Functional characterization of the Beet necrotic yellow vein virus RNA-5-encoded p26 protein: evidence for structural pathogenicity determinants

Didier Link1, Laure Schmidlin1, Audrey Schirmer2, Elodie Klein1, Mathieu Erhardt1, Angèle Geldreich1, Olivier Lemaire2 and David Gilmer1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A Beet necrotic yellow vein virus isolate containing a fifth RNA is present in the Pithiviers area of France. A full-length cDNA clone of RNA-5 was obtained and placed under the control of a T7-RNA-pol promoter that allowed the production of infectious transcripts. ‘Pithiviers' isolate-specific necrotic symptoms were obtained on Chenopodium quinoa when RNA-5-encoded p26 was expressed either from RNA-5 or from an RNA-3-derived replicon. By using haemagglutinin- and green fluorescent protein-tagged constructs, virally expressed p26-fusion proteins induced the same necrotic local lesions on host plants and were localized mainly in the nucleus of infected cells. Deletion mutagenesis permitted identification of two domains, responsible respectively for nuclear export and cytoplasmic retention of the p26 mutated proteins. By using a yeast two-hybrid system, Gal4DB–p26 protein self-activated transcription of the His3 reporter gene. The p26 transcription-activation domain was located within its first 55 aa and has been studied by alanine scanning. Resulting p26 mutants were tested for their capability to induce necrotic symptoms and to localize in the nuclear compartment.

The GenBank/EMBL/DDBJ accession number for the full-length infectious transcript of RNA-5 described in this work is AY823407.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Beet necrotic yellow vein virus (BNYVV; genus Benyvirus) is a positive-stranded RNA virus with rod-shaped particles and an unusual multi-component genome. BNYVV is transmitted by the soil-borne organism Polymyxa betae (recently proposed to be tranferred from the kingdom Fungi to Protozoa). This phytovirus is responsible for rhizomania disease of sugar beet, which is characterized by extensive rootlet proliferation from the main taproot and other abnormalities. BNYVV genes required for basic housekeeping functions, such as genome replication, cell-to-cell movement, packaging and suppression of post-transcriptional gene silencing, reside on RNA-1 (6746 nt) and RNA-2 (4812 nt) (Tamada, 1999; Dunoyer et al., 2002). These genome components are necessary for virus multiplication in hosts of the family Chenopodiaceae and are sufficient for local lesion formation on leaves of a diagnostic species, such as Chenopodium quinoa (Quillet et al., 1989; Tamada et al., 1989). All field isolates of BNYVV contain two additional RNAs. RNA-3 (1775 nt) controls rhizomania symptom expression on the natural host (Tamada et al., 1999) and local lesion phenotype on leaves (Tamada et al., 1989; Jupin et al., 1992), whereas RNA-4 (1467 nt) carries information necessary for vector transmission of the virus (Tamada & Abe, 1989). Some BNYVV isolates contain a fifth RNA (RNA-5, 1342–1347 nt) that can also influence symptom severity in a synergistic fashion with RNA-3 (Tamada et al., 1996a). BNYVV RNA-5 encodes a ~26 kDa protein (p26) that differs between J- and P-type isolates (Koenig et al., 1997; Miyanishi et al., 1999; A. Schirmer, D. Link, V. Cognat, B. Moury, M. Beuve, A. Meunier, C. Bragard, D. Gilmer & O. Lemaire, unpublished data).

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Synthesis of a full-length infectious cDNA clone of BNYVV RNA-5.
A FirstChoice RLM-RACE kit (Ambion) was employed on total RNA extracted from infected sugar beet root containing the ‘Pithiviers' isolate of BNYVV. cDNA corresponding to the 5' part of RNA-5 was amplified by using a sense primer (nt 1–12) containing a T7 promoter and an EcoRI restriction site, coupled with the 26MR reverse primer (nt 967–945). A second PCR was performed to amplify cDNA corresponding to the 3' end of RNA-5 by using the 26F sense primer (nt 605–629) and an Ambion reverse primer containing a BamHI restriction site. PCR products were subsequently cloned in the pGEM-T vector (Promega) to produce pGEM-R55 and pGEM-R53, respectively, and sequenced. The pGEM-R55 EcoRI–BsrGI restriction fragment containing the T7-RNA-pol promoter was cloned in EcoRI–BsrGI-digested pGEM-R53 to produce pGEM-R553. The pGEM-R553 EcoRI–BamHI restriction fragment was subsequently cloned in pUC19 (Fermentas) to obtain pUC-R51, containing the full-length cDNA sequence of RNA-5. Extra nucleotides between the RNA-5 poly(A) sequence and the BamHI site were replaced with a HindIII restriction site by PCR mutagenesis, leading to the vector pB55. This latter was used for the synthesis of full-length infectious transcripts of RNA-5 (GenBank accession no. AY823407).

