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1 Department of Applied Biology, PO Box 27, FIN-00014 University of Helsinki, Finland
2 Mikocheni Agriculture Research Institute, PO Box 6226, Dar es Salaam, Tanzania
3 International Potato Center (CIP), Apartado 1558, Lima 12, Peru
Correspondence
Jari P. T. Valkonen
jari.valkonen{at}helsinki.fi
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the sequences of the 3'-proximal region of RNA1 are EU124491 (isolate Is), EU124490 (m2-47), EU124493 (Mis1), EU124494 (Tug2) and EU124492 (Unj2), and for Hsp70h sequences are EU124487 (Is), EU124488 (m2-47) and EU124489 (Tug2).
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). These viruses share a conserved set of ‘core’ genes, but they also show a high level of variability in gene content at the 3' end of the monopartite genomes in the genera Closterovirus and Ampelovirus and at the 3' end of RNA1 in viruses of the genus Crinivirus, which have bipartite genomes (Dolja et al., 2006). Gene content and organization of the variable 3'-proximal genomic region have evolutionary implications (Karasev et al., 1996; Dolja et al., 2006), which are interesting not least because some of the genes in the 3' region encode RNA silencing suppression (RSS) proteins (Reed et al., 2003; Lu et al., 2004; Kreuze et al., 2005; Chiba et al., 2006). RSS proteins combat the fundamental antiviral resistance that is based on RNA silencing in plants (Lindbo & Dougherty, 2005). The functional similarities of the RSS proteins contrast with their very low or non-significant sequence homology with other viral and host proteins (Lakatos et al., 2006; Mérai et al., 2006). It has been suggested that these characteristics might indicate a relatively recent acquisition of the genes for RSS proteins in viral genomes to counteract evolving eukaryotic host defence responses to viral pathogens (Voinnet, 2005).
Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) is a phloem-limited, whitefly-transmitted, bipartite virus that acts synergistically with several unrelated viruses also infecting sweetpotato (Ipomoea batatas L.) (Gibson et al., 1998; Karyeija et al., 2000; Kokkinos & Clark, 2006; Mukasa et al., 2006; Untiveros et al., 2007). Among the synergistic interactions, co-infection of SPCSV with sweet potato feathery mottle virus (SPFMV; genus Potyvirus, family Potyviridae) causes the severe sweet potato virus disease (SPVD), economically the most devastating disease affecting sweetpotatoes (Milgram et al., 1996; Gibson et al., 1998; Gutiérrez et al., 2003). SPVD is manifested as a drastic increase in symptom severity and yield reduction accompanied by an increase in SPFMV titres of up to 600-fold, whereas the titres of SPCSV are reduced in comparison with plants infected with SPCSV alone (Karyeija et al., 2000; Mukasa et al., 2006). Due to the synergistic interactions with other sweetpotato viruses, further understanding of the molecular biology of SPCSV has become of immense importance. Viral synergism is often found to be associated with the action of RSS proteins (Pruss et al., 1997). In a Ugandan isolate of SPCSV (SPCSV-Ug), two proteins involved in RSS are encoded by genes at the 3' end of RNA1 (Kreuze et al., 2005). They are expressed from subgenomic RNAs (sgRNAs) that accumulate at high levels early in infection in the young, systemically infected leaves (Kreuze et al., 2002). One of these proteins (p22) has been shown to act alone in suppressing silencing of the green fluorescent protein reporter gene mRNA in leaves of Nicotiana benthamiana, but the suppression activity was further enhanced by co-expression of the other SPCSV RNA1-encoded protein, RNase3 (Kreuze et al., 2005). This two-component RSS system is speculated to play a role in the synergism of SPCSV with other sweetpotato viruses and the development of SPVD.
