (Hammond, 1994) had been described, that pre-
vented the structural interactions in the terminally located RY motif. The result-
ing mutant, PSTVd-R
,was not infectious if it was mechanically inoculated onto
tomato leaves, but it was able to replicate in crown galls developing on plants that
had been injected with recombinant
Agrobacterium
carrying the PSTVd-R
+
cDNA.
Systemic infection occasionally occurred in those plants, because of the emergence
of revertants. However, sequence analysis indicated the emergence of only one class
of variant (apart from the wt PSTVd) in which the original base pair interaction at
the RY motif was restored despite sequence alterations in the terminal right loop
(Hammond, 1994).
In vivo
and
in vitro
binding assays have indeed shown that the
R
+
+
mutation impedes interaction of VirP1 with the PSTVd RNA, whereas this in-
teraction is partially restored in variant 1 (Maniataki
et al.
, 2003). Taken together,
these results strongly support the involvement of VirP1 in PSTVd RNA movement.
Note that it was shown that VirP1 is expressed in petals (Martinez de Alba
et al.
,
2003), organs that have been found free of PSTVd (see above). Therefore, a rea-
son other than the lack of VirP1 accounts for the absence of PSTVd unloading in
petals.
At first, the nuclear localization signal of VirP1 and its homology with some
transcription factors was difficult to reconcile with its role in PSTVd movement.
However, PSTVd replication occurs in the nucleus and we have seen in the previous
sections of this chapter that mutations of the NLS found in the maize transcription
factor KN1 significantly inhibit transport of the
KN1
mRNA through plasmodesmata
in mesophyll cells (Lucas
et al.
, 1995; reviewed in Wu
et al.
, 2002). This analogy
could suggest that common mechanisms are involved in KN1-mediated trafficking of
the KN1 mRNA and the VirP1-mediated trafficking of viroid RNAs and is consistent
with the proposal that VirP1 may be broadly implicated in movement of endogenous
transcripts. Based on this idea, it is of note that several viroids induce symptoms
on their host that are reminiscent of the developmental anomalies usually elicited
by mutations in bromodomain-containing proteins. Thus, it is possible that the
developmental aberration of viroid-infected plants result, at least partly, from a
competition between the binding of cellular RNA and of viroid RNA by VirP1.
3.3
Intercellular movement of RNA silencing
3.3.1 Mechanism of RNA silencing
This section highlights the most significant steps that contributed to our current
understanding of the core reactions of RNA silencing in eukaryotes and, more