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 GFP–p26, the GFP termination codon was replaced by CCC and the first p26 initiation codon by GGG. p26 and fragments derived from the GFP–p26 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-GFP–p26 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-HA–peroxidase 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 Murashige–Skoog (MS) medium plates supplemented with 0·25 M mannitol for 2–4 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, 488/505–545 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 488/505–545 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 Gal4BD–p26 fusions (pGBT9, Clontech) on minimal medium depleted of tryptophan and histidine. For two-hybrid assays, Gal4AD–p26 fusions (pGAD424, Clontech) were transferred into yeast strain Y187. Mating was used to obtain diploids expressing both fusion proteins.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A full-length cDNA clone of RNA-5 is able to reproduce ‘Pithiviers' isolate symptoms that are induced by the expression of p26 protein
The first step of this work was the construction of pB55, which was used to produce full-length infectious RNA-5 transcripts (Fig. 1a). The RNA-5-based replicon (Rep-5) was obtained by replacing the pB55-p26 coding sequence by NcoI, EcoRV and BglII sequences (Schmidlin et al., 2005). The p26 sequence, fused in frame with the HA epitope, was cloned into Rep-5 and Rep-3 (Erhardt et al., 2000) to create RNA-5HA and Rep-3-p26HA, respectively (Fig. 1a). When inoculated onto C. quinoa leaves, together with Stras12 helper strain (i.e. RNA-1 and RNA-2) that produces chlorotic local lesions (Quillet et al., 1989), RNA-5, RNA-5HA, Rep-3-p26, Rep-3-p26HA (Fig. 1a) and Rep-3-GFP-p26 (data not shown) were all able to produce similar necrotic local lesions (Nec) characteristic of the BNYVV-‘Pithiviers' isolate. Such necrotic symptoms were indeed due to the expression of p26 protein, as a Rep-3-p26fs frameshift mutant induced not necrosis, but Stras12-like chlorotic spots (CS, Fig. 1a).



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Fig. 1. Analysis of the biological properties of BNYVV upon infection of C. quinoa leaves. (a) Schematic overview of cDNA infectious clones and constructs used. Nucleotide sequences surrounding the p26 translation-initiation sites (shown in bold) are detailed for each type of cDNA clone and the HA epitope is symbolized by a black square. Amino acid residues replaced are shown in bold and denoted by asterisks. {bullet}, Position of the frameshift mutation. The p26 ORF appears in grey. Thin and thick lines represent RNA-5- and RNA-3-derived non-translated sequences, respectively. An, Polyadenylated sequence. Drawings are not to scale. Local lesion symptoms obtained on C. quinoa 6 d.p.i. are detailed. Nec, Necrosis; CS, chlorotic spots; YS, yellow spots. (b, c) Comparison of RNA (upper panel) and protein contents (lower panel) of C. quinoa local lesions inoculated with RNA-1 and -2 supplemented with the specified Rep-3- or RNA-5-derived RNA. (b) The membrane was probed with specific riboprobes complementary to RNA-1, -2, -3 and -5. The star indicates the position of a truncated RNA issued from Rep-3-GFP–p26. (c) The membrane in the upper panel was probed with specific riboprobes complementary to RNA-1, -2 and -3. The middle panel corresponds to the same membrane stripped and probed with a specific p26 cDNA probe. Positions of viral RNAs and proteins are shown. Rep-3-p26HA and RNA-5/-5HA co-migrate. Rib, rRNA; MS, membrane stain; p26HA, immunodetection of HA-tagged proteins; CP, immunodetection of coat protein. Three independent experiments gave identical results.