There is limited information about the genetic variability of SPCSV, all of which is based on analysis of the nucleotide sequences of genes encoding the plant heat-shock protein-like Hsp70h protein and coat protein on RNA2. The Hsp70h gene sequences can be used to place SPCSV isolates into two phylogenetically distinct groups (Fenby et al., 2002; IsHak et al., 2003; Tairo et al., 2005), which also correlate with serological differences (Hoyer et al., 1996b; Gibson et al., 1998; Alicai et al., 1999). According to these molecular criteria, SPCSV isolates Unj2 and Mis1 from distant locations in Tanzania (Tairo et al., 2005) and the Ugandan isolate SPCSV-Ug, for which the complete sequence is available (Kreuze et al., 2002), belong to the so-called East African (EA) strain whose distribution is largely confined to East Africa. Recently, a few SPCSV isolates serologically related to the EA strain were found in Peru (Gutiérrez et al., 2003). However, SPCSV isolates from Nigeria, Israel and the USA have been found to be related to each other (Hoyer et al., 1996b; Pio-Ribeiro et al., 1996; Vetten et al., 1996) and assigned to the strain WA, named according to the original description of SPCSV as a ‘chlorotic stunt’-causing agent in sweetpotatoes in Nigeria, West Africa (Schaefers & Terry, 1976). Closterovirus-like particles were later detected in sweetpotatoes showing chlorotic stunt symptoms in Nigeria (Winter et al., 1992), Israel (Cohen et al., 1992) and Kenya (Hoyer et al., 1996a, b). The virus in these plants was named sweet potato sunken vein virus. However, The International Committee for Taxonomy of Viruses has recommended the name SPCSV (Gibson et al., 1998; Fauquet & Fargette, 2005; Fauquet et al., 2005), which is followed here.
Little is known about the possible sequence variability of RNA1 in SPCSV. The aim of this study was to characterize the 3'-end sequences of RNA1 in a few selected SPCSV isolates of the EA and WA strains with different geographical origins. The data showed that a few but not all EA strain isolates of SPCSV contained the gene for the p22 RSS protein, whereas the other isolates lacked a 767 nt region of RNA1 that included the p22 gene. This first report on intraspecific variability in gene content of members of the family Closteroviridae also indicates that the gene for RSS protein p22 is not essential for synergism between SPCSV and SPFMV, or for the development of SPVD.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and has been used in a few previous studies (Karyeija et al., 2000; Kreuze et al., 2002, 2005). SPCSV-Mis1 was obtained from Misurgwi district in the Lake Victoria basin in Tanzania and SPCSV-Unj2 from Unguja on Zanzibar Island on the Tanzanian coast of the Indian Ocean (Tairo et al., 2005). SPCSV-m2-47 was isolated from an infected sweetpotato plant grown in the Cañete valley, Peru (Gutiérrez et al., 2003). SPCSV-Is, previously described as SPSVV in Israel (Cohen et al., 1992; Milgram et al., 1996), was kindly provided by Professor Gad Loebestein (The Volcani Center, Bet Dagan, Israel). The isolate of SPFMV (SPFMV-Nam1) used in this study was originally obtained from an infected sweetpotato plant in Uganda and belongs to the EA strain of SPFMV (Karyeija et al., 2000; Kreuze et al., 2000). Seeds of Ipomoea setosa and pathogen-free in vitro plantlets of sweetpotato (I. batatas cv. Tanzania) were obtained from the International Potato Center (CIP; Lima, Peru). Viruses were maintained and experiments carried out in these plants in an insect-proof greenhouse (temperature 24–26 °C, relative humidity 70 %) under natural daylight extended to 16 h by illumination with high-pressure sodium lamps (light intensity 150–200 µmol s–1 m–2 at the level of plant height). Viruses were transmitted to new plants by graft inoculation. Infected plants were propagated by rooting stem cuttings.
Serological detection of SPFMV.
SPFMV was detected by double antibody sandwich (DAS)-ELISA as described previously (Gibson et al., 1998). In brief, 150 mg leaf material was ground in a polyvinyl bag with 600 µl extraction buffer [PBS containing 5 % Tween 20 and 2 % polyvinylpyrrolidone (Mr 40 000)]. The homogenate was transferred to a 1.5 ml Eppendorf tube and spun at 6000 g for 2 min. Aliquots of the supernatant (100 µl) were transferred to a microtitre plate (Greiner Laborteknik) previously coated with rabbit polyclonal antibodies to SPFMV coat protein (provided by CIP) and incubated at 4 °C overnight. After washing twice for 3 min each, 100 µl alkaline phosphatase-conjugated anti-SPFMV antibody was added to each well and the plate was incubated at 37 °C for 3 h and washed as before. The colour reaction was developed using p-nitrophenyl phosphate (Sigma) as the substrate. Absorbance (405 nm) was recorded using a Benchmark Microplate reader (Bio-Rad Laboratories).
RNA isolation.
