1 Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
2 University of Dundee, Dundee DD1 4NH, UK
3 University of Warwick HRI, Wellesbourne, Warwick CV35 9EF, UK
Correspondence
Michael Taliansky
mtalia{at}scri.sari.ac.uk
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ABSTRACT |
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These authors contributed equally to this paper.
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MAIN TEXT |
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To explore intracellular localization of the PLRV CP in PLRV-infected plants, we used transgenic Nicotiana benthamiana plants (CW1 line) expressing the full-length PLRV genome as a transgene which is strongly silenced (Barker et al., 2001). To allow PLRV to multiply in cells, these transgenic plants were either challenged with Tobacco mosaic virus (TMV) or heat-shocked as described by Taliansky et al. (2004)
. Immunogold electron microscopy (IGEM) confirmed that both treatments led to a significant increase in PLRV accumulation in big groups of cells, comparable to those detected by tissue immunoprinting (Barker et al., 2001
). Regardless of the means used to induce PLRV multiplication, the cytoplasm of infected cells contained large amounts of PLRV particles labelled with PLRV antibody (Fig. 2b
). The nuclei, and the nucleoli in particular, were also labelled with PLRV antibody, although virus particles were not seen in these structures, suggesting that the CP accumulates in them in a form other than virus particles (Fig. 2a, c
). Although in control (non-transgenic) plants, gold label was occasionally found in nuclei non-specifically associated with chromatin, it was never detected in nucleoli, indicating specific association of the PLRV CP with the nucleolus. In many cells, tubules were found that were labelled with PLRV antibodies. The tubules had a diameter of
1520 nm, were present in all cell types, including phloem parenchyma and companion cells, and resembled the fibrillar structures found by Shepardson et al. (1980)
in potato plants aphid-infected with PLRV or by Nass et al. (1998)
in BYDV-PAV-infected oat plants. These tubules were localized in the cytoplasm (Fig. 2a, d
) and nucleus, specifically targeting and often disturbing the integrity of the nucleolus (Fig. 2a, c
). In another approach, we used Pea enation mosaic virus-2 (PEMV-2) to help PLRV invade mesophyll tissues, as described by Ryabov et al. (2001)
. Intracellular distribution of the PLRV CP in doubly infected (PLRV+PEMV-2) wild-type N. benthamiana plants was similar to that described for CW1 transgenic plants (data not shown) and therefore was not affected by PEMV-2. The fact that the localization of PLRV CP did not depend on a factor used for development of PLRV multiplication strongly suggests that the nucleolar localization of the PLRV CP is intrinsic to the protein itself rather than being induced by other factors used for the induction of PLRV multiplication. Using transgenic Nicotiana tabacum plants expressing the full-length PLRV genome (Franco-Lara et al., 1999
), similar results were obtained, thus demonstrating that nucleolar targeting is not restricted to N. benthamiana (data not shown).
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To confirm that the arginine-rich region of PLRV CP (17PRRRRRQSLRRRANR31) can direct the nucleolar localization of a protein, we prepared a pGreen 0029 vector-based construct [pGR.CP(17-31)-GFP; Fig. 1] in which fragment 17PRRRRRQSLRRRANR31 was fused to the N-terminus of GFP. CLSM demonstrated a clear nucleolar localization of this chimeric protein (Fig. 3d
). We also investigated the ability of the 17PRRRRRQSLRRRANR31 sequence of the PLRV CP to functionally replace the NoLS of another plant virus protein localized to the nucleolus. As with the PLRV CP, the ORF3 protein encoded by Groundnut rosette virus (GRV) has been shown to target the nucleolus (Ryabov et al., 1998
, 2004
; Taliansky & Robinson, 2003
; Kim et al., 2004
). Arginine-to-asparagine substitutions in the region 108RPRRRAGRSGGMDPR122 of this protein abolished its accumulation in nucleoli (Ryabov et al., 2004
), suggesting a role for this domain in nucleolar localization. Therefore, we prepared a construct [pGR.ORF3/CP(17-31)-GFP; Fig. 1
] which expressed a GFP-tagged GRV ORF3 protein containing the PLRV CP 17PRRRRRQSLRRRANR31 sequence (putative NoLS) inserted in place of the GRV ORF3 NoLS (ORF3 108RPRRRAGRSGGMDPR122). CLSM showed that the ORF3/CP(17-31)-GFP protein generated as a result of such a replacement accumulated in the nucleolus (Fig. 3e
). In contrast, the GRV ORF3-GFP protein with deletion of the ORF3 NoLS [GRV ORF3
(108-122); Fig. 1
] used as a control was detected throughout the nucleoplasm and in some bodies associated with the nucleus, but not in the nucleolus (Fig. 3f
). Collectively, these results add further support to the suggestion that the 17PRRRRRQSLRRRANR31 region of the PLRV CP is directly involved in the nucleolar localization of the protein as a NoLS.