 
The p26 open reading frame (ORF) contains three potential initiation codons (Fig. 1a). The first and the last AUGs are in a favourable initiation context (Kozak, 2002) (–3R and +4R), whereas the second AUG presents a non-favourable initiation context (–3Y and +4Y). In order to identify the start codon, we used a p26 sequence tagged with the HA sequence, cloned into the RNA-3-based replicon. The p26HA nucleotide sequence was modified to produce mutants p26HA-2, -3 and -a to -e (Fig. 1a). Nucleotide sequences surrounding the second AUG (Rep-3-p26HA-2) and the third AUG (Rep-3-p26HA-3) were changed to force initiation at the respective AUGs and such proteins still induced necrosis when expressed from the virus (Fig. 1a). A frameshift mutation was created after the second initiation codon (Fig. 1a), which abolishes the synthesis of p26HA proteins initiated at the first and second AUGs. Rep-3-p26HAa induced chlorotic symptoms (Fig. 1a). Rep-3-p26HAb and Rep-3-p26HAc forced the initiation of protein synthesis at the first two AUGs and the first AUG, respectively (Fig. 1a), and such constructs were able to induce necrotic symptoms (Fig. 1a). Finally, if we prevented initiation at the first AUG by a frameshift mutation inserted between the first and second AUG, no necrosis was observed (Fig. 1a, Rep-3-p26HAd and e), although mutant p26HAd induced yellowing symptoms.

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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Fig. 2. Immunoelectron microscopy, using an anti-HA mAb, of the BNYVV RNA-5-encoded p26 fused or not to an HA-epitope tag and expressed in infected C. quinoa leaves 5 d.p.i. Replicons expressing p26 (a) or p26HA (b, c) were inoculated together with RNA-1 and -2 onto C. quinoa leaves. For p26, an average of 11 gold particles were found in the cytoplasm and four in the nucleus (n=9) and for p26HA, 22 gold particles in the nucleus and seven in the cytoplasm (n=24). In rare cases, labelling was also found on virus aggregates (c). Chl, Chloroplast; Cy, cytoplasm; Cw, cell wall; Va, vacuole; M, mitochondrion; N, nucleus; ne, nuclear envelope. Immunodetection was repeated three times with similar statistical results. Bars, 500 nm (a, b); 100 nm (c).

 

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Table 1. Effect of virally expressed p26-MQ1 HA-tagged proteins on symptom production in C. quinoa leaves and comparison of cellular locations for the fusion proteins following viral expression

n/C and N/c refer respectively to predominantly cytoplasmic and predominantly nuclear partitioning of immunogold labelling in infected cells. NA, Not applicable; CS, chlorotic spots; Nec, necrotic spots.

 
To further characterize the domain of p26 responsible for this nuclear targeting, we used the above-described functional GFP–p26 fusion protein, which is still able to induce local lesion necrosis (data not shown). When expressed in the viral context, GFP–p26 was found in both the nucleus and the cytoplasm of infected cells (Fig. 3, GFP–p26, upper panel) but, as mentioned above, Rep-3-GFP–p26 can undergo deletion events that might create truncated GFP–p26 fluorescent molecules undistinguishable from full-length GFP–p26 during CLSM observations. To avoid such a phenomenon, transient-expression vectors based on the pCK plasmid (Reichel et al., 1996; Gaire et al., 1999; Vetter et al., 2004) were also designed to drive expression of full-length or partially deleted p26 proteins fused to GFP (Fig. 3) from a constitutive 35S promoter. Plasmids were introduced into Bright Yellow 2 (BY-2) tobacco cells by means of biolistic gene transfer and fluorescent cells were analysed by CLSM 16 h post-bombardment. All of the biolistic experiments were performed at least twice under the same conditions. In each case, 20 randomly chosen, GFP-expressing BY-2 cells were observed. Minor variations (<=1/20) in cytoplasmic versus nuclear localization were detected for each experiment.



