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Instituto de Biología Molecular y Celular de Plantas (IBMCP), UPV-CSIC, Avda de los Naranjos s/n, 46022 Valencia, Spain
Correspondence
V. Pallás
vpallas{at}ibmcp.upv.es
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Images of the abaxial side of melon cotyledons after agroinfiltration of pMOG(GFP) and a list of primers used for site-directed mutagenesis are available as supplementary material in JGV Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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30 nm), isometric plant virus that has been classified in the genus Carmovirus within the family Tombusviridae (Hibi & Furuki, 1985
; Riviere & Rochon, 1990). MNSV has a narrow range of host plants that is almost restricted to species in the family Cucurbitaceae. Characteristic symptoms of diseased plants include necrotic spots on leaves and stem necrosis. MNSV is transmitted naturally in soil by the zoospores of the chytrid fungus Olpidium bornovanus (Lange & Insunza, 1977; Campbell & Sim, 1994), when it attaches itself to the spore outer covering (Furuki, 1981). Once the virus invades the plant, it can be propagated mechanically during pruning or harvesting. Alternatively, this virus may be seed-propagated, although, in this case, efficient transmission requires the assistance of the fungal vector (Campbell et al., 1996).
The MNSV genome consists of a single-stranded, positive-sense RNA of approximately 4.3 kb (Riviere & Rochon, 1990) and is thought not to be 5'-capped (Rochon & Tremaine, 1989; Hearne et al., 1990; Huang et al., 2000). Several MNSV isolates have been cloned and sequenced [MNSV-Dutch, GenBank accession no. NC_001504
[GenBank]
(Riviere & Rochon, 1990); MNSV-NH and NK, GenBank accession nos AB044291
[GenBank]
and AB044292
[GenBank]
(Ohshima et al., 2000); MNSV-YS and KS, GenBank accession nos AB189944
[GenBank]
and AB189943
[GenBank]
(Kubo et al., 2005)] and infectious transcripts have been obtained [MNSV-M
5, GenBank accession no. AY122286
[GenBank]
(Díaz et al., 2003); MNSV-264, GenBank accession no. AY330700
[GenBank]
(Díaz et al., 2004)]. Molecular analysis of the MNSV genomic sequence revealed the presence of at least five open reading frames (ORFs) flanked by a short 5' untranslated region (UTR) and a non-polyadenylated 3' UTR. Interestingly, an avirulence determinant has been characterized in the 3' UTR of isolate MNSV-264, allowing the infection of non-cucurbit species, in addition to overcoming the resistance conferred by the recessive gene nsv in melon (Díaz et al., 2004; Morales et al., 2005). The 5'-proximal ORF has the potential to encode a protein of 29 kDa (p29) terminating with an amber codon, whose read-through would result in a larger gene product of 89 kDa (p89). Two small, centrally located ORFs encode two consecutive proteins of 7 kDa (p7A and p7B). Interestingly, if read-through of the amber codon located at the end of p7A occurred, both proteins would be joined in frame, resulting in a fusion protein of 14 kDa. Finally, the 3'-proximal ORF encodes a coat protein of 42 kDa (p42), which is related to those of the genus Tombusvirus (Riviere et al., 1989; Riviere & Rochon, 1990; Cañizares et al., 2001). p29 and its read-through protein p89 are expressed from the genomic-length RNA (gRNA), whereas the small p7A and p7B (or p14) proteins and the coat protein are translated from 1.9 and 1.6 kb subgenomic RNAs (sgRNAs), respectively (Riviere & Rochon, 1990).
Function of each MNSV ORF can be deduced from amino acid sequence comparisons with homologue proteins of other well-studied members of the same genus or family. Thus, p29 and p89 are probably viral components of the replicase–transcriptase complex (Hacker et al., 1992; White et al., 1995; Panavien
et al., 2003; Panavas et al., 2005). The two central ORFs, p7A and p7B, could be involved in cell-to-cell movement (Hacker et al., 1992; Li et al., 1998; Marcos et al., 1999; Cohen et al., 2000) and homologues of p42, independently of their structural function as coat protein, have been involved in systemic movement (Cohen et al., 2000), suppression of post-transcriptional gene silencing (PTGS) (Qu et al., 2003; Thomas et al., 2003; Ryabov et al., 2004; Meng et al., 2006) and attachment to the surface of fungus-vector zoospores in natural transmission (McLean et al., 1994; Robbins et al., 1997; Kakani et al., 2001). However, the association between sequence information and biological function of viral proteins has not been demonstrated in the case of MNSV. Therefore, this study reports the synthesis of infectious transcripts corresponding to the MNSV genome of a Spanish isolate (MNSV-Al; Gosalvez et al., 2003) and subsequent mutational analysis by reverse genetics to explore proposed functions of each potential genome-encoded protein during viral infections of melon plants, the natural MNSV host. Furthermore, recombinant virus delivering the green fluorescent protein (GFP) marker was used to monitor viral factors affecting the spread of infection. Additionally, the putative role of each gene product in suppression of PTGS was studied.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) as described previously (Astruc et al., 1996; Covelli et al., 2004) and purified after electrophoretic separation on polyacrylamide gels. Briefly, the dsRNAs were denatured, polyadenylated and reverse-transcribed with a primer containing a 3'-dT25 tail with the last base degenerated. These modified RNAs were PCR-amplified by using an oligo(dT) primer combined with others derived from internal regions of the isolate MNSV-Dutch. Five clones from each terminus were sequenced and used to design MNSV-Al-specific primers corresponding to the 5' and 3' UTR ends. T7 promoter sequence was included in the 5' UTR-specific primer. Full-length cDNA construction was performed by RT-PCR amplification of three overlapping regions covering the entire gRNA of MNSV-Al, using primers corresponding to both MNSV-Al termini combined with others based on the MNSV-Dutch sequence. The three fragments were assembled by using single endonuclease-restriction sites located in the MNSV-Al sequence and ligated into the pUC18 vector (MBI Fermentas) by standard subcloning strategies, giving rise to the pMNSV(Al) clone. Therefore, any MNSV-Dutch specific sequences predetermined by the used primers were removed. All reverse transcriptase and amplification reactions were carried out by using the RevertAid H Minus Moloney murine leukemia virus reverse transcriptase (MBI Fermentas) and Vent DNA polymerase (New England Biolabs), respectively, following the manufacturers' instructions. PCR-amplification conditions included an initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 40 s, annealing at 60 °C for 40 s and extension at 72 °C for 2 min.
