1 Département de Virologie, Institut de Biologie Moléculaire des Plantes du CNRS, 12 rue du Général Zimmer, 67084 Strasbourg, France
2 Fakultät für Biologie, Lehrstuhl für Genomforschung, 33594 Bielefeld, Germany
Correspondence
David Gilmer
david.gilmer{at}ibmp-ulp.u-strasbg.fr
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ABSTRACT |
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Present address: LBMAGM, 42 rue du laboratoire, L1011 Luxembourg.
Present address: IBVM INRA, 71 Avenue E. Bourleaux, BP81, 33883 Villenave d'Ornon Cedex, France.
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INTRODUCTION |
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BNYVV, like virtually all plant RNA viruses, replicates in the cytoplasm but immuno-gold electron microscopy observations have shown that p25 is present both in the nuclei and in the cytoplasm of infected leaf cells (Haeberle & Stussi-Garaud, 1995). In this paper, we have studied sequences that control the movement of p25 in and out of the nucleus. We have used a biolistic approach to deliver transient expression vectors encoding p25 fused to the jellyfish green fluorescent protein (GFP) to cultured plant cells. This study was performed in order to characterize the nuclear sequences that govern the movement of BNYVV p25 in and out of the nucleus and to determine the pathways involved in such nuclear translocations.
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METHODS |
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Point mutations in p25 were created by overlap extension mutagenesis. pCKeGFP-derived constructs were produced by PCR amplification of repGFPp25 using a sense primer corresponding to the 3' end of the GFP gene and XbaI-containing reverse primers complementary to the 3' end of the p25 gene except for pCKGFPp25Ct, which was obtained with a reverse primer providing a stop codon after aa 103, followed by an XbaI restriction site. Fragments were placed between the cauliflower mosaic virus 35S promoter and terminator in pCKeGFP (Gaire et al., 1999
; Reichel et al., 1996
) by using BsrGI and XbaI sites. All constructs and PCR fragments were characterized by restriction enzyme digestion and sequenced with an ABI prism 373 DNA sequencer (Applied Biosystems) or a Hitachi 3100 Genetic Analyser (Applied Biosystems) using a BigDye Terminator sequencing kit (Applied Biosystems) and specific primers.
Infection of leaves and analysis of infection products.
Virus infection procedures were as described previously (Erhardt et al., 2000; Lauber et al., 1998
). Polypeptides containing the sequence of p25 and/or GFP were immuno-detected on Western blots with a p25-specific antiserum (Niesbach-Klosgen et al., 1990
) and mouse monoclonal anti-GFP antibody (Roche), respectively.
BY-2 transient expression.
GFP-fusion proteins were transiently expressed in BY-2 tobacco suspension cells (Nicotiana tabacum cv. Bright Yellow 2) maintained as described (Banjoko & Trelease, 1995). Cells were subcultured every 7 days and harvested 3 days after medium renewal for biolistic bombardment. The harvested cells were filtered onto Whatman discs and placed on 0·8 % agar Murashige-Skoog (MS) media plates supplemented with 0·25 M mannitol for 24 h. Particle preparation and biolistic assays were performed as described (Hunold et al., 1995
) with the following modifications: 4 mg 1·1 µm tungsten particles (Bio-Rad) were sterilized in 1 ml absolute alcohol for 20 min. Particles were then mixed with 10 µg plasmid DNA supplemented with 18 % glycerol, 1·5 M CaCl2 and 90 mM spermidine in a final volume of 180 µl. The firing distance was 11 cm and helium pressure 7 bars. After bombardment, cells were transferred to 0·8 % agar MS media plates and incubated in the dark for 16 h at 28 °C. BY-2 transfected cells were collected under HBO binocular microscope (excitation/emission wavelength 488/505545 nm) 16 h post-bombardment and cultured in MS liquid media prior to further treatment and/or confocal laser scanning microscopy (CLSM) observations.
In vivo detection of GFP by CLSM.
