Cajal bodies and the nucleolus are required for a plant virus systemic infection
2007; Springer Nature; Volume: 26; Issue: 8 Linguagem: Inglês
10.1038/sj.emboj.7601674
ISSN1460-2075
AutoresSang Hyon Kim, Eugene V. Ryabov, Natalia O. Kalinina, Daria V. Rakitina, Trudi Gillespie, Stuart MacFarlane, Sophie Haupt, John W. Brown, Michael Taliansky,
Tópico(s)Plant Disease Resistance and Genetics
ResumoArticle5 April 2007free access Cajal bodies and the nucleolus are required for a plant virus systemic infection Sang Hyon Kim Sang Hyon Kim Scottish Crop Research Institute, Invergowrie, Dundee, UK Search for more papers by this author Eugene V Ryabov Eugene V Ryabov HRI, University of Warwick, Wellesbourne, Warwick, UK Search for more papers by this author Natalia O Kalinina Natalia O Kalinina Scottish Crop Research Institute, Invergowrie, Dundee, UK AN Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Daria V Rakitina Daria V Rakitina Scottish Crop Research Institute, Invergowrie, Dundee, UK AN Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Trudi Gillespie Trudi Gillespie Scottish Crop Research Institute, Invergowrie, Dundee, UK Search for more papers by this author Stuart MacFarlane Stuart MacFarlane Scottish Crop Research Institute, Invergowrie, Dundee, UK Search for more papers by this author Sophie Haupt Sophie Haupt School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author John WS Brown John WS Brown Scottish Crop Research Institute, Invergowrie, Dundee, UK Search for more papers by this author Michael Taliansky Corresponding Author Michael Taliansky Scottish Crop Research Institute, Invergowrie, Dundee, UK Search for more papers by this author Sang Hyon Kim Sang Hyon Kim Scottish Crop Research Institute, Invergowrie, Dundee, UK Search for more papers by this author Eugene V Ryabov Eugene V Ryabov HRI, University of Warwick, Wellesbourne, Warwick, UK Search for more papers by this author Natalia O Kalinina Natalia O Kalinina Scottish Crop Research Institute, Invergowrie, Dundee, UK AN Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Daria V Rakitina Daria V Rakitina Scottish Crop Research Institute, Invergowrie, Dundee, UK AN Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Trudi Gillespie Trudi Gillespie Scottish Crop Research Institute, Invergowrie, Dundee, UK Search for more papers by this author Stuart MacFarlane Stuart MacFarlane Scottish Crop Research Institute, Invergowrie, Dundee, UK Search for more papers by this author Sophie Haupt Sophie Haupt School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author John WS Brown John WS Brown Scottish Crop Research Institute, Invergowrie, Dundee, UK Search for more papers by this author Michael Taliansky Corresponding Author Michael Taliansky Scottish Crop Research Institute, Invergowrie, Dundee, UK Search for more papers by this author Author Information Sang Hyon Kim1, Eugene V Ryabov2, Natalia O Kalinina1,3, Daria V Rakitina1,3, Trudi Gillespie1, Stuart MacFarlane1, Sophie Haupt4, John WS Brown1 and Michael Taliansky 1 1Scottish Crop Research Institute, Invergowrie, Dundee, UK 2HRI, University of Warwick, Wellesbourne, Warwick, UK 3AN Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia 4School of Life Sciences, University of Dundee, Dundee, UK *Corresponding author. Plant Pathology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK. Tel.: +44 1382 562731; Fax: +44 1382 562426; E-mail: [email protected] The EMBO Journal (2007)26:2169-2179https://doi.org/10.1038/sj.emboj.7601674 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The nucleolus and Cajal bodies (CBs) are prominent interacting subnuclear domains involved in a number of crucial aspects of cell function. Certain viruses interact with these compartments but the functions of such interactions are largely uncharacterized. Here, we show that the ability of the groundnut rosette virus open reading frame (ORF) 3 protein to move viral RNA long distances through the phloem strictly depends on its interaction with CBs and the nucleolus. The ORF3 protein targets and reorganizes CBs into multiple CB-like structures and then enters the nucleolus by causing fusion of these structures with the nucleolus. The nucleolar localization