Construction of a recombinant GFP-MNSV(Al) clone.
The p42 ORF from the full-length clone pMNSV(Al) was replaced by the GFP ORF to generate the recombinant pMNSV(Al)-
cp-GFP clone. Nucleotides located between positions 2845 and 4000, corresponding to the almost-complete p42 gene, were removed by reverse-PCR strategies using primers VP598 and VP599 (see Supplementary Table S1, available in JGV Online), leading to the pMNSV-Al-cp clone. The GFP ORF was PCR-amplified by using the pEGFP-C3 vector (Clontech) as a template and primer pair VP543 (5'-GCGGCCGCGGTCGCCACCATGGTGAG-3'; underlined endonuclease-restriction site, NotI) and VP551 (5'-CACAGGGCCCCTACTTGTACAGCTCGTCCA-3'; underlined endonuclease-restriction site, ApaI). Subsequently, the GFP cDNA was ligated to pMNSV-Al-cp by the previously introduced NotI and ApaI restriction sites and T4 DNA ligase (Promega), resulting in the translational fusion of the first 9 aa of p42 to the GFP ORF.
Protein-deficient expression constructs.
MNSV-Al protein-deficient expression constructs used in this study are represented in Fig. 1. Mutants were obtained from both the full-length pMNSV-Al and recombinant pMNSV(Al)-cp-GFP clones by oligonucleotide-directed mutagenesis using a QuikChangeR XL site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol and primer pairs listed in Supplementary Table S1 (available in JGV Online). The plasmid series pMNSV(Al)-89(FS) and pMNSV(Al)-cp-GFP-89(FS), pMNSV(Al)-7A(FS) and pMNSV(Al)-cp-GFP-7A(FS), pMNSV(Al)-7B(FS) and pMNSV(Al)-cp-GFP-7B(FS), in addition to pMNSV(Al)-42(FS), were obtained by introducing a frame-shift mutation into the p89, p7A, p7B and p42 ORFs, respectively. A mutant virus allowing for p89 read-through-product expression, but not the p29 protein, was generated by introducing an amber stop codon into a tyrosine codon mutation at the end of the p29 ORF in the pMNSV(Al)-29(–) and pMNSV(Al)-cp-GFP-29(–) plasmids. Fusing p7A and p7B ORFs yielded p14 expression by changing an amber stop codon into an alanine codon mutation at the end of p7A in the pMNSV(Al)-14(+) and pMNSV(Al)-cp-GFP-14(+) plasmids.
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Total RNA extraction, Northern blot and tissue-blotting assay.
Total nucleic acid extraction from either inoculated melon-plant cotyledons or agroinfiltrated Nicotiana benthamiana leaves was performed as described previously (Navarro et al., 2004). RNAs were electrophoresed through formaldehyde-denatured gel and transferred to positively charged nylon membranes (Roche). Alternatively, symptomatic cotyledons were crushed in the nylon membranes (Roche) for a tissue-blotting assay, as described previously (Más & Pallás, 1995). Northern and tissue-blot membranes were air-dried and nucleic acids were bound by using a UV cross-linker (700x100 µJ cm–2). Viral RNA detection was carried out as described previously (Pallás et al., 1998), using a digoxigenin-labelled riboprobe (Roche) complementary to a region of the MNSV-Al p42 ORF (Gosalvez et al., 2003) or to the complete GFP gene. Hybridization signals were quantified by densitometry analysis of film using 1D Manager v. 2.0 software.
Binary constructs and Agrobacterium tumefaciens-mediated transient-expression assays.
cDNAs corresponding to all putative MNSV-Al ORFs (p29, p89, p7A, p7B, p14 and p42), the modified p42 ORF containing the aforementioned frame-shift mutation for pMNSV(Al)-42(FS), the complete MNSV-Al genome and the helper component proteinase ORF (HC-Pro) from Tobacco etch potyvirus were cloned between the cauliflower mosaic virus 35S promoter and the terminator sequence of the Solanum tuberosum proteinase inhibitor II gene (PoPit) in the binary vector pMOG800 (Knoester et al., 1998). These clones were named pMOG29, pMOG89, pMOG7A, pMOG7B, pMOG14, pMOG42, pMOG42(FS), pMOG-MNSV(Al) and pMOG(HC-Pro), indicating the cloned sequences.