Taking into account the presence of NoLS in the CP portion of the PLRV P5, it could be expected that the P5 would also be localized in the nucleolus. Indeed, when the cDNA construct encoding the P5GFP fusion, with the leaky AUG termination codon deleted and a slightly truncated RT portion (CP-RT-GFP; Fig. 1), was expressed in plant tissues from the pGreen vector (pGR.CP-RT-GFP), green fluorescence was detected in the nucleolus (Fig. 3g
). However, these results seemingly contradict our previous observations, in which no fluorescence was detected in nucleoli of plants expressing the same CP-RT-GFP protein from the full-length PLRV genome (PLRV-GFP; Nurkiyanova et al., 2000
; see also Fig. 1
). In this situation, fluorescence was observed throughout the nucleoplasm, but not in the nucleolus (Nurkiyanova et al., 2000
; see also Fig. 3h
), suggesting that the P5 can be retained outside of, or redirected from, the nucleolus in the presence of the replicating virus. To test this idea, the CP-RT-GFP protein was produced in transgenic plants expressing a full-length PLRV genome (line CW1) using an Agrobacterium-mediated delivery system. In contrast to non-transgenic (control) N. benthamiana plants (Fig. 3g
), in CW1 transgenic plants green fluorescence was not targeted to the nucleolus (Fig. 3i
), confirming the ability of the replicating PLRV to prevent CP-RT-GFP accumulation in the nucleolus. In contrast to CP-RT-GFP, the nucleolar localization of PLRV CP-GFP was not affected by replicating PLRV (Fig. 3j
).
The nucleolus, a prominent subnuclear compartment, is regarded as the site of transcription and modification of rRNA and biogenesis of ribosomal subunits, and also participates in other aspects of cell function, including regulation of the cell cycle, cell growth, and ageing (Reviewed by Lamond & Earnshaw, 1998; Pederson, 1998
; Cockell & Gasser, 1999
; Carmo-Fonseca et al., 2000
; Olson et al., 2000
).
In this work we have shown that the PLRV CP region 17PRRRRRQSLRRRANR31 operates as a NoLS and directs the protein to the nucleolus. Because of the ability of GFP (used as a reporter protein fused to PLRV CP) to target nucleoplasm it is difficult to conclude whether the NoLS can also act as the NLS that targets the PLRV CP from the cytoplasm into the nucleus through the nuclear pore complex, or whether other PLRV CP sequences are involved in this process.
A specific role of nucleolar targeting of PLRV CP may be the modulation of host gene expression (for example by interacting with ribosomal components) to adapt it for the needs of virus infection. It is also possible that PLRV has developed the ability to recruit a nucleolar component(s) to exploit its function(s) in virus replication, assembly or movement.
The NoLS of the PLRV CP is located near the N-terminus of the protein. Based on known structural protein sequences of different icosahedral viruses, the N-terminal arginine-rich domain is likely to be involved in the interaction with viral RNA (Dolja & Koonin, 1991). Thus, the RNA binding domain and the NoLS may overlap and compete with each other to modulate the involvement of the CP in different virus functions.
As with the major CP, the extended P5 version also containing the NoLS is directed to the nucleolus when it is individually expressed in plant tissues using an Agrobacterium delivery system. However, in the presence of replicating PLRV, only the major CP localizes to the nucleolus. We suggest that P5 protein does not accumulate in the nucleolus in the presence of PLRV infection because PLRV RNA or PLRV-encoded or -induced factors retain this protein outside the nucleolus.
The involvement of the nucleolus in virus infections could also be a feature of other viruses. Indeed, a number of animal viruses interact with the nucleolus and its proteins (reviewed by Hiscox, 2002). Many of these interactions are not restricted to any particular type of virus, with examples from retroviruses, DNA viruses and RNA viruses. Furthermore, the plant virus GRV encodes a long-distance movement protein that is also targeted to the nucleolus (Taliansky & Robinson, 2003
; Kim et al., 2004
; Ryabov et al., 2004
).
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ACKNOWLEDGEMENTS |
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Received 11 April 2005;
accepted 27 June 2005.
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