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Journal of Virology, July 2002, p. 6815-6824, Vol. 76, No. 13
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.13.6815-6824.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

P0 of Beet Western Yellows Virus Is a Suppressor of Posttranscriptional Gene Silencing

S. Pfeffer, P. Dunoyer, F. Heim, K. E. Richards, G. Jonard, and V. Ziegler-Graff*

Institut de Biologie Moléculaire des Plantes du CNRS et de l'Université Louis Pasteur, 67084 Strasbourg, France

Received 19 November 2001/ Accepted 8 April 2002


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ABSTRACT
 
Higher plants employ a homology-dependent RNA-degradation system known as posttranscriptional gene silencing (PTGS) as a defense against virus infection. Several plant viruses are known to encode proteins that can suppress PTGS. Here we show that P0 of beet western yellows virus (BWYV) displays strong silencing suppressor activity in a transient expression assay based upon its ability to inhibit PTGS of green fluorescent protein (GFP) when expressed in agro-infiltrated leaves of Nicotiana benthamiana containing a GFP transgene. PTGS suppressor activity was also observed for the P0s of two other poleroviruses, cucurbit aphid-borne yellows virus and potato leafroll virus. P0 is encoded by the 5'-proximal gene in BWYV RNA but does not accumulate to detectable levels when expressed from the genome-length RNA during infection. The low accumulation of P0 and the resulting low PTGS suppressor activity are in part a consequence of the suboptimal translation initiation context of the P0 start codon in viral RNA, although other factors, probably related to the viral replication process, also play a role. A mutation to optimize the P0 translation initiation efficiency in BWYV RNA was not stable during virus multiplication in planta. Instead, the P0 initiation codon in the progeny was frequently replaced by a less efficient initiation codon such as ACG, GTG, or ATA, indicating that there is selection against overexpression of P0 from the viral genome.


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INTRODUCTION
 
Most if not all plants possess a defense mechanism against virus infection known as posttranscriptional gene silencing (PTGS; see reference 44 for a recent review). PTGS is a homology-dependent RNA degradation system to recognize and degrade viral RNA (and any homologous transgene mRNA) in the cytoplasm. Mechanistically similar phenomena exist in animals and fungi (reviewed in references 4, 13, and 35). The mechanism by which a virus infection triggers PTGS in plants is not fully understood, but double-stranded RNA is a strong inducer of PTGS (43) and such a form is produced during replication of an RNA virus. Production of small RNAs of ~21 to 23 nucleotides, called small interfering RNAs (siRNAs), corresponding to both the plus and minus strands of the target RNA is associated with PTGS (12, 13, 45). In plants, gene silencing at one site can trigger PTGS in distant tissues and across a graft union (29, 39a). The mobile signal, which must consist at least in part of homologous RNA, is thought to move from cell to cell through plasmodesmata and systemically via the vasculature.

Some plant viruses have been shown to encode proteins which can counteract PTGS (8, 22, 39, 40, 42). These silencing suppressor proteins may act at different steps in the PTGS pathway. Thus, (i) the potyvirus helper component-proteinase (HCPro) interferes with initiation and maintenance of silencing at a step coincident with or upstream of siRNA production (1, 7, 16, 23, 24), (ii) the 2b protein of cucumber mosaic virus (CMV) prevents initiation of PTGS in new growth by inhibiting the long-range PTGS signaling activity (3, 7, 11), and (iii) p25 from potato virus X (PVX) suppresses production or action of the mobile silencing signal (40). There is evidence that silencing suppressor proteins are encoded by other plant viruses as well (39, 42).

The poleroviruses (family Luteoviridae) are obligately transmitted by aphids in a circulative manner and are phloem restricted in their hosts (27). Important members of the genus include Potato leafroll virus (PLRV; the type member), Beet western yellows virus (BWYV), and Cucurbit aphid-borne yellows virus (CABYV). The polerovirus genome consists of single-stranded plus-sense RNA of ~5.7 kb with six recognized open reading frames (ORFs) (Fig. 1A). The 5'-proximal ORFs 0, 1, and 2 are translated from viral RNA and yield P0, P1, and the translational frameshift protein P1-2. P1 and P1-2 are components of the viral RNA-dependent RNA polymerase and are thought to be translated by ribosomes which scan past the P0 start codon without initiating protein synthesis. The 3'-proximal ORFs 3, 4, and 5 are translated from a subgenomic RNA and yield the viral capsid proteins and a putative movement protein (26, 27).



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  		FIG. 1. Schematic representation of the BWYV genome and of some of the clones used in the study. (A) Genome organization of BWYV RNA. Numbered rectangles represent ORFs. The ORF for P0 is shaded. The diagonal arrow corresponds to the approximate position of translation frameshift to synthesize the P1-2 fusion protein mentioned in the text. The subgenomic RNA (sgRNA) which is used for translation of 3'-proximal genes is represented by a horizontal arrow below the map. (B) Structure of PVX-P0 chimera RNA. The white rectangles (not to scale) represent PVX genes, and the shaded rectangle represents the BWYV P0 gene. The black vertical lines correspond to the duplicated PVX subgenomic RNA promoter sequences, and horizontal arrows below indicate major subgenomic RNAs. The arrow above the P0 gene indicates the position of the frameshift (fs) mutation in PVX-P0fs. (C) Structures of the agro-infection vectors used for expression of different proteins in plants. Only the transcription cassette of each vector is shown with the cauliflower mosaic virus 35S transcription promoter and termination sequences indicated.