Total RNA was isolated from 400 mg fresh Ipomoea leaves using Trizol (Invitrogen) following the manufacturer's instructions. RNA was resuspended in 250 µl sterile Milli-Q water (Sigma-Aldrich). The amount and quality of the RNA were checked using a spectrophotometer (8543 UV-Visible, Agilent Technologies) and agarose gel electrophoresis, respectively.
Real-time PCR.
Primers and real-time PCR conditions for amplification of SPFMV were as described by Mukasa et al. (2006). RNA samples were treated with DNase I (Promega) at 37 °C for 30 min and the reaction was stopped as instructed by the manufacturer. The RNA was then reverse-transcribed in a reaction mix (20 µl) containing 200 ng random hexamer primers, 10 mM dithiothreitol, 0.5 mM dNTPs, 20 U RNasin (Promega) and 400 U Moloney murine leukemia virus reverse transcriptase (Promega) at 37 °C for 1 h. The reaction was stopped by heating at 70 °C for 10 min and diluted fivefold with Milli-Q water. The cDNA (5 µl) was used as template for real-time PCR, which was carried out in a total reaction volume of 25 µl. The reaction mix contained SYBR Green QPCR Master Mix (Finnzymes) and 1.0 µM each primer. The 26S rRNA gene of I. batatas (GenBank accession no. AJ972410) was used as an internal control and amplified using primers as described previously (Mukasa et al., 2006). Each sample was loaded in triplicate on a 96-well optical plate (Applied Biosystems) and scanned using the ABI Prism 7000 Sequence Detection System (Applied Biosystems).
Cloning and sequence analysis.
cDNA was prepared from total RNA extracted from I. setosa leaves of SPCSV-infected plants as described above. The reaction mix (15 µl) for PCR-based amplification of SPCSV sequences contained 200 µM dNTPs, 0.5 µM each primer, 0.02 U high-fidelity Phusion DNA polymerase (Finnzymes) and 1.0 µl cDNA. Amplification was carried out in a Mastercycler gradient thermocycler (Eppendorf). Primers for amplification of regions of RNA1 were designed according to the putatively conserved regions of the RNA-dependent RNA polymerase (RdRp) gene and the 3'-untranslated region (3' UTR) of criniviruses, and the RNA1 sequence of SPCSV-Ug (GenBank accession no. AJ428554), to amplify the region from the RdRp gene to the 3' UTR (Fig. 1a). The forward primer RdRp-F (5'-CAANACNAANGAATTGAACAT-3'; designed according to the sequence of SPCSV-Ug) and the degenerate reverse primer SVV-R3 (5'-TTTTTGAGNTTTTANAATACACAC-3') were used to amplify a region from the 3'-proximal region of the RdRp gene to the middle of the 3' UTR, which corresponded to nt 7197–9277 in SPCSV-Ug.
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. For isolate Is, primers WAhsp-F1 (5'-CKATGAARGTCCTTCGAATC-3') and WAhsp-R1 (5'-TATTCTTCGTAAAGGRAAACCT-3') were used to amplify the corresponding Hsp70h fragment. These primers were designed based on sequences of the WA strain isolates available in databases.
Amplification products were cloned using a pCR-Blunt cloning system (Invitrogen) in Escherichia coli DH5
cells. Two or more clones obtained in independent PCRs were sequenced from each isolate and genomic region at the Haartman Institute DNA sequencing unit (University of Helsinki, Finland). Sequence alignments were done using the CLUSTAL W algorithm available in the AlignX program (VectorNTI-v9 package; Invitrogen).
Northern blot hybridization.
Total RNA (10 µg) was separated in a 5.5 % formaldehyde-containing denaturing agarose gel and blotted onto Hybond-NX membrane (Amersham Biosciences) overnight by capillary transfer. The RNA was fixed to the membrane by exposure to UV light for 1 min and pre-hybridized in a solution containing 50 % formamide (Sigma), 5x SSPE, 5 % SDS, 2.5x Denhart's solution and 1 mg herring sperm DNA (Sigma) ml–1. Probes complementary to the RNase3 and p22 coding sequences were prepared and labelled with 32P[UTP] (Amersham) by in vitro transcription of the genes cloned in plasmids under the T7 or SP6 polymerase promoter. Hybridizations were carried out in a fresh batch of pre-hybridization solution containing 25 µl of the in vitro transcription reaction at 48 °C overnight. The following day, membranes were washed at 68 °C twice in 5x SSC with 0.5 % SDS and twice in 0.2x SSC with 0.5 % SDS. Membranes were exposed to X-ray film (Kodak) for 4, 16 or 48 h before being developed using an X-OMAT 1000 automated developer (Kodak).