The previously described binary constructs, pMOG(GFP) carrying the GFP (Clontech) expression cassette (Herranz et al., 2005) and the pMOG800 plasmid were transformed into A. tumefaciens strain C58C1 by electroporation. Identification of viral silencing suppressors was performed by transient-expression assay on GFP-transgenic N. benthamiana (line 16c, provided by D. C. Baulcombe, The Sainsbury Laboratory, John Innes Centre, Norwich, UK; Ruiz et al., 1998) as described previously (Voinnet et al., 2000; Qu et al., 2003). Three leaves per plant (four plants per construct) were infiltrated into the abaxial side of leaves with a bacterial suspension consisting of an A. tumefaciens strain carrying the construct pMOG(GFP) either alone or in combination (1 : 1 mixture) with each strain containing plasmids described above.
In the case of the trans-complementation of pMNSV(Al)-cp-GFP mutants, fully expanded cotyledons of 1-week-old melon plants were agroinfiltrated following the procedure described above for N. benthamiana. Plants were inoculated 4–5 days later with modified pMNSV(Al)-cp-GFP transcripts.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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); MNSV-NH and NK (Ohshima et al., 2000); M5 (Díaz et al., 2003); MNSV-264 (Díaz et al., 2004); MNSV-YS and KS (Kubo et al., 2005)]. A full-length cDNA clone of MNSV gRNA, including a T7 RNA promoter at the 5' end (pMNSV-Al), was constructed to enable MNSV-Al gRNA synthesis without foreign sequences at the 3' end. These viral transcripts produced a local, and occasionally systemic, infection in susceptible melon plants, independently of the presence of a cap analogue at the 5' terminus, as occurred with biologically active clones of isolates MNSV-M5 and MNSV-264 (Díaz et al., 2003, 2004). Symptoms produced by inoculation of in vitro MNSV-Al transcripts and the systemic spread rate were identical to those observed when viral RNA isolated from virions or purified virions was inoculated (data not shown). Local chlorotic spots became necrotic lesions in the cotyledons at approximately 5–6 days post-inoculation (d.p.i.). When systemic infection had developed, new young leaves emerged, filled almost completely with chlorotic spots.
p42 is a pathogenicity determinant necessary for systemic infection
Frame-shift or non-stop codon mutations of the five viral ORFs were introduced into the pMNSV-Al infectious clone. Run-off transcripts produced from these mutants and from the original pMNSV-Al clone (wild type) were inoculated onto melon-plant cotyledons. Neither local nor systemic symptoms were observed, even at 10 d.p.i., in melon plants inoculated with MNSV(Al)-29(–), MNSV(Al)-89(FS), MNSV(Al)-7A(FS) and MNSV(Al)-7B(FS) RNAs, which had impaired expression of p29, p89, p7A and p7B, respectively. Similar results were obtained with RNAs from pMNSV(Al)-14(+), which lacked individual p7A and p7B, but expressed both proteins fused as p14 (data not shown). However, MNSV(Al)-42(FS) RNAs, containing a frame-shift mutation within p42, induced the appearance of local symptoms on inoculated cotyledons, but never in emerging leaves, unlike the situation observed for wild-type transcripts. Local symptoms included chlorotic spots, unlike the more severe necrotic lesions generated by infection with wild-type RNAs (Fig. 2a). Thus, the viral RNA distribution in both types of local symptoms was analysed by tissue-blotting assay. Hybridization signals of at least five different experiments consistently revealed that, in the absence of p42, infection foci were smaller than those observed in wild-type infections (Fig. 2a). No hybridization signal was detected when mock-inoculated plant cotyledons were also assayed (data not shown). These data suggest that p42 is an important factor controlling symptoms, which is required for systemic transport and also enhances cell-to-cell movement.
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, lane 8). No significant effect was observed with this mutation on either the accumulation level of viral RNAs or the synthesis of both sgRNAs when compared with those observed for the wild-type RNA or even for viral particles, indicating that replication/accumulation are not affected detectably by the absence of p42 (Fig. 2b, compare lane 8 with lanes 1 and 2). To study p42 function further, a deletion mutant was constructed (pMNSV-Al-cp) and tested for its replication ability. Inoculation of these truncated RNAs resulted in an asymptomatic infection where one gRNA and two sgRNAs of approximately 1.2 kb smaller than the corresponding wild-type RNAs were detected by Northern blot hybridization (Fig. 2b, lane 9). Furthermore, the accumulation levels of these MNSV-Al-cp RNAs were comparable to what is found in wild-type infections. These results suggest strongly that symptoms depend on the presence of the p42 coding region and the protein itself.
p29 and p89 are essential for MNSV replication, whereas cell-to-cell movement is controlled by the small p7A and p7B proteins
A GFP-based approach commonly used to monitor virus infections (Baulcombe et al., 1995) was developed to differentiate between replication and cell-to-cell movement-deficient mutants. Thus, a GFP-MNSV recombinant [pMNSV(Al)-cp-GFP], obtained by replacing the p42 ORF from the full-length clone pMNSV(Al) by the GFP ORF, was constructed. The GFP gene was fused in frame with the first 9 aa of the p42 ORF without affecting the p7B overlapping amino acids (Fig. 1b). Transcripts derived from this chimeric construct [MNSV(Al)-cp-GFP RNAs] were inoculated mechanically onto melon ‘Galia’ cotyledons and GFP expression was monitored by confocal microscopy as green fluorescence. At 3 d.p.i., fluorescent infection foci were observed in inoculated cotyledons, indicating that chimeric RNAs were able not only to replicate, as GFP expression is only possible if the corresponding sgRNA 2 is synthesized, but also to move from cell to cell (Fig. 3a). Systemic spread of the fluorescence was not detected and no symptoms were observed, consistent with the results obtained before with MNSV(Al)-cp RNAs.
