1 Institut de Biologie Moléculaire des Plantes, 12 rue du Général Zimmer, 67084 Strasbourg cedex, France
2 INRA, UR-BIVV, 28 rue de Herrlisheim, 68021 Colmar, France
Correspondence
David Gilmer
david.gilmer{at}ibmp-ulp.u-strasbg.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the full-length infectious transcript of RNA-5 described in this work is AY823407.
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INTRODUCTION |
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In this study, we have constructed a full-length infectious cDNA clone of BNYVV RNA-5 and characterized the expression and localization of p26 fused to a haemagglutinin (HA) tag or to the jellyfish green fluorescent protein (GFP) in infected and transfected tobacco BY-2 cells. Expression of BNYVV p26 protein was either performed in an RNA-3-derived replicon (Erhardt et al., 2000) or in full-length RNA-5. A mutagenesis approach was used to identify the p26 start codon and the domains responsible for symptom production and p26 subcellular localization. Self-activation of transcription was observed by using a yeast two-hybrid screen. We sought to delimit the domain(s) responsible for such transcription activation by alanine scanning and, finally, tested the effect of mutants on p26 subcellular localization and symptom production.
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METHODS |
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Clones and infectious transcripts.
Clones and procedures for production of run-off transcripts have been described previously (Quillet et al., 1989; Lauber et al., 1998a
). Fusions between GFP and p26 (Reichel et al., 1996
) were generated by overlap-extension mutagenesis (Ho et al., 1989
) of cloned cDNA by using PCR. In the replicon containing GFPp26, the GFP termination codon was replaced by CCC and the first p26 initiation codon by GGG. p26 and fragments derived from the GFPp26 construct were amplified by the use of sense primers containing an EcoRI site and reverse primers containing a SalI site and cloned into pGBT9, pGAD424, pLexA and pB42AD vectors (all from Clontech) between the EcoRI and the SalI/XhoI sites. Deletions and point mutations in p26 were created by overlap-extension mutagenesis as described above. pCK-EGFP-derived constructs were obtained by PCR amplification of Rep-3-GFPp26 by using an NcoI-containing sense primer corresponding to the 5' end of the GFP gene and BamHI-containing reverse primers complementary to the 3' end of the p26 gene. Fragments were placed between the cauliflower mosaic virus 35S promoter and terminator in pCK-EGFP (Reichel et al., 1996
; Gaire et al., 1999
; Vetter et al., 2004
), using the NcoI and BamHI sites. All constructs and PCR fragments were characterized by restriction-enzyme digestion and sequenced with 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.
Viral infection procedures were as described previously (Lauber et al., 1998b; Erhardt et al., 2000
). Polypeptides containing the p26 and GFP sequences were immunodetected on Western blots by using a mouse monoclonal anti-HAperoxidase antibody (Roche, 1/20 000) and the GFP-specific rabbit antiserum anti-GFP-IV (1/60 000), respectively. BNYVV coat protein (CP) was immunodetected with rabbit polyclonal antibody anti-CP-VI (1/120 000). Polyclonal antibodies were previously incubated for 2 h in 1·5 ml PBS buffer containing 1 % Tween 20, 5 % skimmed milk and 5 mg healthy C. quinoa acetonic powder and centrifuged for 1 min at 2000 g. Goat anti-rabbit antiserum coupled to peroxidase (Molecular Probes, 1/10 000) was used for the chemiluminescent detection of primary complexes by using a Roche detection kit and a Bio-Rad ChemDoc apparatus, which allows quantification using Quantity One Bio-Rad software. Stripping was performed by soaking membranes for 10 min in 0·2 M NaOH followed by extensive washes in water and PBS buffer before further immunodetections.
BY-2 transient expression.
GFP-fusion proteins were expressed transiently in BY-2 tobacco suspension cells (Nicotiana tabacum cv. Bright Yellow 2) maintained as described by Banjoko & Trelease (1995). Cells were subcultured for 7 days and harvested 3 days after medium renewal for biolistic bombardment. The harvested cells were filtered onto filter-paper discs and placed on 0·8 % agar MurashigeSkoog (MS) medium plates supplemented with 0·25 M mannitol for 24 h. Particle preparation, biolistic assays and leptomycin B (LMB) treatments were performed as described previously (Vetter et al., 2004
). After bombardment, cells were transferred to 0·8 % agar MS medium plates and incubated in the dark for 16 h at 28 °C. Transfected BY-2 cells were collected under HBO binoculars (excitation/emission wavelengths, 488/505545 nm) 16 h post-bombardment and cultured in MS liquid medium prior to further treatment and/or confocal laser-scanning microscopy (CLSM) observations.