Although P0 has never been detected in polerovirus-infected plants, there is genetic evidence that it is functionally important. Thus, null mutations in the P0 gene of BWYV and PLRV strongly diminish or abolish viral RNA accumulation in protoplasts and plants (34, 46). In this paper we show that P0 of BWYV displays strong silencing suppressor activity in a transient expression assay based upon its ability to inhibit PTGS of green fluorescent protein (GFP) when expressed in agro-infiltrated leaves of Nicotiana benthamiana containing a GFP transgene. Silencing suppressor activity was also observed for the P0s of CABYV and PLRV. The poor expression of P0 from BWYV viral RNA is in part a consequence of the poor initiation codon context of the P0 ATG, although other factors are important as well. Experiments using a BWYV mutant in which the P0 initiation codon context was optimized indicate that overexpression of P0 is deleterious to viral RNA accumulation and that second-site mutations predicted to lower the efficiency of translation initiation at the P0 start codon were abundant in the progeny RNA.


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MATERIALS AND METHODS
 
Plasmids and transgenic plants. Full-length BWYV cDNA in pBW0 (20) was used as template for PCR amplification of the BWYV P0 ORF using appropriate specific primers. A nonviral EcoRV restriction site was added to the 5' extremity of each primer. The EcoRV-digested PCR fragment was ligated into EcoRV-digested pP2C2S (2) to yield pPVX-P0. pPVX-P0fs was produced by filling in of the XhoI site in the P0 ORF (nucleotide [nt] 255 in BWYV RNA) and recircularization. The aforesaid PCR fragment was also ligated into SmaI-digested pBin61 (40) to produce pBin-P0. pBin-P0CAB and pBin-P0PL were produced in a similar fashion by PCR of cloned cDNA from CABYV (31) and the Dutch isolate of PLRV (a gift of Frank van der Wilk) using appropriate specific primers. In mutant pBin-P0-, a DNA fragment containing a knockout mutation in the BWYV P0 ORF was obtained by PCR using DNA of the previously described P0 knockout mutant BW1.6346 (46) as template. pBin-P05'wt and pBin-P05'opt were constructed by PCR using an antisense primer complementary to the 3'-terminal sequence of the ORF and a sense primer with the desired additional 5'-terminal sequences built in.

pBin-BW was obtained by inserting a HindIII fragment containing the 35S terminator sequence from pBin61 into the SalI site of pBinBW0 (5). pBin-BW5'opt and pBin-BWP0- were constructed by PCR mutagenesis (14) to generate a PCR fragment containing the 35S promoter and the mutated BWYV cDNA sequence extending to the XhoI site at nt 255. This fragment was used to replace the corresponding sequence in the wild-type clone pBin-BW. The various plasmids described above were characterized by restriction site analysis, and all the regions generated by PCR were completely sequenced. Agrobacterium tumefaciens strains containing pBin-GFP (7) and pBin-HCPro, a pBin61 derivative harboring the HCPro coding region of potato virus Y, were obtained from D. C. Baulcombe.

Virus infection and agro-infiltration. N. benthamiana line 16c, which is homozygous for the GFP transgene, and the Agrobacterium infiltration method have been described previously (41). For coinfiltrations, each A. tumefaciens culture was grown to an optical density at 600 nm of 1 and mixed in equal volumes prior to infiltration. Plants were observed and photographed under UV light as described previously (41). Capped in vitro bacteriophage T7 RNA polymerase runoff transcripts were prepared (7) from SpeI-linearized pP2C2S, pPVX-P0, and pPVX-P0fs and mechanically inoculated to celite-dusted lower leaves of N. benthamiana.

RNA and protein analysis. High- and low-molecular-weight RNA fractions were extracted from plant tissues, separated by agarose or polyacrylamide gel electrophoresis, and transferred to nitrocellulose (Schleicher-Schuell or HyBond-NX [Amersham] membranes) (9, 12). RNA samples for electrophoresis were adjusted to the same concentration by spectrophotometry, and equal loading was verified by visualization of the bands of ethidium bromide-stained rRNA (high-molecular-weight RNA) or tRNA (low-molecular-weight RNA) on the gels. Specific RNAs were detected by hybridization with antisense 32P-labeled RNA probes corresponding to nt 240 to 664 of the GFP gene, the 3'-terminal 150 nt of PVX RNA, and either nt 1 to 376 or the 3'-terminal 196 nt of BWYV RNA. Radioactive signals were detected by autoradiography and quantified using a FUJIX BAS1000 phosphorimager and MacBAS image analysis software.

Reverse transcriptase PCR (RT-PCR) was performed essentially as described previously (6). Reverse transcription was primed with an oligonucleotide complementary to the 3'-terminal 20 nt of ORF 0 5'-terminally tagged with a nonviral HindIII site. PCR on the reverse transcript was carried out using the above-mentioned primer and a primer corresponding to the first 20 nt of the BWYV RNA sequence plus a 5'-terminal nonviral HindIII site. The PCR fragment was digested with HindIII and cloned into HindIII-cleaved pBS- (Stratagene), and the inserts of randomly selected clones were sequenced.

For Western (immuno)blot analysis, leaf material was ground to a powder in liquid nitrogen and 200 mg of the powder was further ground with 500 µl of concentrated (2x) gel loading buffer (18). Samples were boiled for 5 min and then centrifuged for 5 min at 6,000 x g before gel loading. Proteins were separated by electrophoresis through a denaturing 12% polyacrylamide gel (18) by inserting an XmnI-EcoRI fragment (nt 104 to 1005) containing most of ORF 0 into pEA305{Delta}HindIII-3 (28) downstream and in frame with the bacteriophage {lambda} cI cistron. The resulting recombinant plasmid, pBW.P9.8, overproduced the CI-P0 fusion protein in bacteria, and the protein was purified and used to raise a P0-specific antiserum essentially as previously described (28). Immunoreactive proteins were visualized by enhanced chemiluminescence (Pierce or Roche).