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cp-GFP mutants, similar to those generated in pMNSV(Al) (Fig. 1b), to discriminate between mutants that cannot replicate from those that have their cell-to-cell movement impaired. Therefore, the modified MNSV(Al)-cp-GFP RNAs were inoculated onto melon cotyledons and, at 3, 6 and 8 d.p.i., a total of 100 cotyledons were monitored for green fluorescence expression. RNAs from pMNSV(Al)-cp-GFP-89(FS) and pMNSV(Al)-cp-GFP-29(–), which were defective in the putative replicase p29 and p89 proteins, respectively, were unable to induce fluorescence (data not shown). Those from pMNSV(Al)-cp-GFP-7A(FS) and pMNSV(Al)-cp-GFP-7B(FS), which were defective in putative cell-to-cell movement proteins p7A and p7B, respectively, led to GFP expression in individual cells (approx. 10 single cells per cotyledon) (Fig. 3b, panels 1 and 2, respectively) until 8 d.p.i. Afterwards, the green fluorescence disappeared. Transcripts from pMNSV(Al)-cp-GFP-14(+) provided similar results, indicating that viral RNA may not reach adjacent cells by using the p14 fusion protein alone (Fig. 3b, panel 3). These data suggest strongly that p29 and p89 are essential for virus RNA replication, whilst p7A and p7B are involved in cell-to-cell movement, but probably do not function as a p14 fusion protein.
Different complementation assays were performed to gain insight into the mechanism of cell-to-cell movement involving both p7A and p7B. Unlike the results reported for Turnip crinkle virus (TCV) in Arabidopsis thaliana (Li et al., 1998; Cohen et al., 2000), movement was not restored when RNAs from pMNSV(Al)-cp-GFP-7A(FS) and pMNSV(Al)-cp-GFP-7B(FS) were inoculated together. Transgenic melon plants overexpressing MNSV proteins remain unavailable and experimental plants like A. thaliana or N. benthamiana are not MNSV host plants (except for isolate MNSV-264; Díaz et al., 2003). Thus, we developed a different complementation approach using individual inoculation onto melon cotyledons of either MNSV(Al)-cp-GFP-7A(FS) or MNSV(Al)-cp-GFP-7B(FS) RNAs and providing each of the functional proteins by transient expression using A. tumefaciens infiltration. Transient expression in melon-plant cotyledons was assessed previously by agroinfiltration of binary vectors carrying GFP or p42 ORFs. Major expression of either GFP or p42, assessed as green fluorescence appearance or by Western blot analysis, respectively, was observed at 5 d.p.i. Unlike in other experimental systems, such as N. benthamiana, a non-uniform green fluorescence distribution among melon-cotyledon cells was observed (see Supplementary Fig. S1, available in JGV Online). Therefore, 10-day-old cotyledons were agroinfiltrated with binary vectors carrying non-mutated p7A or p7B ORFs (pMOG7A and pMOG7B) and, 5 days later, coincident with the peak of transient expression, they were inoculated with MNSV(Al)-cp-GFP-7A(FS) or MNSV(Al)-cp-GFP-7B(FS) RNAs, respectively. A putative model involving p7A/p14 or p7B/p14 was evaluated by inoculating MNSV(Al)-cp-GFP-14(+) RNAs onto either pMOG7A- or pMOG7B-agroinfiltrated cotyledons. Fifty cotyledons from each complementation assay were monitored by confocal microscopy. Individual cells and small fluorescent groups of cells were observed in similar proportions when MNSV(Al)-cp-GFP-7A(FS) and MNSV(Al)-cp-GFP-7B(FS) RNAs were complemented with transient p7A expression (groups of no more than three to four cells) (Fig. 3c, panel 4) and p7B (groups of no more than six cells) (Fig. 3c, panel 5), respectively. By contrast, fluorescence was only detected in single cells in complementation assays performed with MNSV(Al)-cp-GFP-14(+) RNAs (Fig. 3c, panel 6). Although we cannot rule out the possibility that small foci originated by unconnected events of viral infection on adjacent cells, this seems very unlikely, as these results were never observed when movement mutants were inoculated onto pMOG800-agroinfiltrated cotyledons (data not shown).
Both transiently expressed proteins 7A and 7B were able to complement in trans the corresponding mutant transcripts, although the replicating mutant viruses never reached the levels of infection foci produced when both movement proteins were provided by MNSV(Al)-cp-GFP RNA (Fig. 3a). This was probably because the agroinfection was not distributed homogeneously into the cotyledon as described before (see Supplementary Fig. S1, available in JGV Online). Hence, the local spread of movement-deficient RNAs was completely dependent on the location of the initial virus-infected cells inside a region expressing the corresponding movement protein. In addition, a new difficulty must be circumvented, as proteins must be agro-expressed at levels able to support viral movement, coinciding in time with the presence of the viral RNA inside the cell.