In vivo detection of GFP by epifluorescence and 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 20 GFP-expressing BY-2 cells were observed in each case. No more than 5 % of transfected cells displayed variation of subcellular localization. Such cells were not taken into consideration. Laser scanning was performed by using identical settings for single-track mode, and excitation/emission wavelengths were 488/505545 nm for GFP. Image processing was carried out with LSM510 version 2.5 (Zeiss), ImageJ 1.32j (http://rsb.info.nih.gov/ij/) with LSM plug-in and Adobe Photoshop 7.
Immunolocalization of p26HA by transmission electron microscopy.
Leaf-tissue samples were taken from the leading edge of local lesions at 5 days post-infection (d.p.i.), fixed for 2 h in 2 % (v/v) glutaraldehyde, treated for 12 h at 4 °C in 10 % (w/v) picric acid and stained for 2 h in 0·1 % (v/v) osmium tetroxide and for 12 h in 2 % (w/v) uranyl acetate. All treatments were performed in 150 mM sodium phosphate buffer, pH 7·2. Samples were then dehydrated through an ethanol series and infiltrated with EPON 812 medium-grade resin (Polysciences Inc.). Polymerization was for 60 h at 60 °C. Ultrathin sections (90 µm) were cut by using an Ultracut E microtome (Reichert) and collected on grids coated with Formvar (EMS). Immunolocalization of p26HA was performed by incubating the sections with a mouse anti-HA antibody diluted 1/50 in 1 % BSA in PBS for 2 h at room temperature. The secondary goat anti-mouse serum was diluted 1/50 in 0·1 % BSA and coupled to 15 nm gold-conjugated antibody (Aurion EM reagents; EMS).
One- and two-hybrid assays.
One-hybrid and two-hybrid experiments were performed by using the Matchmaker two-hybrid system based on the Yeast Protocol Handbook (BD Biosciences Clontech) and also as described by Haasen et al. (1999) and Szurek et al. (2001)
. For one-hybrid experiments, His3 reporter-gene activation was tested in yeast strain HF7c transformed with Gal4BDp26 fusions (pGBT9, Clontech) on minimal medium depleted of tryptophan and histidine. For two-hybrid assays, Gal4ADp26 fusions (pGAD424, Clontech) were transferred into yeast strain Y187. Mating was used to obtain diploids expressing both fusion proteins.
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RESULTS |
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The ability of all the constructs to replicate and express p26 was monitored respectively by Northern blot and Western blot analysis of RNA and proteins from standardized pools of local lesions. Equal amounts of total RNA (from three 2·5 mm2 discs, each centred on a local lesion) were analysed (Fig. 1b and c, upper panel, Rib). Viral RNAs were detected by antisense RNA-1-, -2-, -3- and -5-specific riboprobes (Fig. 1b
, upper panel) and RNA-1-, -2- and -3-specific riboprobes (Fig. 1c
, upper panel) to estimate the accumulation of tagged or untagged RNA constructs. The presence of the HA and GFP sequences did not alter the replication capabilities of RNA-5- or Rep-3-derived molecules (Fig. 1b
, compare RNA-5 with RNA-5HA and Rep-3-p26 with Rep-3-p26HA). Rep-3-GFP-p26 also replicated efficiently, but to a lower extent; the appearance of a truncated form of the RNA was noted (Fig. 1b
). Rep-3-p26HA constructs were all able to accumulate (Fig. 1c
, upper and middle panels) and did not influence RNA-1 and -2 accumulation greatly. By using a specific p26 cDNA probe (Fig. 1c
, middle panel), detection of RNA-containing p26 sequence showed that Rep-3-p26 derivatives accumulated in similar amounts to RNA-5HA, with exceptions for Rep-3-p26HAa and -e, which displayed lower signals.
Because a specific p26 antiserum is not available, only GFP (data not shown), HA-tagged p26 and CP proteins were detected on Western blots (Fig. 1b and c, lower panels). Equal amounts of total protein extracts from local lesions were analysed (Fig. 1b and c
, MS) by using specific antibodies. No major variation of CP accumulation was detected between the different samples (Fig. 1b and c
, lower panels). RNA-5-derived expression of p26HA (Fig. 1b and c
) was four- to fivefold lower than when the protein was expressed from Rep-3-p26HA RNA (Fig. 1b and c
), as described previously (Schmidlin et al., 2005
). For this reason, we decided to further express p26HA protein and its derivatives in the Rep-3 vector.
p26HAb and p26HAc induced similar necrotic local lesions to p26HA, which all led to the detection of HA-tagged proteins (Fig. 1c, lower panel). No necrosis was observed when p26HAa, -d and -e were expressed, where only a trace amount of p26HAa was detected (Fig. 1c
, lower panel). p26HA-2 and p26HA-3 were found in similar amounts to p26HA within necrotic local lesions with lower molecular masses (Fig. 1c
, lower panel). Such comparative analysis permitted us to conclude that p26 translation initiation occurred at the first AUG (Fig. 1c
, compare RNA-5HA and Rep-3-p26HA with Rep-3-p26HA-2 and -3).