of the ORF3 protein is essential for subsequent formation of viral ribonucleoprotein (RNP) particles capable of virus long-distance movement and systemic infection. We provide a model whereby the ORF3 protein utilizes trafficking pathways involving CBs to enter the nucleolus and, along with fibrillarin, exit the nucleus to form viral ‘transport-competent’ RNP particles in the cytoplasm. Introduction The nucleolus is a prominent subnuclear domain and is the site of transcription of rRNA, processing of pre-rRNAs and biogenesis of preribosomal particles. In addition, the nucleolus also participates in other aspects of RNA processing and cell function such as stress responses and the cell cycle (Rubbi and Milner, 2003; Olsen, 2004). The nucleolus is structurally and functionally linked to Cajal bodies (CBs) that are conserved structures found in both animals and plants (Beven et al, 1995; Cioce and Lamond, 2005). CBs contain small nuclear and small nucleolar ribonucleoprotein particles (snRNPs and snoRNPs) as well as a range of different proteins including coilin, a protein essential for CB formation, and some nucleolar proteins such as fibrillarin, dyskerin and Nopp140 (Ogg and Lamond, 2002; Cioce and Lamond, 2005; Matera and Shpargel, 2006). They are involved in the maturation of spliceosomal snRNPs and snoRNPs where newly assembled snRNPs and snoRNPs traffick through CBs before accumulating in splicing speckles and the nucleolus, respectively (Sleeman and Lamond, 1999; Sleeman et al, 2001). CBs are dynamic structures that can move within the nucleus and interact with chromatin and the loci of specific genes (Boudonck et al, 1999; Platani et al, 2002). The multifunctional nature of the nucleolus and CBs has recently been extended to RNA silencing: in Arabidopsis, the production of heterochromatic small interfering RNAs involved in transcriptional silencing occurs in CBs or processing foci in the nucleolus (Li et al, 2006; Pontes et al, 2006). Finally, a number of animal and plant viruses including the RNA-containing tobacco etch virus and the DNA-containing tomato yellow leaf curl virus have a nucleolar phase in their life cycles (see Hiscox, 2002, 2007 for reviews). However, the specific role of the nucleolus and other subnuclear bodies in virus infections remains elusive. Umbraviruses differ from most other viruses in that they do not encode a capsid protein (CP) such that conventional virus particles are not formed in infected plants. Umbraviral genomes encode at least three proteins (Figure 1A). In the umbravirus, groundnut rosette virus (GRV), two open reading frames (ORFs) at the 5′-end of the RNA are expressed by a frameshift mechanism as a single protein that appears to be an RNA replicase (Figure 1A; Taliansky and Robinson, 2003). The other ORFs (ORF3 and ORF4) overlap with each other. ORF4 encodes the movement protein that mediates the cell-to-cell movement of viral RNA via plasmodesmata (Ryabov et al, 1998). ORF3 protein is the long-distance movement factor that facilitates trafficking of viral RNA through the phloem, the specialized vascular system used by plants for the transport of assimilates and macromolecules (Ryabov et al, 1999). Figure 1.ORF3 protein domains involved in nuclear localization and cRNP formation. (A) Schematic representation of the GRV genome with protein sequences of the wild-type (WT) and mutant ORF3 R- and L-rich domains. (B) Representation of the TMV-based vector for expression of WT and mutated ORF3 (ORF3*)-GFP fusion proteins or GFP alone replacing the TMV CP gene. (C–G) Intracellular localization of free GFP (C), ORF3-GFP (D) and the mutant GFP fusions: RA-GFP (E), L153A-GFP (F) and L149A-GFP (G) expressed from a TMVΔCP vector in epidermal cells and determined by CLSM. Each set of GFP images presents a whole cell at low magnification (LM) and a nucleus at high magnification (HM) showing GFP image (left-hand panel), DAPI staining (centre panel) and overlaid GFP and DAPI images (right-hand panel). Free GFP localizes to the nucleus (largely excluded from the nucleolus) and cytoplasm (C) whereas ORF3-GFP localizes to the nucleolus and cytoplasmic inclusions (indicated by arrows) (D). RA-GFP is largely excluded from the nucleus (E), L153A-GFP is nuclear with strong localization to the nucleolus (F) and L149A-GFP is nuclear with strong accumulation in small nuclear bodies (G). No, nucleolus; N, nucleus (shown by dashed line). Scale bars, 20 μm (LM) and 5 μm (HM). Download figure Download PowerPoint Umbraviral ORF3 proteins (26–29 kDa) show no significant similarity with any other recorded or predicted proteins (Taliansky and Robinson, 2003). The GRV ORF3 protein interacts with viral RNA in vivo to form filamentous ribonucleoprotein (RNP) particles, which have elements of regular helical structure, but not the uniformity typical of virus particles (Taliansky et al, 2003). The RNPs accumulate in cytoplasmic inclusion bodies, which have been detected in all cell types and, more importantly, were abundant in phloem-associated cells (Taliansky et al, 2003). The RNPs protect the viral RNA and move long distances through the phloem, thereby determining the ability of umbravirus to cause systemic infection. Finally, in addition to its presence in the cytoplasm, the ORF3 protein was also found in nuclei and predominantly in nucleoli (Ryabov et al, 1998, 2004). In this paper, we demonstrate that once the GRV ORF3 protein has been imported into the nucleus, it interacts with and reorganizes CBs into multiple CB-like structures (CBLs) and enters the nucleolus by causing fusion of the CBLs with the nucleolus. The ORF3 protein also causes the redistribution of some of the pool of the major nucleolar protein, fibrillarin, to the cytoplasm. More importantly, the nucleolar phase of the ORF3 protein is essential for the formation of viral RNPs and consequently virus long-distance movement, providing a model where CBs and the nucleolus are integrally required for these processes. Results Protein domains of ORF3 involved in nuclear localization ORF3 proteins from different umbraviruses contain two conserved domains: a basic arginine-rich sequence (positions 108–122; R-rich domain) and a hydrophobic leucine-rich region (amino acids 148–156; L-rich domain) showing invariant leucine residues in a motif 149-LXXLL-153 (Figure 1A) (Taliansky and Robinson, 2003; Ryabov et al, 2004). To examine the effect of these conserved regions on the detailed localization of ORF3 protein, ORF3 constructs were introduced into a tobacco mosaic virus (TMV) vector where the TMV CP (normally essential for virus long-distance movement) had been removed (TMVΔCP). The CP was replaced by a fusion of the ORF3 protein to the N-terminus of GFP (TMVΔCP.ORF3-GFP—abbreviated to ORF3-GFP) (Figure 1B). Following inoculation of Nicotiana benthamiana plants, the ORF3-GFP fusion complemented the inability of TMVΔCP to move long distances through the phloem (data not shown). For localizations, infection sites were allowed to develop on inoculated leaves for about 7 days (late stage of infection in inoculated leaves) before fluorescence was monitored. Free GFP expressed from TMVΔCP (TMVΔCP-GFP) was clearly visible in the nucleus (but largely excluded from the nucleolus) and cytoplasm, which is distributed as a thin layer appressed to the highly convoluted cell wall of the epidermal cells (Figure 1C). In leaves inoculated with ORF3-GFP, the fusion protein localized to the nucleolus and in the vast majority of cells to large cytoplasmic inclusions (Figure 1D). The nucleolar localization is clearly shown in the higher magnification images of individual nuclei below the whole cell image confirming our previous observations obtained by electron microscopy (Ryabov et al, 2004) and confocal laser scanning microscopy (Ryabov et al, 1998). The fluorescent cytoplasmic inclusions varied in number and size (sometimes as large as 10 μm). The pattern of fluorescence in the inclusions also varied from uniform, intense labelling to diffuse labelling with numerous more heavily labelled speckles (compare ORF3-GFP in Figures 1D and 6A), presumably reflecting different levels of ORF3 protein accumulation in different cells. Figure 2.Electron micrographs showing localization, morphology and structure of inclusions formed by ORF3-GFP or its mutants (RA-GFP and L149A-GFP) in palisade mesophyll cells of N. benthamiana plants. (A) Sections showing typical general views