GFP fluorescence in epidermal cells of infected C. quinoa leaves and in transfected BY-2 cells was visualized by CLSM with an LSM510 Zeiss laser scanning confocal microscope equipped with an inverted Zeiss Axiovert 100M microscope and a 63x, 1·2 water-immersion objective. For each construct, experiments were reproduced at least twice and 30 GFP-expressing BY-2 cells were observed in each case. No more than 3 % of transfected cells displayed variation of subcellular localization. Such cells were not taken into consideration. Laser scanning was performed using identical settings for single-track mode and excitation/emission wavelengths (488/505545 nm) for GFP. Image processing was carried out with LSM510 version 2.5 (Zeiss) and Photoshop 5.5 (Adobe system).
In vitro GST pull-down assays.
Rice GST-importin- and -
were provided by A. Baba, E. Herzog and T. Hohn (Friedrich Miescher Institute, Basel, Switzerland). Pepper GST-importin-
was provided by U. Bonas and B. Szurek (Institut fur Genetik, Martin-Luther Universität Halle-Wittenberg, Halle, Germany). All constructs were cloned into pGEX (Pharmacia Biotech) and expressed in E. coli BL21 as described (Herzog et al., 2000
). A rapid translation system (RTS100; Roche Molecular Biochemicals) was used, as described by the supplier, to produce identical amounts of wild-type or mutated 35S-radiolabelled p25 to perform the test. In vitro GST-pull-down assay conditions were as described (Herzog et al., 2000
) using identical amounts of GST-tagged proteins and 1 % Tween and 1 mM DTT in PBS buffer to perform the assay. Aliquots of the bound protein were separated on 12 % SDS-PAGE and the 35S-labelled proteins were visualized by autoradiography.
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RESULTS |
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To map a putative NLS within p25, the subcellular localization of different p25 deletion mutants was analysed. The deletion mutants GFPp25Ct (aa 104219 deleted), GFPp25
BA (aa 104196 deleted), GFPp25
Nt (aa 1102 deleted) and GFPp25
SSt (aa 170 deleted) were tested as described above. In GFPp25
Ct (Fig. 1b
) and GFPp25
BA (data not shown) the fusion proteins were localized exclusively in the nuclei of expressing cells, indicating that an NLS is present within the first 102 aa of p25. GFPp25 was never observed in the nucleolus of transfected cells. These data also suggest that a sequence in the C-terminal region either acts to retain part of the GFPp25 in the cytoplasm or promotes export of GFPp25 from the nucleus to the cytoplasm.
Deletion of the N-terminal domain of p25 (GFPp25Nt) resulted in a mainly cytoplasmic localization of the fusion protein (Fig. 1b
), although detection of a faint green fluorescent signal in the nucleus indicated that some passive diffusion of the 39 kDa protein may have taken place. However, even if some passive diffusion of GFPp25
Nt occurred the fusion protein no longer accumulated abundantly in the nucleus (compare GFPp25 and GFPp25
Nt, Fig. 1b
).
Deletion mutant GFPp25SSt (
170) was also tested in order to map precisely the putative NLS within the first 102 aa of p25. Deletion of the first 70 aa (GFPp25
SSt) eliminated the NLS-like activity and the protein was only observed in the cytoplasm of the transfected cells (Fig. 1b
).
The sequence KRIRFR is responsible for nuclear targeting of p25 and can partially drive the nuclear localization of a GFP-GUS reporter gene
p25 contains the sequence motif 57KRIRFR62 (Haeberle & Stussi-Garaud, 1995), which resembles the basic NLS motifs of the simian virus 40-type (Gorlich & Kutay, 1999
; Kalderon et al., 1984
). Mutations resulting in short deletions or substitutions of basic residues for alanine were created in this putative NLS (Fig. 2
a) in the context of the constitutively nuclear-targeted protein GFPp25
Ct. The resulting constructs were tested in transient expression assays as described above. Replacement of all of the basic amino acids within the putative NLS by alanine (57AAIAFA62 in GFPp25
Ct-m1, Fig. 2a
) strongly inhibited nuclear targeting (Fig. 2b
). Single alanine substitution of each basic amino acid residue within the NLS motif distinguished three classes of mutants: (i) the mutations K57A in GFPp25
Ct-m2 and R58A in GFPp25
Ct-m3 (Fig. 2b
) partially blocked nuclear import of the protein, resulting in a distribution of the proteins between the nucleus and the cytoplasm of the cells; (ii) the mutation R60A in GFPp25
Ct-m4 (Fig. 2b
) did not interfere with nuclear targeting, whereas (iii) the mutation R62A in GFPp25
Ct-m5 completely blocked nuclear import (Fig. 2b
), indicating that this particular fusion protein was also unable to diffuse into the nucleus.