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RESULTS
 
Enhanced pathogenicity of a PVX chimera expressing BWYV P0. Previous work has assigned tentative functions to all of the recognized BWYV proteins except for P0, the protein encoded by the 5'-proximal ORF on the viral RNA (Fig. 1A). An earlier study showed that the BWYV P0 null mutants were viable but accumulated 5- to 10-fold lower levels of progeny RNA than the wild type in inoculated plants and protoplasts (46). The stimulatory effect of P0 on virus accumulation levels could indicate that the protein acts as a nonessential enhancer factor for BWYV RNA replication. Alternatively, P0 could augment virus accumulation indirectly, for example, by counteracting a host defense mechanism such as PTGS.

If P0 counters a PTGS-related host defense, it would be expected to augment pathogenicity and/or virus accumulation levels not only of the cognate virus but of other, unrelated viruses as well. This has been shown to be the case with HCPro and CMV 2b, for which expression of each silencing suppressor protein from a PVX or tobacco mosaic virus chimera strongly exacerbated symptoms compared to infection with the virus alone (7, 21, 32). To determine whether expression of P0 can similarly enhance the pathogenicity of another virus, the P0 coding region was inserted into the PVX-based vector pP2C2S (2) to produce pPVX-P0 (Fig. 1B). Inoculation of N. benthamiana with pPVX-P0 transcripts produced necrotic lesions on the inoculated leaves by 4 days postinfection (Fig. 2A, panel 3). The appearance of viral RNA in the upper leaves (Fig. 2B, lane 3) was accompanied by necrosis in the petioles and veins and then in mesophyll tissue (Fig. 2A, panel 3). Death of the upper leaves and eventually of the entire plant resulted. Northern blot analysis of RNA extracted from the upper leaves with an ORF 0-specific probe revealed that the P0 coding sequence was retained in the progeny viral RNA (Fig. 2B, lane 7).



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  		FIG. 2. A PVX-P0 chimera is hypervirulent on N. benthamiana. (A) N. benthamiana plants which have been mock inoculated (panel 1) or inoculated with wild-type PVX transcript (panel 2), PVX-P0 (panel 3), or PVX-P0fs (panel 4). In panel 3, the arrow indicates an inoculated leaf and the arrowhead indicates an upper leaf with severe systemic necrosis. Photographs were taken 15 days postinfection. (B) Northern (RNA) blot analysis of viral RNA extracted from upper leaves of the plants shown in panel A and probed with either a PVX RNA-specific probe (lanes 1 to 4) or a probe specific for the BWYV P0 coding region (lanes 5 to 8). The upper band in each lane corresponds to genomic RNA (g). The lower bands in lanes 2 to 4 are the 3'-proximal subgenomic RNAs sg1, sg2, and sg3 (see Fig. 1B). The extra sg1 band in lane 4 compared to lane 3 indicates that a short deletion occurred in a fraction of the PVX-P0fs progeny RNA in this experiment.

In contrast, plants inoculated with the transcript of a vector containing a frameshift-mutated version of the P0 gene (pPVX-P0fs; Fig. 1B) developed faint chlorotic lesions on the inoculated leaves and a mild mosaic on the upper leaves (Fig. 2A, panel 4), accompanied by the appearance of chimeric viral RNA (Fig. 2B, lanes 4 and 8). These symptoms are similar to those observed following infection with the empty vector pP2C2S (Fig. 2A, panel 2). We conclude that expression of P0 can enhance the pathogenicity of an unrelated virus and that this activity is independent of other BWYV genes.

BWYV P0 is a suppressor of PTGS. The foregoing observations prompted us to further test P0 for its ability to suppress PTGS. This was done by use of a pBin19-based binary vector to transiently express P0 (pBin-P0; Fig. 1C) in N. benthamiana line 16c (7), which contains a transgene encoding GFP. The leaves and stems of 16c plants normally fluoresce green under long-wavelength UV light. However, expression of the GFP transgene can be posttranscriptionally silenced by infiltrating a lower leaf of a 16c plant with A. tumefaciens harboring a binary vector (pBin-GFP; Fig. 1C) designed to transiently express a second copy of the GFP transcript (41). For simplicity, we shall refer below to each A. tumefaciens culture used in such infiltrations by the name of the binary vector that it harbors.

The GFP silencing provoked in line 16c by infiltration of pBin-GFP can be blocked by simultaneous infiltration of a vector expressing a silencing suppressor protein (40; also see reference 15). Thus, the infiltrated zone (the patch) on 16c leaves coinfiltrated with pBin-GFP and pBin-HCPro (pBin-GFP + pBin-HCPro) (Fig. 1C), where HCPro is the known silencing suppressor of potato virus Y (7), became bright fluorescent green 5 days postinfiltration (p.i.) (Fig. 3A, panel 2). Patches which had received pBin-GFP plus the empty vector pBin61 had undergone silencing of the GFP signal by this time and appeared deep red (Fig. 3A, panel 1). Similarly, when 16c leaves where coinfiltrated with pBin-GFP plus pBin-P0, the infiltrated patch also appeared bright fluorescent green by 5 days p.i. (Fig. 3A, panel 3), indicating that P0, like HCPro, has silencing suppressor activity. Use of a vector (pBin-P0-) containing a nontranslatable version of the P0 cistron in the infiltration mix did not provoke suppression of GFP silencing in the patch (Fig. 3A, panel 4).