of healthy cells and cells infected with ORF3-GFP after IGL with antibodies against the ORF3 protein. Cells infected with the ORF3 mutants, RA-GFP and L149A-GFP, are essentially similar. All cells contain normal organelles such as nucleus (N) nucleolus (No), chloroplasts (Chl) and vacuole (V). In addition, virus-infected cells (exemplified by ORF3-GFP) also contain TMV-specific X-bodies (XB) containing electron-dense (gold-labelled) ORF3 protein-related inclusions (shown by arrows). Scale bars, 5 μm. (B) Higher magnification of X-bodies and cytoplasmic inclusions of ORF3-GFP and its mutants, RA-GFP and L149A-GFP. XB, X-bodies; arrows, inclusions. Scale bars, 2 μm. (C) High-resolution EM sections of cytoplasmic inclusions formed by ORF3-GFP, RA-GFP and L149A-GFP labelled with antibody against the ORF3 protein (IGL) and with an RNA probe specific for the positive strand of the ORF3 gene (ISH). ORF3-GFP inclusions are composed of complexes of filamentous RNP particles (c-RNP) containing the ORF3 protein and viral RNA. The inclusions of the ORF3 mutants, RA-GFP and L149A-GFP did not have a filamentous structure; they contained mutant ORF3 proteins (IGL) but did not contain viral RNA (ISH). All of the inclusions appear as large fluorescence masses (up to 10 μm) in confocal images (Figure 1D–G). Scale bars, 100 nm. Download figure Download PowerPoint Mutations to the R- and L-rich domains were introduced into ORF3-GFP to monitor effects on intracellular localization. Replacement of all six arginine residues (amino-acid positions 108, 110, 111, 112, 115 and 122) in the R-rich domain by alanine residues gave RA-GFP (Figure 1A), which was unable to enter the nucleus (Figure 1E), confirming previous observations that the R-rich domain is involved in nuclear import (Ryabov et al, 2004). Fluorescence was also observed in large inclusions in the cytoplasm of infected cells (Figure 1E). None of the single arginine–alanine substitutions affected nucleolar localization of the ORF3 protein or inclusion formation (Figure 3C). Figure 3.Protein domains of ORF3 involved in virus long-distance movement. (A, B) Effect of ORF3 protein mutations on the accumulation of TMVΔCP.ORF3 in inoculated (in) and uninoculated (u) leaves of N. benthamiana analyzed by RT–PCR (A) and infectivity assay (B). (A) Ethidium bromide-stained agarose gels show RT–PCR products (after 50 cycles of amplification) corresponding to fragments of GFP gene (270 bp) and the control ubiquitin gene (176 bp) (indicated by arrows). (B) Accumulation of the virus was determined by inoculation of RNA isolated from inoculated (in) and uninoculated (u) leaves on test N. tabacum ‘Xanthi-nc’ plants leading to the production of visible lesions. The data are the average number of lesions per half leaf. Only ORF3-GFP shows the presence of viral RNA (A) and infectious virus (B) in uninoculated leaves of N. benthamiana. (C) Summary of localization, RNP formation and long-distance movement data showing the correlation between the ability of the ORF3 protein to traffic through the nucleolus to the cytoplasm, its capacity to form viral movement-competent c-RNPs (detected by IGL and ISH, see Figure 2C), and their systemic transport (long-distance movement, LDM). N and No, nuclear and nucleolar localization of the ORF3 protein, respectively; N-exp, nuclear export; LDM, long-distance movement. *, all of the mutants with single arginine–alanine substitutions in the R-rich domain did not affect nuclear localization and c-RNP formation and were able to rescue long-distance virus movement (data not shown) and are exemplified by the R108A-GFP mutant. (D) Typical print hybridization patterns of inoculated and uninoculated leaves of N. benthamiana infected with PEMV-2 and PEMV-2 containing the L149A mutation in its ORF3. Viral RNA was detected with a 32P-labelled cDNA probe corresponding to PEMV-2 ORF3. Download figure Download PowerPoint Mutations to the leucine-rich region replaced either all the invariant leucine residues (positions 149, 152 and 153) or individual leucines with alanine residues. Mutation of L152 or L153 did not affect the nucleolar localization of ORF3-GFP but led to a higher accumulation of the protein in the nucleoplasm