MNSV movement protein p7B and coat protein (p42) delayed RNA silencing in transient-expression experiments
An A. tumefaciens-mediated transient-expression assay on transgenic N. benthamiana plants expressing GFP (lane 16c; Ruiz et al., 1998) was performed as reported previously (Voinnet et al., 2000; Qu et al., 2003) to study the capacity of all MNSV genome-encoded proteins to act as potential RNA-silencing suppressors (see Methods for detailed description of constructs). At 2 d.p.i., the transient GFP expression induced an evident increase of green fluorescence in all infiltrated leaves when compared with the fluorescence observed in non-agroinfiltrated (transgenically expressing GFP) or pMOG800-agroinfiltrated (data not shown) leaves. As expected, the increase in GFP mRNA levels rapidly triggered the PTGS process in pMOG(GFP)-agroinfiltrated leaves and, at 4 d.p.i., the fluorescence almost disappeared as a result of specific GFP mRNA degradation (Fig. 4a). Nevertheless, when leaves were co-infiltrated with the mixture of bacterial strains carrying pMOG(GFP) and pMOG(HC-Pro), early GFP expression or GFP mRNA accumulation was much higher than in leaves infiltrated with pMOG(GFP) alone (Fig. 4a, b, respectively). Moreover, fluorescence was maintained until 11 d.p.i., as expected for the activity of the potyviral HC-Pro, a strong RNA-silencing suppressor (Kasschau & Carrington, 1998; Anandalakshmi et al., 2000), and then started to decline (Fig. 4a). p7B or p42 proteins were able to delay complete GFP silencing at least up to 7 d.p.i. as monitored by green fluorescence intensity, whereas the expression of MNSV-Al gene products p29, p89, p14 and p7A had no obvious consequence on PTGS (data not shown; Fig. 4). This effect was clearly protein- rather than RNA-mediated, as no PTGS suppression occurred when the MOG vector carried the complete MNSV-Al genome. As N. benthamiana is a non-host of the MNSV-Al isolate, no sgRNAs are produced and p7A, p7B and p42 proteins are not expressed (Riviere & Rochon, 1990) (Fig. 4a). Therefore, expression of 7B as well as p42 contributed to the stabilization of GFP mRNA (Fig. 4b), which further led to elevated GFP fluorescence (Fig. 4a). The effect of p7B and p42 proteins on GFP silencing was approximately 10-fold weaker than that generated by HC-Pro as measured by the GFP mRNA accumulation levels at 5 d.p.i. (Fig. 4b).
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cp-GFP RNAscp-GFP RNAs were inoculated at day 5 post-infiltration. Local-spread progress was assessed at 3, 5, 8, 12 and 14 d.p.i. by monitoring green fluorescence. The differences observed from three independent experiments (eight cotyledons per assay) demonstrated clearly that the presence of p42 and HC-Pro produced an enhancing effect (higher in the case of HC-Pro) on infection-focus size, clearly obvious at 5 d.p.i. At this point, the mean diameter of infection foci in the presence of either p42 or HC-Pro was 750±53 and 820±45 µm, respectively (Fig. 5a, panels 1 and 2, respectively), whereas in the absence of both proteins it was 300±13 µm (Fig. 5a, panel 3). Additionally, viral RNA accumulation/replication rates were maintained when p42 or HC-Pro was expressed, as all foci in infected cotyledons fused until the inoculated cotyledons were invaded completely at 14 d.p.i. (data not shown). Unlike these results, the smaller fluorescence foci produced in the absence of both proteins continued to spread slowly until 8–10 d.p.i., and then spreading stopped [and the fluorescence began to decay gradually (Fig. 5b)]. Green fluorescence was never observed in vascular tissues.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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cp-GFP-89(FS) and pMNSV(Al)-cp-GFP-29(–). Consequently, these overlapping proteins are essential for MNSV replication and are probably part of the replication complex, as has been described for related viruses such as TCV (Hacker et al., 1992; Rajendran et al., 2002) and Tomato bushy stunt virus (Rajendran & Nagy, 2003, 2004; Panavien et al., 2003; Panavas et al., 2005).
Following intracellular replication, cell-to-cell movement of carmoviruses (Cohen et al., 2000; García-Castillo et al., 2003) seems to be controlled by two small proteins working in trans (Hacker et al., 1992; Li et al., 1998; Cohen et al., 2000), an RNA-binding protein (Marcos et al., 1999; Vilar et al., 2001) and a membrane protein (Vilar et al., 2002), referred to as double-gene-block proteins or DGBps (Hull, 2002). Interestingly, the homologous proteins from TCV have overlapping regions (Carrington et al., 1989), whilst MNSV-Al DGBps (p7A and p7B) are exceptional among sequenced carmoviruses, as both proteins are arranged in frame in such a way that a fusion protein consisting of the complete p7A–p7B ORFs (p14) could be synthesized by a read-through process (Riviere & Rochon, 1990). However, the results reported here demonstrated that p7A and p7B operating in trans are sufficient to move viral RNAs among neighbouring cells of the natural host plant. Complementation in trans of homologous TCV proteins was demonstrated on experimental plants (Li et al., 1998; Cohen et al., 2000), although indirect evidence was reported in its natural host Brassica campestris (Hacker et al., 1992). p14 was, by contrast, unable to promote cell-to-cell movement, even in the presence of either p7A or p7B. This last observation was also suggested by the presence of a strong termination codon at the end of p7A ORF that avoids p14 synthesis in isolate MNSV-M5 (Díaz et al., 2003). Consequently, if this protein is still expressed in some isolates, it is unlikely to play a role in local spread.