p26 protein is partially targeted to the nuclear compartment of infected cells
Immunoelectron microscopy was performed on C. quinoa ultrathin sections infected with RNA-1 and -2 supplemented with Rep-3-p26 or Rep-3-p26HA. Samples were taken at 5 d.p.i., before the appearance of severe necrosis. By using specific HA monoclonal immunogold detection, 74 % of gold particles were located mainly within the nuclear compartment (Fig. 2b; Table 1
), whereas only 27 % of nuclear labelling was observed in cells expressing the non-tagged form of p26 (Fig. 2a
; Table 1
). In this latter situation, gold particles detected within the nuclear compartment were mainly due to well-known colloidal gold affinity to chromatin. In rare cases, gold particles were found on virus aggregates (Fig. 2c
).
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LMB inhibition of nuclear export performed on GFPp26 fusion proteins did not modify the subcellular localization of GFP-RQ3, GFP-FQ3, GFP-D7 or GFP-D8 species, which were present predominantly in the cytoplasmic compartment (Table 2). However, GFP-D9, GFP-FQ1 and GFP-FQ2 presented a strong nuclear localization after LMB treatment (Table 2
), indicating that these fusion proteins were able to enter the nuclear compartment, but were exported back and accumulated in the cytoplasm as shown previously for BNYVV-p25 (Vetter et al., 2004
). These findings suggest that aa 55110 may act as a cytoplasmic-retention sequence, as no passive diffusion or nuclear accumulation of GFP-fusion proteins containing this domain were detected in LMB-treated BY-2 cells, whereas residues 110172 may contain a nuclear-export signal sensitive to LMB treatment (Table 2
). Analysis of the p26 amino acid sequence did not detect any known nuclear-localization or -export signals.
p26 does not interact directly with the CP or readthrough (RT) domain, but its N-terminal sequence is responsible for transcriptional-activation activity in yeast
To determine whether p26 can interact directly with viral particles (Fig. 2c), we used full-length or truncated p26, CP and RT domains as bait and prey in a yeast two-hybrid screen. Yeast strain HF7c carrying pGBT9-derived (Gal4BD) vectors were selected on tryptophan-depleted minimal medium, whereas yeast strain Y187 carrying pGAD424-derived vectors (Gal4AD) were selected on leucine-depleted minimal medium. After mating, diploids were selected on LeuTrp-depleted minimal medium and equal numbers of diploids were plated onto both LeuTrpHis-depleted and LeuTrp-depleted minimal medium to detect interactions and to control yeast diploid growth.
We only observed a direct interaction of CP with itself and with the RT domain (data not shown), as expected (Tamada et al., 1996b), and also a strong transcription activation mediated by Gal4BDp26 and -RQ1 (aa 155) fusion proteins, whichever protein was used as prey (data not shown). When tested in yeast strain EGY48, LexAp26 fusion protein also strongly induced lacZ reporter-gene transcription (data not shown).
We further characterized this Gal4BDp26-mediated transcriptional activation. Eighteen sets of contiguous amino acid residues were replaced sequentially by alanine in order to cover the N-terminal domain constituted by aa 254 in the full-length Gal4BDp26 fusion-protein context (Fig. 4a). HF7c recombinant clones expressing such fusion proteins were selected on Trp-depleted minimal medium. Yeast cells were then plated onto TrpHis-depleted minimal medium for a His3 transcription-activation assay (Fig. 4b
). Yeast expressing full-length Gal4BDp26 was able to grow in the absence of histidine, as were the Gal4BDp26 alanine mutants MQ1-A, -B, -D to -F, -M and -P to -R, indicating that transcription activation of the His3 reporter gene occurred (Fig. 4b
). By contrast, mutants MQ1-C, -G to -L, -N and -O were not able to activate transcription efficiently (Fig. 4b
).
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DISCUSSION |
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Immunoelectron microscopy has permitted us to the determine the localization of HA-tagged p26 mainly in the nucleus of infected cells. A similar localization was observed by using a GFPp26 fusion protein. We concluded that this nuclear targeting is due to active import because a 97 kDa GFPp26CHS fusion protein was also targeted to the nucleus (Fig. 3). GFP-fused, deleted forms of p26 were produced in BY-2 cells to map the domain responsible for nuclear targeting. Out of the nine GFP-fused constructs, only GFP-RQ1 was able to localize in the nucleus (Fig. 3
) and this was due to passive diffusion. Taken together, our results thus suggest that the nuclear localization of the p26 protein depends upon the structure, rather than a discrete nuclear-localization signal. Possible nuclear targeting is driven by nuclear-localization motifs similar to those that mediate nuclear addressing of the capsid proteins of Minute virus of mice (Lombardo et al., 2000
, 2002
).