than was observed with unmutated ORF3-GFP (Figure 1F). On the other hand, replacement of either all three leucines (LA-GFP) or the single leucine L149 (L149A-GFP) caused the mutant fusion proteins to be excluded from the nucleolus and to accumulate in the nucleus in multiple small structures in the nucleoplasm (Figure 1G). Thus, L149A, in addition to the R-rich domain, is essential for nucleolar targeting of the ORF3 protein. The higher level of nuclear accumulation of all of the leucine mutants compared to ORF3-GFP (Figure 1D) suggests that the leucine-rich region acts as a nuclear export signal (Ryabov et al, 2004) and that the ORF3 protein traffics between the nucleus (nucleolus) and cytoplasm in infected cells. As with the wild-type ORF3-GFP or RA-GFP mutant large fluorescent cytoplasmic inclusions were found in cells expressing all of the leucine mutants (Figure 1F and G). Viral RNP particles are present only in large cytoplasmic inclusions formed by the wild-type ORF3 protein Following expression of GFP-tagged ORF3 and its mutant proteins, fluorescence was observed in large cytoplasmic inclusions. The majority of plant viruses produce cytoplasmic inclusion bodies, reflecting the high level of production of viral RNA and proteins (see Hull, 2002 for review). For GRV, we showed previously by immunogold labelling (IGL) and in situ hybridization (ISH) that the cytoplasmic inclusions were composed of filamentous RNP particles (referred to as cytoplasmic RNPs (c-RNP)) containing the ORF3 protein and viral RNA (Taliansky et al, 2003). To characterize the large fluorescent cytoplasmic inclusions formed by the TMV-expressed ORF3-GFP construct and its mutants at late stages of infection, we analyzed cells by electron microscopy, IGL and ISH. Healthy palisade mesophyll cells showed the cytoplasm containing protoplasts around the periphery of the central vacuole with a distinct nucleus and nucleolus (Figure 2A). In addition to these normal organelles, cells infected with TMV vector expressing ORF3-GFP showed the presence of X-bodies or viroplasms, large virus-induced amorphous inclusion bodies typical of TMV infection (Esau and Cronshaw, 1967; Hull, 2002) and frequently (but not always) found in the proximity of the nucleus. The X-bodies formed by ORF3-GFP also contained multiple electron-dense cytoplasmic inclusions (e.g., Figure 2A and B; Taliansky et al, 2003). Similar multiple inclusions were observed on closer examination of X-bodies formed in cells expressing the RA-GFP and L149A-GFP mutants (Figure 2B). The inclusions varied in size from 300 nm to 4 μm, and as many as 10 μm, separate inclusions could be found in a single cell section (Figure 2A; see also Taliansky et al, 2003). Such inclusion bodies were not detected in cells infected with the TMV vector alone or in healthy cells (Figure 2A). Figure 4.Effect of ORF3 protein on the integrity and localization of CBs. (A, B) Confocal images of epidermal cells infected with TMVΔCP expressing ORF3-GFP, L149A-GFP and free GFP (as a control) and immunostained using antibodies to coilin (A) and U2B″ (B). In ORF3-GFP-infected cells, coilin and U2B″ localize with the ORF3 protein to nucleoli, whereas in L149A-GFP-infected cells, these proteins are found in multiple small bodies (CBLs). CBs are detected in GFP-expressing cells but not in ORF3-GFP cells. Panels: I—GFP images; II—fluorescent (red) antibody labelling; III—overlay images. CB, Cajal bodies; CBL, CB-like structures (shown by arrowheads); No, nucleoli; N, nuclei (indicated by dashed lines according to DAPI staining). Chl, chloroplasts showing natural red autofluorescence. Scale bars, 5 μm. (C) Intranuclear localization of the ORF3-GFP protein in a pseudo-time course of ORF3-GFP infection. The left panel (LES, for lesion) shows a GFP image of a whole virus-infected lesion with cells corresponding to different stages of infection. Cells at the front of the infection site represent early infection events (E), whereas those cells in the centre of the infection site represent late events (L). ‘Early’ event cells show fluorescence mainly in the perinuclear region; intermediate (middle) stage cells (M) show accumulation of fluorescence in multiple subnuclear bodies, similar