On the other hand, the absence of p42 led to localized infections as well as a reduced infection-focus size, even though it has been reported that coat proteins are dispensable for carmovirus cell-to-cell movement (type I movement; Scholthof, 2005). However, it is possible that these coat protein effects on local and systemic spread are associated with their RNA silencing-suppression ability, as suggested for other plant viral systems (Qu & Morris, 2005). From data presented here and elsewhere, it is revealed that p42 has several functions throughout the MNSV life cycle. Primarily, this protein is responsible for the capsid structure (Riviere et al., 1989; Riviere & Rochon, 1990), but it is also a pathogenicity determinant (protein that increases viral symptoms) enhancing local spread and an essential factor for systemic infection. Moreover, p42 and p7B are weak silencing suppressors, as they delayed, but did not prevent, PTGS in transient-expression experiments on GFP-transgenic plants. Nevertheless, similar results were observed for other plant viral suppressors when they were analysed initially by using this transient-expression method (p25 of Potato virus X; p20 and CP encoded by Citrus tristeza virus; Lu et al., 2004). Analogous combinations of p42 functions have been reported for other viral proteins such as potyviral HC-Pro and cucumber mosaic virus 2b, including the related TCV coat protein (Brigneti et al., 1998; Qu et al., 2003; Thomas et al., 2003; Roth et al., 2004; Ryabov et al., 2004; Qu & Morris, 2005). In this sense, p42 can contribute to development of lesions indirectly by facilitating virus replication and spreading. Interestingly, and in parallel to our observations with regard to the p7B component of the DGBp, a weak RNA-suppressor activity was initially described in agroinfiltration experiments for potato virus X p25, protein 1 from the triple-gene block of proteins that are required for cell-to-cell movement of potexviruses (Voinnet et al., 2000; Lough et al., 2001). These data suggest that PTGS could be controlled by a component of the cell-to-cell movement machinery as a common feature among a number of plant viruses whose silencing suppression might be linked to viral transport (Verchot-Lubicz, 2005). Although the PTGS mechanism stage (initiation, local or systemic spread of a silencing signal or maintenance) affected by both MNSV suppressors was not studied in this work, the transient expression of p42 was relevant at an early infection stage by stimulating cell-to-cell movement and maintaining RNA replication. However, further studies on transgenic melon plants or, alternatively, the use of MNSV isolates infecting experimental plants (MNSV-264; Díaz et al., 2003) are necessary.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Astruc, N., Marcos, J. F., Macquaire, G., Candresse, T. & Pallás, V. (1996). Studies on the diagnosis of hop stunt viroid in fruit trees: identification of new hosts and application of a nucleic acid extraction procedure based on non-organic solvents. Eur J Plant Pathol 102, 837–846.[CrossRef]
Baulcombe, D. C., Chapman, S. & Santa Cruz, S. (1995). Jellyfish green fluorescent protein as a reporter for virus infections. Plant J 7, 1045–1053.[CrossRef][Medline]
Brigneti, G., Voinnet, O., Li, W.-X., Ji, L.-H., Ding, S.-W. & Baulcombe, D. C. (1998). Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J 22, 6739–6746.[CrossRef]
Campbell, R. N. & Sim, S. T. (1994). Host specificity and nomenclature of Olpidium bornovanus (=Olpidium radicale) and comparisons to Olpidium brassicae. Can J Bot 72, 1136–1143.
Campbell, R. N., Wipf-Scheibel, C. & Lecoq, H. (1996). Vector-assisted seed transmission of melon necrotic spot virus in melon. Phytopathology 86, 1294–1298.
Cañizares, M. C., Marcos, J. F. & Pallás, V. (2001). Molecular variability of twenty-one geographically distinct isolates of carnation mottle virus (CarMV) and phylogenetic relationships within the Tombusviridae family. Arch Virol 146, 2039–2051.[CrossRef][Medline]
Carrington, J. C., Heaton, L. A., Zuidema, D., Hillman, B. I. & Morris, T. J. (1989). The genome structure of turnip crinkle virus. Virology 170, 219–226.[CrossRef][Medline]
Cohen, Y., Gisel, A. & Zambryski, P. C. (2000). Cell-to-cell and systemic movement of recombinant green fluorescent protein-tagged turnip crinkle viruses. Virology 273, 258–266.[CrossRef][Medline]
Covelli, L., Coutts, R. H. A., Di Serio, F., Citir, A., Aç
kgöz, S., Hernández, C., Ragozzino, A. & Flores, R. (2004). Cherry chlorotic rusty spot and Amasya cherry diseases are associated with a complex pattern of mycoviral-like double-stranded RNAs. I. Characterization of a new species in the genus Chrysovirus. J Gen Virol 85, 3389–3397.
Díaz, J. A., Bernal, J. J., Moriones, E. & Aranda, M. A. (2003). Nucleotide sequence and infectious transcripts from a full-length cDNA clone of the carmovirus Melon necrotic spot virus. Arch Virol 148, 599–607.[CrossRef][Medline]
Díaz, J. A., Nieto, C., Moriones, E., Truniger, V. & Aranda, M. A. (2004). Molecular characterization of a Melon necrotic spot virus strain that overcomes the resistance in melon and nonhost plants. Mol Plant Microbe Interact 17, 668–675.[Medline]
Furuki, I. (1981). Epidemiological Studies on Melon Necrotic Spot (Technical Bulletin 14). Shizuoka Agricultural Experiment Station, Shizuokaken, Japan.