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  		FIG. 3. Expression of BWYV P0 in N. benthamiana line 16c suppresses PTGS of the GFP transgene. (A) Leaves of line 16c plants agro-infiltrated with bacteria mixtures harboring pBin-GFP plus one of the following: empty vector pBin61 (panel 1), pBin-HCPro (panel 2), pBin-P0 (panel 3), or pBin-P0- (panel 4). Photographs were taken with long-wavelength UV light 5 days p.i. (B) Northern (RNA) blot analysis of high-molecular-weight RNA (upper panel) and low-molecular-weight RNA (lower panel) extracted from the agro-infiltrated zone (patch) of 16c leaves such as those shown in panel A. The patches had received bacterial mixtures harboring pBin-GFP plus one of the following: empty vector pBin61 (lane 2), pBin-HCPro (lane 3), pBin-P0 (lane 4), or pBin-P0- (lane 5). The RNA loaded in lane 1 came from a noninfiltrated 16c leaf. The blots were hybridized with a probe specific for GFP mRNA. In the lower panel, the mobilities of 32P-labeled oligodeoxynucleotides of the indicated length are shown to the left. (C) Line 16c plants 15 days p.i. of bacterial mixtures harboring pBin-GFP plus one of the following: pBin61 (panel 1), pBin-HCPro (panel 2), or pBin-P0 (panel 3). Upper leaves in panels 1 and 3 show vein-proximal silencing of the GFP signal. The inserts show an agro-infiltrated leaf from each plant harvested at the same time.

Northern blot analysis of GFP transcript levels confirmed the visual observations of P0-mediated silencing suppression. GFP transcript was abundant in the RNA of patches that had received pBin-GFP + pBin-HCPro (Fig. 3B, top, lane 3) and pBin-GFP + pBin-P0 (Fig. 3B, top, lane 4), but its level was at least 25-fold lower in patches treated with pBin-GFP + pBin61 (Fig. 3B, top, lane 2) or pBin-GFP + pBin-BWP0- (Fig. 3B, top, lane 5). Conversely, GFP-specific siRNAs (21- to 23-mer) were readily detected by Northern blotting of RNA from patches which had received pBin-GFP + pBin61 and pBin-GFP + pBin-P0- (Fig. 3B, bottom, lanes 2 and 5) but were 7 to 10 times less abundant in the patches which had received pBin-GFP + pBin-HCPro and pBin-GFP + pBin-P0 (Fig. 3B, bottom, lanes 3 and 4).

The high-intensity green fluorescence of the patches which had received pBin-GFP plus either pBin-P0 or pBin-HCPro is due to the fact that the GFP transcript in the patch is produced from pBin-GFP as well as from the transgene and is protected from PTGS by the presence of the silencing suppressor at 5 days p.i. Observations at later times, however, revealed that there were significant differences between the behaviors of P0 and HCPro in the coinfiltration assay. Thus, patches which received the pBin-GFP + pBin-P0 mixture remained bright fluorescent green for up to 25 days (Fig. 3C, panel 3 insert), whereas the green fluorescence of patches that had received pBin-GFP + pBin-HCPro had significantly faded by this time (Fig. 3C, panel 2 insert). Another difference between P0 and HCPro concerned the effect on systemic spread of PTGS. Plants infiltrated with pBin-GFP + pBin-HCPro did not display GFP silencing in the upper leaves at 15 days p.i. (Fig. 3C, panel 2) or later times up to 25 days (data not shown). In contrast, the upper leaves of 16c plants that had received pBin-GFP + pBin-P0 had undergone vein-proximal PTGS of the GFP signal by 15 days p.i. (Fig. 3C, panel 3) and GFP silencing spread to the entire leaf thereafter (not shown). This rate of systemic silencing is similar to that observed in plants which had received pBin-GFP + pBin61 (Fig. 3C, panel 1).

Silencing suppressor activity of P0 from other poleroviruses. P0 is the most divergent protein among the poleroviruses (26). Of sequenced poleroviruses, P0 of PLRV displays the lowest (14%) sequence identity with P0 of BWYV while P0 of CABYV has the highest (23%), with most of the aligned residues being in the C-terminal region (Fig. 4A). To determine if P0 from other poleroviruses can suppress GFP silencing in N. benthamiana line 16c, binary constructs expressing P0 of CABYV and PLRV were created. Both pBin-P0CAB and pBin-P0PL suppressed GFP silencing in the coinfiltration patch assay, although the relative intensities of the GFP fluorescence in the patches (not shown) suggested that P0 of PLRV was a much less efficient suppressor than the P0s of BWYV and CABYV. Northern blot analysis confirmed the visual impression: the GFP transcript in the patch infiltrated with pBin-P0PL + pBin-GFP (Fig. 4B, lane 5) accumulated 5 to 10 times less abundantly than those in the patches which had received pBin-P0CAB + pBin-GFP (Fig. 4B, lane 4) and pBin-P0 + pBin-GFP (Fig. 4B, lane 3). Note that the difference in silencing suppressor activity of the different P0s does not correlate with compatibility between the virus and the host used in the patch coinfiltration assay, since PLRV, like BWYV, infects N. benthamiana, whereas CABYV does not (19).



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  		FIG. 4. The P0s of the poleroviruses CABYV and PLRV have silencing suppressor activity. (A) Sequence similarity between the different P0s. The P0 sequences of CABYV and PLRV were aligned pairwise to the BWYV P0 sequence by using the MultAlign interface. (B) Northern (RNA) blot analysis of high-molecular-weight RNA extracted from the infiltrated patch of 16c leaves which had received a mixture of bacteria harboring pBin-GFP plus one of the following: empty vector pBin61 (lane 2), pBin-P0 (lane 3), pBin-P0CAB (lane 4), or pBin-P0PL (lane 5). The RNA loaded in lane 1 came from a noninfiltrated 16c leaf. The blot was hybridized with a probe specific for GFP mRNA.