All of the GFPp26-deleted forms that we studied had a molecular mass lower than the exclusion limit of nuclear-pore complexes and could thus have entered the nucleus by passive diffusion. As demonstrated previously for BNYVV p25 protein (Vetter et al., 2004), such diffusible proteins can be exported actively from the nucleus back to the cytoplasm under the control of a nuclear-export signal. We have shown here that GFP-D9 accumulated in the nucleus after inhibition of the CRM-1 export pathway with LMB (Table 2
), suggesting that it is exported actively in the absence of the inhibitor. LMB treatment had no effect on the cytoplasmic localization of GFP-RQ2, -RQ3, -D7 or -D8, suggesting that the D8 sequence domain was responsible for retention of these proteins in the cytoplasm. LMB-mediated nuclear accumulation of GFP-FQ1, a protein that contains the D8 domain, demonstrated that cytoplasmic retention was sensitive to structural features of the polypeptide used. Such structural implications were also observed in planta, as none of the p26-deletion mutants were able to induce necrosis of local lesions (Table 2
), indicating that the entire protein is required, rather than a specific domain.
Retention of nuclear-targeted proteins in the cytoplasm is a means of regulating protein function, as demonstrated for the tumour-suppressor protein p53, which is anchored in the cytoplasm by Parc protein (Nikolaev et al., 2003). Similarly, the
-amyloid precursor protein anchors the Fe65 protein in the cytosol (Minopoli et al., 2001
) and prevents its nuclear regulation of transcription (Bruni et al., 2002
; Telese et al., 2005
). Cytoplasmic retention of certain p26-fusion proteins might also explain the lack of transcription activation in yeast obtained with constructs Gal4BD-RQ2 and -RQ3. Interestingly, full-length p26 protein retained transcriptional activity, indicating that structural features are required for the activity. Although transcriptional-activation activity of p26 has not yet been demonstrated in planta, the gain of function for both LexA and Gal4BD fused to p26 nevertheless provides strong circumstantial evidence for the presence of a functional transcription-activation domain. Numerous examples of authentic transcription activators that display such activity have been reported: for example, the Herpes simplex virus VP16 C-terminal domain (Sadowski et al., 1988
; Cousens et al., 1989
), the Xanthomonas campestris AvrBs3 protein (Szurek et al., 2001
), the STAT6 transcription activator (Yang et al., 2002
), the Hepatitis C virus-encoded NS5A protein (Kato et al., 1997
; Tanimoto et al., 1997
; Song et al., 2000
) and the Gal4AD fused to LexA (Rhee et al., 2000
).
Alanine scanning of the N-terminal domain of the p26 protein implicated residues 2037 and 4143 (p26-MQ1-G to -L and -M) in the transcription-activation mechanism and residues 2328 and 3235 (p26-MQ1-H, -I and -K) as having an effect on nuclear targeting. The R26A and R33A replacements within the two latter mutants might explain the decrease, but not the loss, of nuclear import of the p26 protein and might therefore be constitutive of the nuclear-localization motif. It should be noticed that, in the yeast one-hybrid system, all full-length Gal4BDp26-MQ1 fusion proteins are thought to be addressed to the nucleus by Gal4BD nuclear-localization signals but, in planta, such transcription activation would require the presence of the aforesaid protein in the nucleus. Thus, we could not directly correlate transcription activation in yeast with the necrosis observed in planta, as some of the mutants were affected in their subcellular localization. Thus, p26-MQ1-N was able to induce necrosis and was targeted to the plant-cell nucleus, but did not activate transcription in yeast. The p26-MQ1-K protein was deficient in transcription activation and its nuclear addressing in planta was affected, but it was still able to induce necrosis (Table 1). Thus, our data indicate that all of the phenomena are distinct. Therefore, further experiments will be needed to identify more precisely the mechanisms that regulate nuclear and cytoplasmic partitioning of the protein, to determine whether p26 indeed activates transcription in planta and to further map pathogenicity determinants on the p26 structure. Structural modifications after post-translational modification have been reported to affect nucleo-cytoplasmic partitioning, as shown e.g. for the ERK2 protein kinase (Canagarajah et al., 1997
; Khokhlatchev et al., 1998
; Cobb & Goldsmith, 2000
) and the NFAT1 transcription factor (Okamura et al., 2000
; Salazar & Höfer, 2003
, 2005
).
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ACKNOWLEDGEMENTS |
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Received 31 January 2005;
accepted 11 March 2005.