RNA as a signalling molecule - Intercellular Communication in Plants - page 55

could result either from a failure of replication in these organs or from an inhibition
of transport into those tissues. To distinguish between these possibilities, Zhu et al.
(2002) used transgenic Nicotiana benthamiana that constitutively expressed the
PSTVd cDNA under the control of the cauliflower mosaic virus (CaMV) 35S pro-
moter (Hu et al. , 1997). In situ hybridization analysis using an RNA probe specific
for the minus strand of PSTVd (diagnostic of its replication) showed that PSTVd
could in fact replicate in all floral organs. Therefore its specific absence in ovaries,
stamens and petals in mechanically inoculated plants was probably due to restriction
of its trafficking in parts of the floral apex (Zhu et al. , 2002).
Restriction of long-distance viroid movement has also been indicated with hop
stunt viroid (HSVd). HSVd-free plants could be regenerated by culture of an 0.2-mm
shoot tip excised from infected plants, whereas plants generated from larger shoot
tips contained HSVd, suggesting that HSVd is absent from the SAM of infected hop
plants (Momma & Takahashi, 1983). Indeed, in situ hybridization failed to detect
PSTVd in the SAM, even if the viroid was present in the vascular tissues (most
likely the procambium and/or the protophloem) immediately below the SAM (Zhu
et al. , 2001).
3.2.3 Cellular factors involved in viroid movement
3.2.3.1 Phloem Lectin 2
Only two proteins that may play a role in viroid RNA trafficking have been char-
acterized so far. The first one is the phloem lectin 2 (PP2), one of the two most
abundant proteins found in C. maxima phloem exudates (Beyenbach et al. , 1974).
In vitro , this protein binds to HSVd RNA, PSTVd RNA as well as to other larger and
less structured RNAs (including RNA with a polyA tail) but not to DNA (Gomez &
Pallas, 2001; Owens et al. , 2001). Using genus-specific probes/antibodies and inter-
genic grafts, it was shown that PP2, but not its mRNA, is capable of long-distance
trafficking through the phloem. PP2 was synthesized within the companion cells of
the rootstocks, from which it moved into the SE and was subsequently transported
in the phloem stream towards the scion tissues. Upon delivery PP2 was found to ac-
cumulate in CCs, indicating movement out of the scions' SE (Golecki et al. , 1999).
Interestingly, the PP2 of Cucurbitaceae spp. has the capacity to modify plasmodes-
mata SEL and to move from cell to cell when it is microinjected into mesophyll cells
of cucurbit cotyledons (Balachandran et al. , 1997). Altogether, these observations
indicate that PP2 has the attributes that would be normally required for translocation
of not only viroids but also plant RNAs. Further experiments in vivo are necessary
to demonstrate this unequivocally.
3.2.3.2 VirP1
The second protein, named VirP1, was isolated using an RNA-ligand screening
procedure from an expression library of Lycopersicum esculentum (Martinez de
Alba et al. , 2003). VirP1 contains a bipartite nuclear localization signal, a bro-
modomain found in several eukaryotic transcription factors (Haynes et al. , 1992;
Jeanmougin et al. , 1997) and an RNA-binding domain. It was shown that this