Full-length BWYV RNA does not provoke strong silencing suppression in the coinfiltration assay. P0 is encoded by the 5'-proximal gene of BWYV RNA, and the protein is readily detected when viral RNA is translated in vitro (38). Therefore, we asked whether full-length BWYV RNA can elicit silencing suppression in the patch coinfiltration assay. We have shown previously that agro-inoculation of plants with pBinBW0, a pBin19-based binary construct containing full-length BWYV cDNA behind a 35S promoter, leads to production of viral RNA transcripts which can then auto-replicate to give rise to a typical virus infection (5, 20). For use in the patch assays, pBinBW0 was slightly modified by addition of a 35S transcription termination sequence downstream of the BWYV cDNA to produce pBin-BW (Fig. 1C). Preliminary experiments demonstrated that infiltration of N. benthamiana leaves with A. tumefaciens harboring pBin-BW also triggered a virus infection. The infiltrated plants developed typical symptoms of BWYV infection, viral RNA and capsid proteins were detected in the patches and upper leaves, and the plants could serve as an inoculum source for aphid-mediated transmission of BWYV to other plants (data not shown).

Coinfiltration of line 16c leaves with pBin-GFP plus pBin-BW produced suppression of GFP silencing (Fig. 5A, panel 3). However, the intensity of GFP fluorescence in the patches was much lower than in patches treated in parallel with the pBin-GFP + pBin-P0 combination (Fig. 5A, panel 1). This difference was borne out by Northern hybridization analysis of GFP transcript levels in the patches. The level of GFP transcript in the RNA from the pBin-GFP + pBin-BW patches (Fig. 5B, lane 3) was reproducibly 2 to 3 times higher than in patches infiltrated with pBin-GFP + pBin-P0- (Fig. 5B, lane 2) or with pBin-GFP + pBin-BWP0-, a construct in which the wild-type P0 sequence in the viral cDNA was replaced with the P0- sequence (Fig. 5B, lane 4). However, the level of GFP transcript accumulation in the pBin-GFP + pBin-BW patches was 15- to 20-fold lower than in patches coinfiltrated with pBin-GFP + pBin-P0 (Fig. 5B, lane 1). No suppression of GFP silencing was visible in the upper leaves of 16c plants infiltrated with pBin-GFP + pBin-BW at 2 to 3 weeks p.i. (data not shown), although it should be noted that very weak silencing suppressor activity confined to cells of the phloem compartment would have been difficult to detect. We conclude that the silencing suppressor activity displayed by P0 is much lower when the P0 gene is introduced into the patch in its "normal" environment in the viral genome than when it is translated from a monocistronic construct.



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  		FIG. 5. Down-regulation of P0 levels and silencing suppressor activity from genome-length BWYV RNA. (A) Leaves of 16c plants agro-infiltrated with bacteria mixtures harboring pBin-GFP plus one of the following: empty vector pBin-P0 (panel 1), pBin-P0- (panel 2), pBin-BW (panel 3), or Pbin-BWP0- (panel 4). Photographs were taken with long-wavelength UV light 5 days p.i. (B) Northern (RNA) blot analysis of high-molecular-weight RNA extracted from the agro-infiltrated patches of the leaves shown in panel A. The blot was hybridized with a probe specific for GFP mRNA. (C) Western (immunoblot) analysis of total protein from the infiltrated patches of the leaves shown in panel A. The blot was probed with a polyclonal antiserum specific for BWYV P0. The position of P0 is indicated to the right.

P0 is poorly expressed from full-length BWYV RNA. In view of the failure of pBin-BW to support strong GFP silencing in the agro-infiltrated zone, we next attempted to measure the level of P0 accumulation in leaves that were agro-infiltrated with pBin-BW. To do this, total protein was extracted from patch tissue 5 days p.i. and analyzed by Western (immuno)blot analysis for P0 accumulation using a P0-specific polyclonal antiserum. An ~29-kDa band that comigrated with a P0 in vitro translation product (data not shown) was readily immunodetected in protein extracted from patches which had received pBin-GFP + pBin-P0 (Fig. 5C, lane 1) but could not be detected in protein of patches which had received pBin-GFP + pBin-BW (Fig. 5C, lane 3). No P0 was detected in similar experiments when tissue that was agro-infiltrated with pBin-GFP + pBin-BW was sampled at other times (1 to 7 days p.i.; data not shown).

To obtain a measure of the sensitivity of our immunodetection assay for P0, an experiment was carried out in which different amounts of a protein extracted from a pBin-GFP + pBin-P0 patch extract were diluted into a healthy leaf extract and then carried through the P0 Western blot procedure. The results (not shown) indicated that the limit of detection for P0 in our assay is one-tenth the amount of P0 present in the undiluted extract. Thus, the level of P0 accumulation in patches that have been infiltrated with pBin-BW is at least 10 times lower than the level in leaves infiltrated with pBin-P0.