to the CBLs that merge with the nucleolus at later stage (L). Scale bars, 5 μm, except for lesion in (C) (left panel)—200 μm. Download figure Download PowerPoint To examine whether the cytoplasmic inclusions contained viral protein and RNA, sections of inclusion bodies from cells expressing ORF3-GFP, RA-GFP and L149A-GFP were analyzed by IGL and ISH. For ORF3-GFP, masses of filamentous structures were observed in the inclusion bodies that were labelled by antibodies against the ORF3 protein (IGL) and by an RNA probe to the ORF3 region of GRV RNA (ISH) (Figure 2C). Thus, the filamentous structures contained both the ORF3 protein and viral RNA, indicative of the formation of viral RNPs. These cytoplasmic filamentous RNP particles (c-RNP) were very similar to the ORF3 RNP complexes formed in GRV infection whose molecular structure was described in detail previously (Taliansky et al, 2003). Formation of the c-RNP particles did not depend on the TMV vector background, as they were also formed during native umbravirus (GRV) infection (Supplementary Figure S1A). On the other hand, the RA-GFP and L149A-GFP mutants showed little structure in the inclusion bodies, contained mutant ORF3 protein (as shown by IGL) but did not contain viral RNA (as shown by ISH) (Figure 2C). Thus, wild-type ORF3 protein generates cytoplasmic inclusions containing viral RNPs (c-RNP), whereas the amorphous inclusions formed by the ORF3 mutants did not contain viral RNA and, thereby, RNP particles, but only accumulated protein. These cytoplasmic inclusions were, therefore, termed cytoplasmic protein aggregates (c-PAs). In addition to the above mutants, the other leucine mutants also did not produce inclusions with viral RNA/RNPs, forming only ORF3 protein aggregates (c-PA) (data not shown). Thus, although ORF3-GFP and its mutants formed cytoplasmic inclusions, which were readily visible by confocal laser scanning microscopy (Figure 1C–G) and electron microscopy (Figure 2), they differed in their molecular structure and composition, with only the wild-type ORF3-GFP producing viral RNPs (c-RNPs). However, in confocal images (Figure 1D–G), all of the inclusions appear similar as large fluorescent masses in the cytoplasm. The differences in intracellular localization and ability to form RNP particles among ORF3-GFP and its mutants are unlikely to reflect differences in infectivity (replication and cell-to-cell movement) of the TMV vector or in ORF3 protein accumulation because the size of infection foci and intensity of GFP fluorescence were approximately the same for all constructs (data not shown). The differential localizations also did not depend on the virus vector background as experiments using Agrobacterium-mediated transient expression of the corresponding constructs showed similar localization patterns (data not shown). The R- and L-rich domains affect long-distance virus movement The TMVΔCP vector does not move rapidly between lower and upper leaves and allows an assessment of whether the ORF3 mutants can rescue long-distance movement of TMVΔCP by looking for the spread of ORF3-GFP into uninoculated leaves. ORF3-GFP rescued rapid long-distance movement of TMVΔCP from inoculated to upper uninoculated leaves as shown by the presence of ORF3-GFP RNA in uninoculated leaves (Figure 3A) and the ability of extracts from these leaves to produce infectious lesions on test plants (Figure 3B, summarized in C). In contrast, no long-distance movement was observed with RA-GFP, suggesting that the same R-rich domain required for nucleolar localization and production of c-RNPs is essential for long-distance virus movement (Figure 3A–C) (single arginine substitutions did not affect nuclear localization and c-RNP formation and were able to rescue long-distance virus movement; Figure 3C). Similarly, no long-distance viral movement was observed with any leucine-rich region mutant (Figure 3A–C). The phenotype of the L149A mutation was further confirmed by inserting the same critical mutation into ORF3 of a full-length clone of pea enation mosaic virus-2 (PEMV-2), an umbravirus closely related to GRV, resulting in the complete blockage of long-distance movement of this virus (Figure 3D). Thus, the R- and