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All of the aforesaid mutations were then analysed in the context of full-length p25 fused to GFP. Analysis of the subcellular localization following bombardment revealed that, in every case, the nuclearcytoplasmic partitioning was as observed in the GFPp25Ct context, except that there was an even stronger cytoplasmic localization of the mutant GFPp25-m1, which exhibited no detectable fluorescent signal in the nuclei (Fig. 2b
).
To provide further evidence for active nuclear import of p25, we generated a cDNA construct (GFPp25Ct-GUS; Fig. 2a
) in which the GUS sequence was fused to the C terminus of GFPp25 deleted of its C-terminal domain. This was done in order to create a very large fusion protein (about 106 kDa) that should be totally blocked from entering the nucleus by passive diffusion. As a control, we used the nuclear import-deficient construct GFPp25
Ct-m7-GUS (Fig. 2a
). The N-terminal region of p25 containing the putative NLS relocated a significant portion of the GFPp25
Ct-GUS fusion protein to the nucleus of the transfected cells (Fig. 2b
), whereas the GFPp25
Ct-m7-GUS fusion protein was exclusively localized in the cytoplasm (Fig. 2b
). Western blotting analysis conducted on protein extracted from BY-2 cells expressing GFPp25
Ct-GUS and GFPp25
Ct-m7-GUS revealed that both proteins were of the expected size (not shown). Thus, these results represent additional evidence that p25 enters the nucleus by an active NLS-dependent process.
p25 binds importin- in vitro
Import of proteins into the nucleus is often mediated by the importin--related transport pathway. This pathway involves either a direct interaction of the nuclear-targeted protein with importin-
or -
, which binds to importin-
. To obtain additional information on the p25 nuclear import mechanism, possible interactions between p25 and pepper (Szurek et al., 2001
) or rice (Jiang et al., 1998
) importin-
and -
(Matsuki et al., 1998
) were assayed by in vitro pull-down experiments using GST-tagged importins. Radiolabelled wild-type p25 expressed in E. coli was able to bind to E. coli-expressed GST-importin-
from rice and pepper, whereas p25 carrying the m1 mutation described above was unable to bind importin-
from either species (Fig. 3
). Neither protein interacted with the matrix (R), with GST (G) or with rice GST-importin-
(rI
) (Fig. 3
). Similarly, no interaction was observed between identical amounts of p25 carrying the m5 mutation (R62A) and the pepper importin-
(pI
). Co-immunoprecipitation assays using an anti-GST serum gave identical results (data not shown). Thus, these data are consistent with the hypothesis that nuclear entry of p25 is mediated by importin-
and involves the p25 sequence 57KRIRFR62.
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LMB treatment of BY-2 cells expressing GFP did not significantly influence its subcellular distribution between the nuclear and cytoplasmic compartments (Fig. 4). On the other hand, when the cells were bombarded with a construct expressing GFP fused to the well-characterized HIV rev nuclear export sequence ( GFPNESrev+; Haasen et al., 1999
), the fluorescent signal was stronger in the cytoplasm than in the nucleus and LMB treatment caused the GFPNESrev+ to localize predominantly in the nuclear compartment (Fig. 4
). A GFP construct fused to the rev NES, which had been mutated to abolish its export activity (GFPNESrev; Haasen et al., 1999
), displayed similar subcellular distribution in the presence and the absence of LMB. We conclude that LMB inhibits CRM1/Exportin1-mediated nuclear export in BY-2 cells in our experimental conditions.
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Characterization of p25 nuclear export sequence
Nuclear export sequences are rich in hydrophobic residues although there is not a strict consensus motif (for review see Macara, 2001). Analysis of the C-terminal p25 sequence revealed a region rich in hydrophobic residues between aa 164 and 196. To study the effect of this hydrophobic domain (HD, Fig. 5
) upon nuclear export, we fused it to the C-terminal part of the GFP sequence, leading to GFPHD. When expressed in bombarded BY-2 cells, GFPHD behaved exactly as did GFPNESrev+ (see Fig. 4
), i.e. only low levels of GFPHD were detected in the nucleus in the absence of LMB but the protein accumulated strongly in the nucleus following LMB treatment (Fig. 5
). We conclude that the HD domain (aa 164196) contains an NES.