García-Castillo, S., Sánchez-Pina, M. A. & Pallás, V. (2003). Spatio-temporal analysis of the RNAs, coat and movement (p7) proteins of Carnation mottle virus in Chenopodium quinoa plants. J Gen Virol 84, 745–749.
Gosalvez, B., Navarro, J. A., Lorca, A., Botella, F., Sánchez-Pina, M. A. & Pallás, V. (2003). Detection of melon necrotic spot virus in water samples and melon plants by molecular methods. J Virol Methods 113, 87–93.[CrossRef][Medline]
Hacker, D. L., Petty, I. T. D., Wei, N. & Morris, T. J. (1992). Turnip crinkle virus genes required for RNA replication and virus movement. Virology 186, 1–8.[CrossRef][Medline]
Hearne, P. Q., Knorr, D. A., Hillman, B. I. & Morris, T. J. (1990). The complete genome structure and synthesis of infectious RNA from clones of tomato bushy stunt virus. Virology 177, 141–151.[CrossRef][Medline]
Herranz, M. C., Sanchez-Navarro, J.-A., Sauri, A., Mingarro, I. & Pallás, V. (2005). Mutational analysis of the RNA-binding domain of the Prunus necrotic ringspot virus (PNRSV) movement protein reveals its requirement for cell-to-cell movement. Virology 339, 31–41.[CrossRef][Medline]
Hibi, T. & Furuki, I. (1985). Melon necrotic spot virus. In CMI/AAB Descriptions of Plants Viruses (no. 302). Wellesbourne, UK: Association of Applied Biologists.
Huang, M., Koh, D. C.-Y., Weng, L.-J., Chang, M.-L., Yap, Y. K., Zhang, L. & Wong, S.-M. (2000). Complete nucleotide sequence and genome organization of Hibiscus chlorotic ringspot virus, a new member of the genus Carmovirus: evidence for the presence and expression of two novel open reading frames. J Virol 74, 3149–3155.
Hull, R. (2002). Virus movement through the plant and effects on plant metabolism. In Matthews' Plant Virology, 4th edn, pp. 373–436. San Diego: Academic Press.
Kakani, K., Sgro, J.-Y. & Rochon, D. (2001). Identification of specific cucumber necrosis virus coat protein amino acids affecting fungus transmission and zoospore attachment. J Virol 75, 5576–5583.
Kasschau, K. D. & Carrington, J. C. (1998). A counterdefensive strategy of plant viruses: suppression of posttranscriptional gene silencing. Cell 95, 461–470.[CrossRef][Medline]
Knoester, M., van Loon, L. C., van den Heuvel, J., Hennig, J., Bol, J. F. & Linthorst, H. J. M. (1998). Ethylene-insensitive tobacco lacks nonhost resistance against soil-borne fungi. Proc Natl Acad Sci U S A 95, 1933–1937.
Kubo, C., Nakazono-Nagaoka, E., Hagiwara, K., Kajihara, H., Takeuchi, S., Matsuo, K., Ichiki, T. U. & Omura, T. (2005). New severe strains of Melon necrotic spot virus: symptomatology and sequencing. Plant Pathol 54, 615–620.[CrossRef]
Lange, L. & Insunza, V. (1977). Root-inhabiting Olpidium species: the O. radicale complex. Trans Br Mycol Soc 69, 377–384.
Li, W.-Z., Qu, F. & Morris, T. J. (1998). Cell-to-cell movement of turnip crinkle virus is controlled by two small open reading frames that function in trans. Virology 244, 405–416.[CrossRef][Medline]
Lough, T. J., Emerson, S. J., Lucas, W. J. & Forster, R. L. S. (2001). Trans-complementation of long-distance movement of White clover mosaic virus triple gene block (TGB) mutants: phloem-associated movement of TGBp1. Virology 288, 18–28.[CrossRef][Medline]
Lu, R., Folimonov, A., Shintaku, M., Li, W.-X., Falk, B. W., Dawson, W. O. & Ding, S.-W. (2004). Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome. Proc Natl Acad Sci U S A 101, 15742–15747.
Marcos, J. F., Vilar, M., Pérez-Payá, E. & Pallás, V. (1999). In vivo detection, RNA-binding properties and characterization of the RNA-binding domain of the p7 putative movement protein from carnation mottle carmovirus (CarMV). Virology 255, 354–365.[CrossRef][Medline]
Más, P. & Pallás, V. (1995). Non-isotopic tissue-printing hybridization: a new technique to study long-distance plant virus movement. J Virol Methods 52, 317–326.[CrossRef][Medline]
McLean, M. A., Campbell, R. N., Hamilton, R. I. & Rochon, D. M. (1994). Involvement of the cucumber necrosis virus coat protein in the specificity of fungus transmission by Olpidium bornovanus. Virology 204, 840–842.[CrossRef][Medline]
Meng, C., Chen, J., Peng, J. & Wong, S.-M. (2006). Host-induced avirulence of hibiscus chlorotic ringspot virus mutants correlates with reduced gene-silencing suppression activity. J Gen Virol 87, 451–459.