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Fig. 3. Subcellular localization of GFP (white rectangles)–p26 (grey rectangles), fused or not with chalcone synthase (CHS, dark-grey rectangles) in vivo. Broken lines correspond to deleted regions of the p26 protein. Numbers indicate the position of p26 amino acid boundaries. Drawings are not to scale. GFP–p26 fusion protein expressed during infection of C. quinoa (upper left only) and GFP–p26 fusion proteins expressed in biolistically transfected BY-2 cells are shown. Chloroplasts (Chl) appear in red in epidermal cells. N, Nucleus; Nu, nucleolus; Va, vacuole. This experiment was repeated three times with similar results. Bars, 10 µm.

 
Expression of GFP alone in BY-2 cells (data not shown) resulted in equal localization of the fluorescent protein in both the cytoplasm and the nucleus of the cell by passive diffusion between the two compartments (Chiu et al., 1996; Vetter et al., 2004). When the 53 kDa GFP–p26 protein was expressed either virally (Fig. 3, GFP–p26, upper panel) or transiently (Fig. 3, GFP–p26, lower panel), fluorescence localized to the nucleus and the cytoplasm, with the GFP–p26 localization being predominantly nuclear when expressed transiently. The diffusion-exclusion limit of nuclear pores has been shown to be around 9 nm (Görlich et al., 1996; Görlich & Kutay, 1999), which corresponds to a diffusion limit of up to ~30–50 kDa for a globular protein (Paine et al., 1975; Bonner, 1978; Mattaj & Englmeier, 1998). We fused the chalcone synthase sequence (CHS) in frame with the 3' extremity of the GFP–p26 sequence to produce a ~97 kDa GFP–p26–CHS fusion protein that was still able to access the nuclear compartment (Table 2; Fig. 3). Therefore, GFP–p26–CHS must be transported actively into the nucleus.


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Table 2. Effect of virally expressed GFP-fused and unfused p26 proteins on symptom production in C. quinoa leaves and comparison of cellular locations for the fusion proteins following viral or BY-2 transient expression

N/C, n/C, N/c and C refer respectively to equal nuclear and cytoplasmic, predominantly cytoplasmic, predominantly nuclear and cytoplasmic partitioning of GFP fluorescence in expressing cells. NA, Not applicable; NT, not tested; CS, chlorotic spots; Nec, necrotic spots. Italicized symbols refer to unstable Rep-3-GFP–p26 constructs that could not be interpreted. LMB treatments were reproduced twice with similar results.

 
The subcellular localizations and pathogenicity of different p26-deletion mutants were analysed in both the viral context and BY-2 transient expression. All of the GFP–p26-deletion mutants and p26-deletion mutants tested failed to induce p26-specific necrotic symptoms when inoculated onto C. quinoa leaves (Table 2). The N-terminally deleted forms of GFP–p26, GFP-FQ1 to -FQ3 (Fig. 3), were tested as described and were not able to enter the nuclear compartment abundantly, as did full-length GFP–p26 (Table 2; Fig. 3, compare GFP–p26 lower panel with GFP-FQ1, -FQ2 and -FQ3). Of the C-terminally truncated forms of GFP–p26 tested, GFP-RQ1 to -RQ3 (Fig. 3), only GFP-RQ1 was located abundantly in the nuclei of transfected cells (Table 2; Fig. 3). This is due to passive diffusion, as a 77 kDa GFP–p26-RQ1–CHS fusion protein (Fig. 3) was not localized in the nucleus. We also constructed GFP-D7 (aa 55–171), -D8 (aa 55–109) and -D9 (aa 110–171). Surprisingly, all three fusion proteins remained in the cytoplasmic compartment, even though they have a molecular mass of ~33 kDa (Table 2; Fig. 3). Taken together, these results suggest that the entire sequence of p26 is involved in the nuclear targeting of the protein and internal domains might be responsible for cytoplasmic retention and/or nuclear export of the protein.

LMB inhibition of nuclear export performed on GFP–p26 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 55–110 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 110–172 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 Leu–Trp-depleted minimal medium and equal numbers of diploids were plated onto both Leu–Trp–His-depleted and Leu–Trp-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 Gal4BD–p26 and -RQ1 (aa 1–55) fusion proteins, whichever protein was used as prey (data not shown). When tested in yeast strain EGY48, LexA–p26 fusion protein also strongly induced lacZ reporter-gene transcription (data not shown).