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Sequence alignment of the p25-HDb domain with several well-characterized NES sequences revealed a NES-like motif consisting of the hydrophobic residues V169, V172, V175 and V178 (Fig. 6a). A set of HDb mutants was produced in which each of the aforesaid hydrophobic residues was replaced by an alanine (Fig. 6b
). Alanine substitution mutants targeting three other hydrophobic residues (L174, V181 and L182; Fig. 6b
) that were not part of the NES-like motif were produced as well. The mutant HDb sequences were fused to GFP and the effect of LMB treatment on the subcellular distribution of fluorescence in bombarded BY-2 cells was studied as before. GFPNESrev+ and GFPNESrev provided positive and negative controls, respectively.
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Altered subcellular localization of p25 is associated with changes in virus symptoms
The foregoing experiments were all carried out by transient expression of p25-based constructs in single cells and in the absence of other viral proteins. To assess the subcellular distribution of p25 in the context of a BNYVV infection of whole plants, we produced a transcription vector in which the p25 gene of BNYVV RNA-3 was replaced by the sequence encoding the GFPp25 fusion protein. Previous experiments have shown that BNYVV RNA-3 transcripts encoding GFP fusion proteins can multiply in leaves when co-inoculated with BNYVV RNA-1 and -2 (Stras12) and that the fusion protein is expressed (Erhardt et al., 2000). Inoculation of C. quinoa leaves with Stras12 plus an RNA-3 transcript encoding GFP fused to the N terminus of p25 (repGFPp25) produced numerous fluorescent rings (23 mm diameter) on the inoculated leaves by 45 days post-inoculation (d.p.i.) (data not shown). The rings appeared at positions where local lesions subsequently became visible in natural light.
CLSM observations of epidermal cells in the fluorescent rings produced by inoculation with Stras12 plus repGFPp25 revealed that fluorescence was present in both the nuclear and cytoplasmic compartments (Fig. 7c, lower panel), as observed when GFPp25 was expressed independently in BY-2 cells (Table 1
). Thus, these experiments illustrate that neither the method of introduction of the fusion protein into plant cells (biolistics of BY-2 cells versus mechanical inoculation of leaves) nor the expression vector used (DNA plasmid versus a virus replication-dependent RNA replicon) influences the subcellular distribution of p25. We can also conclude that p25's subcellular localization is not altered by the presence of other BNYVV proteins during its expression.
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As noted above, RNA-3 is an important determinant of symptoms on BNYVV-infected hosts. Thus, on leaves of C. quinoa an inoculum containing only BNYVV RNA-1 and -2 produces mild chlorotic local lesions [chlorotic spot (CS) or CS symptoms similar to those presented in Fig. 7a], whereas addition of RNA-3 provokes formation of strongly chlorotic local lesions known as yellow spot (YS) symptoms (Fig. 7b
; Quillet et al., 1989
; Tamada & Abe, 1989
). The YS phenotype is dependent on expression of p25 (Jupin et al., 1992
; Tamada & Abe, 1989
). Thus, CS symptoms were observed following inoculation with Stras12 plus RNA-3 in which the p25 gene had been deleted (rep0) or replaced with the GFP gene (repGFP; Fig. 7a
, Table 1
). Importantly, inoculation of leaves with Stras12 plus repGFPp25 produced YS local lesions (Fig. 7c
) similar to those provoked by wild-type RNA-3, illustrating that the presence of the N-terminal GFP moiety does not interfere with the effect of p25 on leaf symptoms (compare Fig. 7b and c
).