Morales, M., Orjeda, G., Nieto, C. & 10 other authors (2005). A physical map covering the nsv locus that confers resistance to Melon necrotic spot virus in melon (Cucumis melo L.). Theor Appl Genet 111, 914–922.[CrossRef][Medline]
Navarro, J. A., Botella, F., Maruhenda, A., Sastre, P., Sánchez-Pina, M. A. & Pallás, V. (2004). Comparative infection progress analysis of Lettuce big-vein virus and Mirafiori lettuce virus in lettuce crops by developed molecular diagnosis techniques. Phytopathology 94, 470–477.[Medline]
Ohshima, K., Ando, T., Motomura, N., Matsuo, K. & Sako, N. (2000). Comparative study on genomes of two Japanese melon necrotic spot virus isolates. Acta Virol 44, 309–314.[Medline]
Pallás, V., Sánchez-Navarro, J. A., Más, P., Cañizares, M. C., Aparicio, F. & Marcos, J. F. (1998). Molecular diagnostic techniques and their potential role in stone fruit certification schemes. Options Méditerr 19, 191–208.
Panavas, T., Hawkins, C. M., Panavien, 
. & Nagy, P. D. (2005). The role of the p33 : p33/p92 interaction domain in RNA replication and intracellular localization of p33 and p92 proteins of Cucumber necrosis tombusvirus. Virology 338, 81–95.[CrossRef][Medline]
Panavien, ., Baker, J. M. & Nagy, P. D. (2003). The overlapping RNA-binding domains of p33 and p92 replicase proteins are essential for tombusvirus replication. Virology 308, 191–205.[CrossRef][Medline]
Qu, F. & Morris, T. J. (2005). Suppressors of RNA silencing encoded by plant viruses and their role in viral infections. FEBS Lett 579, 5958–5964.[CrossRef][Medline]
Qu, F., Ren, T. & Morris, T. J. (2003). The coat protein of turnip crinkle virus suppresses posttranscriptional gene silencing at an early initiation step. J Virol 77, 511–522.
Rajendran, K. S. & Nagy, P. D. (2003). Characterization of the RNA-binding domains in the replicase proteins of tomato bushy stunt virus. J Virol 77, 9244–9258.
Rajendran, K. S. & Nagy, P. D. (2004). Interaction between the replicase proteins of Tomato bushy stunt virus in vitro and in vivo. Virology 326, 250–261.[CrossRef][Medline]
Rajendran, K. S., Pogany, J. & Nagy, P. D. (2002). Comparison of Turnip crinkle virus RNA-dependent RNA polymerase preparations expressed in Escherichia coli or derived from infected plants. J Virol 76, 1707–1717.
Riviere, C. J. & Rochon, D. M. (1990). Nucleotide sequence and genomic organization of melon necrotic spot virus. J Gen Virol 71, 1887–1896.
Riviere, C. J., Pot, J., Tremaine, J. H. & Rochon, D. M. (1989). Coat protein of melon necrotic spot carmovirus is more similar to those of tombusviruses than those of carmoviruses. J Gen Virol 70, 3033–3042.
Robbins, M. A., Reade, R. D. & Rochon, D. M. (1997). A cucumber necrosis virus variant deficient in fungal transmissibility contains an altered coat protein shell domain. Virology 234, 138–146.[CrossRef][Medline]
Rochon, D. M. & Tremaine, J. H. (1989). Complete nucleotide sequence of the cucumber necrosis virus genome. Virology 169, 251–259.[CrossRef][Medline]
Roth, B. M., Pruss, G. J. & Vance, V. B. (2004). Plant viral suppressors of RNA silencing. Virus Res 102, 97–108.[CrossRef][Medline]
Ruiz, M. T., Voinnet, O. & Baulcombe, D. C. (1998). Initiation and maintenance of virus-induced gene silencing. Plant Cell 10, 937–946.
Ryabov, E. V., van Wezel, R., Walsh, J. & Hong, Y. (2004). Cell-to-cell, but not long-distance, spread of RNA silencing that is induced in individual epidermal cells. J Virol 78, 3149–3154.
Scholthof, H. B. (2005). Plant virus transport: motions of functional equivalence. Trends Plant Sci 10, 376–382.[CrossRef][Medline]
Thomas, C. L., Leh, V., Lederer, C. & Maule, A. J. (2003). Turnip crinkle virus coat protein mediates suppression of RNA silencing in Nicotiana benthamiana. Virology 306, 33–41.[CrossRef][Medline]
Verchot-Lubicz, J. (2005). A new cell-to-cell transport model for potexviruses. Mol Plant Microbe Interact 18, 283–290.[Medline]
Vilar, M., Esteve, V., Pallás, V., Marcos, J. F. & Pérez-Payá, E. (2001). Structural properties of carnation mottle virus p7 movement protein and its RNA-binding domain. J Biol Chem 276, 18122–18129.
Vilar, M., Saurí, A., Monné, M., Marcos, J. F., von Heijne, G., Pérez-Payá, E. & Mingarro, I. (2002). Insertion and topology of a plant viral movement protein in the endoplasmic reticulum membrane. J Biol Chem 277, 23447–23452.
Voinnet, O., Lederer, C. & Baulcombe, D. C. (2000). A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana. Cell 103, 157–167.[CrossRef][Medline]
White, K. A., Skuzeski, J. M., Li, W., Wei, N. & Morris, T. J. (1995). Immunodetection, expression strategy and complementation of turnip crinkle virus p28 and p88 replication components. Virology 211, 525–534.[CrossRef][Medline]
Received 22 December 2005;
accepted 27 March 2006.
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