It may be argued that the low accumulation of P0 in the pBin-BW patches relative to that in the pBin-P0 patches could be due to differences in the amounts of transcript produced from the two vectors. To test this hypothesis, Northern blots of total RNA from patches which had received pBin-GFP + pBin-BW or pBin-GFP + pBin-P0 were hybridized with an antisense RNA probe specific for the P0 ORF. In three independent experiments there was 240 to 275 times more radioactivity associated with the genome-length (~5.7-kb) RNA species in the patches which had received pBin-BW (Fig. 6C, lane 6) than with the ~0.8-kb transcript in the pBin-P0-infiltrated tissue (Fig. 6C, lane 3). We do not know what fraction of the 5.7-kb species corresponds to primary transcript (produced by transcription of pBin-BW DNA) and what fraction corresponds to BWYV RNA produced by replication of the primary transcript. Nor do we know if the 5.7-kb primary transcript and BWYV RNA are translated with similar efficiency (see Discussion). Nevertheless, the aforesaid observations rule out the possibility that the poor accumulation of P0 in the patches infiltrated with pBin-BW relative to the P0 level in the pBin-P0-infiltrated patches is related in a straightforward manner to poor accumulation of the longer messenger species.



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  		FIG. 6. Effect of initiation codon context on expression of P0. (A) Sequence of the region immediately surrounding the P0 initiation codon in the constructs. The first 33 residues of each transcript (designated n30...ATC) is derived from the vector. The first residue of the 31-nt viral 5'-noncoding region in pBin-P05'wt and pBin-P05'opt is indicated by a dot above the sequence. Underlined residues correspond to positions -3 and +4 relative to the A residue in the P0 initiation codon. Residues modified in pBin-P05'opt are shown in bold. (B) Northern (RNA) blot analysis of high-molecular-weight RNA extracted from agro-infiltrated patches of 16c leaves which had received bacterial mixtures harboring pBin-GFP plus one of the following: empty vector pBin61 (lane 2), pBin-P0 (lane 3), pBin-P05'wt (lane 4), pBin-P05'opt (lane 5), pBin-BW (lane 6), or pBin-BW5'opt (lane 7). The RNA loaded in lane 1 came from a noninfiltrated 16c leaf. The blot was hybridized with a probe specific for GFP mRNA. (C) Northern (RNA) blot analysis of the same samples shown in panel B, using a probe specific for the P0 coding region. The bands corresponding to 5.7-kb BWYV RNA (genomic RNA) and 0.8-kb P0 mRNA are labeled to the right. (D) Western (immunoblot) analysis of total protein from the infiltrated patches. The blot was probed with a polyclonal antiserum specific for BWYV P0. The position of P0 is indicated to the right. Lane 5 was loaded with one-third the amount of protein extract loaded in the other lanes.

The 5'-noncoding region of BWYV RNA regulates P0 expression levels. The foregoing observations illustrate that P0 accumulation levels are low when the protein is expressed from full-length viral RNA compared to the levels attained from the monocistronic construct pBin-P0. A significant difference between pBin-BW and pBin-P0 is that the transcribed sequence immediately upstream of the P0 initiation codon in the former corresponds to the BWYV 5'-noncoding region. In pBin-P0, on the other hand, the corresponding sequence is an artificial sequence of nonviral origin (see Fig. 6A for 5'-noncoding sequences of the different constructs). To determine the effect of the upstream sequence on P0 expression, the 31-residue viral 5'-noncoding region was inserted into pBin-P0 immediately upstream of the P0 initiation codon to produce pBin-P05'wt (Fig. 6A). Leaf patches that were agro-infiltrated with pBin-GFP + pBin-P05'wt accumulated GFP transcript to levels similar to those found in patches infiltrated with pBin-GFP + pBin-P0 (Fig. 6B, lanes 3 and 4). However, P0 was not detectable in the pBin-GFP + pBin-P05'wt patches by Western blot analysis (Fig. 6D, lane 4), although it was readily detected in the pBin-GFP + pBin-P0 patches (Fig. 6D, lane 3). Thus, insertion of the viral 5'-noncoding sequence upstream of the P0 cistron reduced accumulation of P0 to below the level of detection by Western blotting, although the amount of the protein produced was still sufficient to efficiently suppress GFP silencing (Fig. 6B, lane 4).

The sequence immediately surrounding an initiation codon has a strong influence on the efficiency of translation initiation in eukaryotes. A purine (usually A) in position -3 relative to the first nucleotide of the AUG is of primary importance for efficient translation initiation, while a G in position +4 has a lesser but still significant stimulatory effect (17). Examination of the different 5'-noncoding sequences (Fig. 6A) reveals that the initiation codon in pBin-P0 is in a fairly good context with an A at position -3 but that the initiation codon context in pBin-P05'wt (and in viral RNA) is poor (TTGATGC; P0 initiation codon underlined). To determine if the poor expression of P0 from the latter is a consequence of the suboptimal initiation codon context, a P0-expressing construct in which the poor context was replaced with the optimal sequence ACCATGG was created (pBin-P05'opt; Fig. 6A). Leaf patches infiltrated with pBin-GFP + pBin-P05'opt accumulated GFP transcript to high levels (Fig. 6B, lane 5), and P0 was readily detected by Western blot analysis (Fig. 6D, lane 5).

A mutation to optimize P0 translation initiation efficiency was not stable during virus multiplication. The optimal P0 initiation codon context described above was also introduced into full-length BWYV cDNA to produce pBin-BW5'opt. Leaf patches infiltrated with this construct plus pBin-GFP accumulated GFP transcript to high levels (Fig. 6B, lane 7), indicating that more P0 was produced than when the wild-type construct pBin-BW was used in the infiltration mix (compare lanes 6 and 7 in Fig. 6B). Nevertheless, P0 still did not accumulate in amounts permitting its detection by Western blot analysis (Fig. 6D, lane 7).