Table 1 summarizes the lesion types provoked by inoculation of C. quinoa leaves with Stras12 plus repGFPp25 containing various p25 mutant forms. It can be seen (Table 1
) that all of the mutant constructs in which the GFPp25 was localized principally in the cytoplasm produced CS symptoms. Three of the constructs tested, repGFPp25
Ct, repGFPp25
BA and repGFPp25nes- (GFPp25 containing the mutated NES sequence 169AYMACLVNTV178), localized strongly to the nuclear compartment when inoculated along with RNA-1 and -2 to leaves (see Table 1
and Fig. 7d, e
). RepGFPp25nes- induced CS local lesions, whereas repGFPp25
Ct induced necrotic local lesions (Nec; Table 1
). The reason why repGFPp25
Ct and repGFPp25
BA but not repGFPp25nes- provoked necrotic lesions is not known, but one possibility is that non-specific binding to nuclear DNA of repGFPp25
Ct and repGFPp25
BA, which (unlike repGFPp25nes-) have a high pI, causes cell death. However, whatever the cause of the toxicity of GFPp25
Ct, our findings show that alteration of the wild-type distribution of GFPp25 between the nuclear and cytoplasmic compartments is accompanied by alterations in p25-related symptoms during virus infection.
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DISCUSSION |
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We also showed that nuclear export of GFPp25Nt involves a domain containing hydrophobic residues (164196) and is sensitive to the export inhibitor LMB. Thus, sensitivity to LMB indicates that the CRM1 pathway actively exports p25. The subcellular distribution of wild-type GFP was not affected by the export inhibitor (Fig. 4
). Although wild-type GFP localized almost equally to both the nucleus and cytoplasm due to the diffusion of the protein, GFP fused to the C-terminal domain of p25 (GFPp25
Nt) or to the HIV rev NES (GFPNESrev+), which is recognized by the plant CRM1/Exportin1 receptor (Haasen et al., 1999
), was localized mainly in the cytoplasm and only weakly in the nucleus (Fig. 4
). These results suggest that the low molecular masses of GFPp25
Nt (40 kDa) and GFPNESrev+ (30 kDa) permit their diffusion into the nucleus (Bonner, 1978
; Gorlich & Kutay, 1999
) but, once inside the nucleus, most of the protein is exported back into the cytoplasm by the CRM1/Exportin1 pathway. Our findings indicate that the CRM1/Exportin1-dependent export sequence is localized in the C-terminal part of p25 and encompassed within the sequence 169VYMVCLVNTV178. When this latter was replaced by the sequence 169AYMACLVNTV178, virally expressed GFPp25nes- protein accumulated abundantly in the nucleus (Fig. 7d
).
Transport of bacterial proteins into the nucleus of eukaryotes (i.e. nuclear targeting of pathogen-encoded proteins) has been demonstrated in several instances. Thus, the AvrBs3 protein of Xanthomonas campestris, which localizes to the host cell nucleus after infection (Szurek et al., 2002), carries two NLS that interact with the nuclear receptor machinery (Szurek et al., 2001
) and a transcription activation domain required for mesophyll cell hypertrophy in susceptible plants (Marois et al., 2002
); the nuclear-targeted Agrobacterium tumefaciens 6b protein induces phytohormone-independent division of cells and alteration of leaf morphology by interacting with the putative host transcription activator NtSIP1 (Kitakura et al., 2002
). Plant virus proteins such as CMV 2b (Lucy et al., 2000
) and TEV NIb (Li et al., 1997
) have also been shown to be addressed actively to the nucleus of infected cells. However, to our knowledge, together with GRV ORF3 protein (Ryabov et al., 2004
), p25 is one of the first proteins encoded by a plant RNA virus, which has been shown to shuttle between the nucleus and the cytoplasm. Furthermore, our findings suggest that p25 belongs to the family of proteins whose activity is regulated by its cellular localization.
When p25 is able to access both the cytoplasm and the nuclear compartment, increase of symptom severity on leaves is observed. Production of the necrotic symptom phenotype (Nec) is poorly understood but may reflect interference with normal nuclear processes such as those described for AvrBs3 (Marois et al., 2002) or generalized toxicity of the highly basic fusion protein in the nucleus of C. quinoa cells as no necrosis was observed on Tetragonia expansa (unpublished results). However, whatever the mechanism, the nec symptoms must be because of the nuclear localization of the fusion protein, as no such symptoms were observed when the NLS sequence was mutated (compare Fig. 7e and f
) or when the export sequence was present (compare Fig. 7e and c
). Future studies of the p25 nucleo-cytoplasmic shuttling upon the BNYVV infection will aim at understanding the functions of such a protein in the rhizomania process.
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ACKNOWLEDGEMENTS |
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Received 29 March 2004;
accepted 5 May 2004.