We further characterized this Gal4BD–p26-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 2–54 in the full-length Gal4BD–p26 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 Trp–His-depleted minimal medium for a His3 transcription-activation assay (Fig. 4b). Yeast expressing full-length Gal4BD–p26 was able to grow in the absence of histidine, as were the Gal4BD–p26 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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Fig. 4. Transcription activation of the His3 reporter gene in the yeast one-hybrid system. Haploids expressing Gal4BD–p26 protein mutant constructs (a) were selected (b) on minimal medium depleted of tryptophan (–T) and tested for the activation of the His3 reporter gene by growth on minimal medium depleted of tryptophan and histidine (–TH). The amino acids replaced by alanine within the first 54 residues are shaded. This assay was repeated three times with identical results.

 
N-terminal mutation affected p26-induced necrosis and nuclear targeting in C. quinoa
The above-described p26 alanine mutants were analysed both for their ability to induce necrotic local lesions and for their subcellular localization on C. quinoa leaves. HA-tagged p26 alanine mutants were expressed in the viral context by using an RNA-3-derived replicon. When inoculated onto C. quinoa leaves together with Stras12, local lesions appeared 4–6 d.p.i. Necrotic spots were found with replicons expressing p26-MQ1-C, -F, -J, -K, -L and -N, whereas Stras12-like chlorotic spots were obtained with p26-MQ1-G, -H, -I and -M (Table 1); the chlorotic spots had necrotic centres for -H and -I (Table 1). RNA and protein accumulation was monitored as described previously and no differences in replication or protein-expression levels were found (data not shown). Analysis of the subcellular localization of the mutated forms of p26HA was also performed on local lesions (Table 1). The p26-MQ1-H, -I and -K mutations were found to alter the subcellular localization of p26, so that <50 % of total gold particles were counted within nuclear compartments. The other mutants behaved as did wild-type p26HA, where >50 % of gold particles were nuclear. Thus, no direct correlation was found between nuclear localization, yeast transcription activation and induction of necrosis.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, we have examined the role of p26-induced necrosis of infected C. quinoa leaves and extended our analysis to characterize the subcellular-localization determinants of this protein and its implications on BNYVV pathogenesis. Earlier reports described BNYVV RNA-5 as a pathogenicity enhancer of rhizomania-affected sugar beets (Tamada et al., 1996a; Heijbroek et al., 1999; Miyanishi et al., 1999). Our results reveal that p26 is also a pathogenicity enhancer in C. quinoa. RNA-3 trans expression and RNA-5 cis expression of p26 led to similar necrosis of C. quinoa local lesions, both in the absence and in the presence of an HA tag, and these properties were used to investigate which of the three potential initiation codons was used to express functional p26. Our results demonstrate clearly that the first AUG in the p26 ORF is used for expression of p26. For a similar RNA-replication level, p26 accumulation was five times lower when expressed from its cognate RNA. Such lower accumulation is probably be due to the presence of three short ORFs (not shown) within the 400 nt long 5' leader sequence of RNA-5, which can downregulate ribosome scanning and initiation at the first p26 AUG (Ryabova et al., 2002).

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 GFP–p26 fusion protein. We concluded that this nuclear targeting is due to active import because a 97 kDa GFP–p26–CHS 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 GFP–p26-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 {beta}-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 20–37 and 41–43 (p26-MQ1-G to -L and -M) in the transcription-activation mechanism and residues 23–28 and 32–35 (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 Gal4BD–p26-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).


   ACKNOWLEDGEMENTS
 
We thank Malek Alioua for sequence analysis and Delphine Squiban and Daniele Scheidecker for technical support. D. L. was supported by ITB and Region Alsace under a Bourse Régionale convention, L. S. was supported by SES-Advanta under the CIFRE programme and A. S. was supported by Agrinord (France). The Inter-Institute Confocal Microscopy Platform used in this study was co-financed by the Région Alsace, the CNRS, the Université Louis Pasteur and the Association pour la Recherche sur le Cancer (ARC).


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 31 January 2005; accepted 11 March 2005.