Northern blot analysis of the RNA from the patches (at 5 days p.i.) and from upper leaves (21 days p.i.) of plants infiltrated with pBin-BW5'opt revealed the presence of viral RNA in both locations (Fig. 7A). The level of accumulation of viral RNA in the patches infiltrated with pBin-BW and pBin-BW5'opt (Fig. 7A, lanes 2 and 3) was ~100 times higher than in the upper leaves (Fig. 7A, lanes 4 and 5; note that a longer autoradiographic exposure time was used for lanes 4 and 5). The difference in viral RNA accumulation levels in the patches and upper leaves is presumably due to the fact that agro-infiltration transfers DNA to almost all cells in the infiltrated zone (23; O. Voinnet, personal communication), whereas virus infection in the upper leaves is confined to cells of the phloem compartment. The pBin-BW patches contained 5 times more viral RNA than the pBin-BW5'opt patches (Fig. 7A, lanes 2 and 3; see also Fig. 6C, lanes 6 and 7). In the upper leaves the relative accumulation levels were 2:1 (Fig. 7A, lanes 4 and 5). Thus, optimization of the P0 initiation codon context does not enhance viral RNA accumulation but, on the contrary, has the opposite effect.



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  		FIG. 7. BWYV RNA in which the P0 translation initiation codon context has been optimized undergoes second-site mutations in the initiation codon. (A) Northern (RNA) blot analysis of viral genome-length RNA in N. benthamiana 16c plants agro-infiltrated with pBin-BW (lanes 2 and 4) or pBin-BW5'opt (lanes 3 and 5). The RNA shown in lane 1 was extracted from a healthy leaf. High-molecular-weight RNA was isolated from patches 5 days after infiltration (lanes 2 and 3) or from upper leaves 21 days after infiltration (lanes 4 and 5). The blot was hybridized with a radioactive probe complementary to the 3'-terminal 196 nt of BWYV RNA. The positions of the genomic RNA and a subgenomic RNA (sgRNA) are shown to the left. An autoradiographic exposure time of 1 h was used to visualize lanes 1 to 3, and an exposure time of 16 h was used for lanes 4 and 5. The subgenomic RNA was difficult to detect in lanes 4 and 5 due to rRNA interference (30). (B) Sequence in the vicinity of the P0 initiation codon for cloned RT-PCR products obtained from viral progeny RNA in the upper leaves of three plants agro-infiltrated with pBin-BW5'opt. The number of clones containing the indicated sequence relative to the number of clones analyzed is shown to the right. Positions modified to optimize codon context are underlined, and second-site mutations in the progeny RNA are in bold type.

To assess the stability in planta of the codon context mutations, total RNA was isolated from the upper leaves of three plants infected with pBin-BW5'opt and the region around the P0 initiation codon was cloned after amplification by specific primer-directed RT-PCR. Sequence analysis of a number of randomly selected clones revealed that the ACC triplet upstream of the ATG codon was conserved in all cases but that the ATG codon itself had frequently undergone mutation (Fig. 7B). Similar analysis on progeny virus RNA from plants that had been infiltrated with the wild-type construct pBin-BW revealed no such modifications (data not shown). For plant 1, the ATG codon had been mutated to ACG in all 11 clones analyzed. For plant 2, the P0 ATG had been replaced with either ACG, ATA, or GTG in 11 of the 14 clones analyzed and 3 clones retained the BW5'opt sequence (Fig. 7B). ACG and GTG have been reported to initiate translation in plants but with only about 15% the efficiency of translation initiation at an ATG codon, while the translation initiation efficiency of ATA was 5% that of ATG (10).

Analysis of the progeny viral RNA in plant 3 produced a somewhat different result. Twelve of fourteen clones analyzed contained a GTG or TTG substitution in the P0 initiation codon, but, unexpectedly, the G residue in position +4 had been replaced by T in all 14 clones (Fig. 7B). This creates a TAA termination codon immediately following the P0 start site, which will presumably terminate any translation that is initiated there. Taken together, our findings thus indicate that there is selection during propagation of virus derived from pBin-BW5'opt for appearance of second-site mutations at the P0 initiation codon (or immediately downstream) which will strongly down-regulate accumulation levels of P0.


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DISCUSSION
 
In this paper we have demonstrated that P0 of BWYV has PTGS suppressor activity, a property shared by the P0s of at least two other poleroviruses. The silencing suppressor activity of P0 appears to be of the same basic type as that of the paradigm silencing suppressor HCPro, although several possibly significant differences were noted. Thus, the suppression induced by pBin-P0 in the patch assay is long lasting, with the infiltrated patch remaining bright fluorescent green for as long as 25 days. In contrast, the silencing suppression induced by HCPro faded over a period of 8 to 15 days p.i. This observation suggests that P0 either is a more efficient suppressor of silencing or is more stable than HCPro in planta. Another distinction between P0 and HCPro concerns their effects on systemic silencing in the coinfiltration patch assay. In our hands, HCPro blocked the appearance of PTGS in upper leaves of infiltrated 16c plants, whereas BWYV P0 had no effect on the rate of GFP silencing in upper leaves (Fig. 3C). Our findings thus suggest that P0 interacts with a component of the silencing machinery responsible for cell-autonomous silencing but (unlike HCPro) does not interfere with production and delivery of the mobile silencing signal. It should be pointed out, however, that the behavior of a silencing suppressor in the coinfiltration patch assay does not necessarily reflect its behavior in other systems. Thus, it has been observed that ß-glucuronidase-transgenic tobacco in which PTGS of ß-glucuronidase had been lifted by crossing with tobacco expressing an HCPro transgene from tobacco etch potyvirus still produced a mobile silencing signal which could operate across a graft junction (24). (However, the reported ability of HCPro to traffic from cell to cell [33] could complicate the interpretive comparison of data from the two types of experiment.) Nevertheless, it is evident that characterization of P0-transgenic plants will be of interest in the future, although production of such plants may be hindered by the reported cytopathic effects of P0 when expressed in this manner (37).