L-rich regions are both required for long-distance viral movement and the mutations reveal a strong correlation between the localization of the ORF3 protein to the nucleolus, its capacity to form c-RNP with viral RNA and the ability of the virus to move through the plant (Figure 3C). The trafficking of the ORF3 protein to the nucleolus and ultimately to the cytoplasm is therefore key to successful GRV infection. L149A-GFP protein accumulates in multiple CBLs To identify the small subnuclear bodies in which the L149A-GFP protein accumulates (Figure 1G), infection sites produced by L149A-GFP and ORF3-GFP were labelled with fluorescent (red) antibodies to the CB protein markers, coilin (Arabidopsis thaliana coilin—Atcoilin; Collier et al, 2006) and U2B″ (Beven et al, 1995; Boudonck et al, 1999) (Figure 4A and B). In control (healthy (data not shown) or infected with TMVΔCP-GFP (Figure 4A)) N. benthamiana cells, the antibodies detected usually one and up to three CBs. In contrast, in cells containing L149A-GFP, the antibodies colocalized with multiple subnuclear bodies showing L149A-GFP fluorescence (Figure 4A and B). The number of such bodies per cell was significantly increased (ca. 6–12) compared to control cells. Thus, the L149A-GFP mutant protein is unable to localize to the nucleolus and instead accumulates in multiple subnuclear structures, which contain CB marker proteins and therefore are termed here CBLs. In cells containing ORF3-GFP, the Atcoilin and U2B″ antibodies colocalized with ORF3 protein in the nucleolus, but unexpectedly did not detect any CBs (Figure 4A and B) in any of the 50 infected cells analysed in each of the five experiments. Thus, the ORF3 protein accumulates in the nucleolus and disrupts or alters CB integrity, relocalizing coilin and U2B″ to the nucleolus. Figure 5.Fusion of CBLs with the nucleolus in GRV-infected plants. (A) Localization of Atcoilin-GFP delivered by Agrobacterium in a pseudo-time course of GRV-YB infection from early (E), middle (M) and late (L) stages of the infection compared to a healthy cell. GRV-YB infection induces yellow blotch symptoms (left panel) at the front of systemic infection and therefore acts as a visible marker of infection progression. Atcoilin-GFP fluorescence in cells at different stages of GRV infection shows multiple CBLs merging with the nucleolus, whereas in healthy cells, CBs remains distinct from the nucleolus (right panel). (B) Subnuclear colocalization of Atcoilin-GFP and AtFib2-mRFP delivered by Agrobacterium into healthy or GRV-YB—infected plant cells at early (E) and late (L) stages of GRV infection. In healthy plant cells, AtFib2 labels both the CBs (usually 1–3 per nucleus) and the nucleolus, whereas Atcoilin colocalizes with AtFib2 only in CBs. At early stages of GRV infection (E), AtFib2 colocalizes with Atcoilin in multiple CBLs, which merge with the nucleolus at late stage (L). CB, Cajal bodies; CBL, CB-like structures (shown by arrowheads); No, nucleoli; N, nuclei (shown by dashed lines according to DAPI staining). Scale bars, 5 μm. Download figure Download PowerPoint ORF3 protein causes CBLs to fuse with nucleoli To investigate the effect of expressing ORF3-GFP on CBs, its distribution in the nucleus was examined in a pseudo-time course of infection experiment. Virus infection spreads from the initial infected cell to generate a lesion on the leaf where the cells in the lesion are at different stages of infection (Figure 4C, left panel): cells at the leading edge of the growing (3 days post-inoculation) infection site represent early infection events whereas those cells in the centre of the infection site represent late events (Figure 4C). Three days after inoculation with ORF3-GFP, ‘early’ event cells showed fluorescence mainly in the perinuclear region with weak labelling of the nucleolus (Figure 4C). Intermediate (middle) stage cells showed significant accumulation of fluorescence in multiple subnuclear bodies, similar to the CBLs produced in the presence of the L149A mutant, that appeared to merge with the nucleolus such that, at later stages, only the nucleolus was intensely labelled (Figure 4C). Thus, it appears that
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