When the monocistronic construct pBin-P0 was used in agro-infiltration, P0 accumulated in amounts that were readily detectable by immunoblot analysis of patch proteins and strong PTGS suppressor activity was observed. Patches infiltrated with the full-length viral cDNA construct pBin-BW, on the other hand, displayed low but detectable PTGS suppressor activity and did not accumulate amounts of P0 which we could detect by immunoblot analysis. Our findings indicate that the inefficient accumulation of P0 in the latter case is due in part to the suboptimal context of the P0 initiation codon in the viral RNA sequence, since replacement of the near-optimal P0 initiation context in pBin-P0 with the viral sequence in pBin-P05'wt reduced P0 accumulation levels to below the level of detection, i.e., at least 10-fold (Fig. 6D, lane 4). Nevertheless, enough P0 was still produced from pBin-P05'wt to strongly suppress PTGS of GFP (Fig. 6B, lane 4), which was not the case for infiltration with pBin-BW (Fig. 6B, lane 6). Since the P0 initiation codon context is identical in the two constructs, an additional factor or factors must contribute to further diminish P0 accumulation levels in the pBin-BW patches.

There are several (not necessarily exclusive) possibilities which could account for the above observations. First, since the pBin-BW transcript is infectious, cells in the infiltrated patches will contain other viral gene products in addition to P0. Possibly, one of these gene products down-regulates the translation or stability of P0. Another possibility is that the viral replication process itself is responsible for the inefficient accumulation of P0. For example, most of the viral RNA in patches infiltrated with pBin-BW could be sequestered in replication complexes and be inaccessible to the translation machinery. Finally, we have shown that abundant amounts of viral genome-length RNA accumulate in the patches infiltrated with pBin-BW. The bulk of this RNA is probably authentic viral RNA (formed by replication of the primary transcript) and hence will possess a 5'-terminal genome-linked protein (VPg [25, 36]). The presence of VPg could impede ribosome access to the P0 initiation codon on the viral RNA relative to the nonreplicating transcripts produced from constructs such as pBin-P0 and pBin-P05'wt, which are presumably capped. To summarize, our observations indicate that the unfavorable P0 initiation codon context is partially responsible for the poor expression of P0 from full-length viral RNA but that an additional factor (or factors), probably linked to the viral replication cycle, is important as well.

As noted in the introduction, null mutations in the P0 gene strongly diminish polerovirus RNA accumulation levels during infection (34, 46), supporting the idea that P0 at least partially counteracts the host's PTGS-like virus defense mechanism. In view of the low accumulation levels of P0 in plants infected with wild-type BWYV, we expected that alteration of the viral RNA sequence to improve P0 expression (by optimization of the P0 initiation codon context) would augment progeny viral RNA accumulation relative to the wild type. This proved not to be the case. Instead, plants infiltrated with pBin-BW5'opt contained less progeny viral RNA than plants infiltrated in parallel with wild-type virus (pBin-BW) and, even more significantly, variants containing modifications in the P0 initiation codon were abundant in the mutant virus population (Fig. 7B). In two pBin-BW5'opt-infected plants that were subject to further analysis, an ACG, GTG, or ATA codon had replaced the P0 ATG with high frequency. These non-ATG triplets can initiate translation in plants, but with only 5 to 15% the efficiency of an ATG (10). In a third plant, the P0 initiation codon underwent mutation with high frequency and an additional mutation creating a termination codon occurred just downstream.

At first thought, it may seem surprising that there is selection in the pBin-BW5'opt progeny for sequence variants bearing mutations that target the P0 initiation codon (or the adjacent downstream codon) rather than for mutations that restore the poor initiation codon context characteristic of the wild type. It should be pointed out, however, that reversion to the wild-type context would require mutations at two positions (-3 and +4) and, furthermore, the necessary substitutions involve nucleotide transversions, which are known to occur with lower frequency than transitions during viral RNA replication (see reference 6 and references therein). The observed P0 initiation codon mutations, on the other hand, involve only one base change, and in most cases the change is a transition.

Why overexpression of P0 is unfavorable to virus multiplication has not yet been established, but it should be noted that enhanced initiation of P0 translation on viral RNA, attained by decreasing the number of ribosomes available to scan downstream, will lower production of P1 and P1-2, which would be expected to result in diminished virus replication rates. Another possibility is that high-level expression of P0 during the infection perturbs host cell metabolism in a manner which is deleterious for virus multiplication. Further experimentation will be aimed at understanding how P0 expression levels are regulated during a normal virus infection.


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ACKNOWLEDGMENTS
 
S. Pfeffer and P. Dunoyer contributed equally to this work.

We thank Olivier Voinnet and Christiane Fritsch for numerous helpful discussions, David Baulcombe for furnishing the GFP-transgenic plant line and constructs, and Frank van der Wilk for a clone containing the PLRV P0 gene. Philippe Hammann was responsible for DNA sequence analysis and Daniele Scheidecker provided technical assistance.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institut de Biologie Moléculaire des Plantes, 12 Rue du Général Zimmer, 67084 Strasbourg Cedex, France. Phone: (33) 388 417257. Fax: (33) 388 614442. E-mail: veronique.ziegler-graff{at}ibmp-ulp.u-strasbg.fr. Back


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Journal of Virology, July 2002, p. 6815-6824, Vol. 76, No. 13
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.13.6815-6824.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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