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RESULTS |
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). This region in SPCSV-Ug and SPCSV-Tug2 starts 29 nt upstream from the beginning of the sgRNA for the p22 RSS protein and ends 38 nt downstream from the stop codon of the p22 open reading frame (ORF) (Fig. 1b). Hence, these isolates were lacking the gene for the p22 RSS protein. Otherwise, the genetic structure of the sequenced part of RNA1 was conserved among the six isolates. All contained the ORFs for RNase3 and p7 (Fig. 1a) and no additional sequence insertions or deletions were detected.
To rule out any possible ambiguities due to RT-PCR or cloning, viral RNA was detected by Northern blot analysis using probes for RNase3 (Fig. 2a), p22 (Fig. 2b) and RdRp (data not shown). The youngest systemically infected leaves of I. setosa were tested, as a previous study on SPCSV-Ug showed that these tissues contained the highest amounts of the sgRNAs for RNase3 and p22 early in infection (Kreuze et al., 2002). Detection with the probe for RNase3 revealed a shift in the sizes of the genomic RNA1 and sgRNA for RNase3 in isolates Unj2, Mis1 and Is (Fig. 2a; lanes 1, 3 and 4, respectively) compared with SPCSV-Ug (Fig. 2a, lane 2), which indicated the absence of a genomic region downstream from the RNase3 gene in isolates Unj2, Mis1 and Is. Subsequent rehybridization of the membrane with a probe for p22 showed a signal in isolate Ug but no detectable signal in isolates Unj2, Mis1 and Is (Fig. 2b). These data were consistent with a lack of the p22-containing region in the three isolates. The lack of signal for p22 was not due to smaller amounts of viral RNA in the RNA samples as neither overexposing the X-ray films nor using higher RNA concentrations revealed a signal (data not shown). Taken together, the data indicated that the p22 gene was present only in the two Ugandan isolates of SPCSV.
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3.5 kb) was consistently detected using the probe for p22 (Fig. 2b, filled arrowhead) but not RdRp (data not shown) or RNase3 (Fig. 2a) in plants of I. setosa (Fig. 2) and sweetpotato (data not shown) infected with SPCSV-Ug. Thus, it did not correspond to the sgRNA for RdRp or RNase3 (1.9 kb) (for genomic organization, see Fig. 1a and Kreuze et al., 2002). Accumulation of the dRNA was higher than that of the sgRNA for p22 (Fig. 2b). This putative dRNA was also observed in a previous study on SPCSV-Ug (Kreuze et al., 2002). dRNAs have been reported in infections with several members of the family Closteroviridae and have been suggested as recombining modules with a role in the evolution of these viruses (Che et al., 2002, 2003; Simon et al., 2004; Eliasco et al., 2006). The presence of a dRNA homologous to p22, but not RNase3, in infections with SPCSV-Ug may suggest that this genomic region is active in recombination.
Genetic variability of SPCSV isolates
Phylogenetic analysis of partial Hsp70h gene sequences is used to assign isolates of SPCSV to the two relatively distantly related strains, EA and WA. Isolates Ug, Unj1 and Mis1 included in this study have been shown to belong to the EA strain (Tairo et al., 2005). Strain identification of isolates Is, m2-47 and Tug2 was carried out in this study and showed that m2-47 and Tug2 belonged to strain EA, whereas isolate Is belonged to strain WA (Fig. 3a).
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) of the sequences that flanked the unique p22-containing insert in isolates Ug and Tug2 (Fig. 1b) and also of the differences in the RNase and p7 genes (Table 1) and the 3'-proximal region of the RdRp gene and the 3' UTR sequences (data not shown) of RNA1. The nucleotide and deduced amino acid sequences of RNase3 in the EA strain isolates Ug, Tug2, Unj2, Mis1 and m2-47 were almost identical (98–99 and 97–100 %, respectively) compared to isolate Is (83 and 80–82 %, respectively). The nucleotide and amino acid sequences of the putative p7 ORF were more variable, showing identities of 97–98 and 92–98 % among the EA strain isolates, which in turn showed identities of 74–76 and 59–61 %, respectively, with the WA strain isolate Is (Table 1).
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) revealed that the p22-encoding isolate SPCSV-Ug accumulated in much higher titres in the young leaves of I. setosa than the isolates Mis1, Unj2 and Is lacking the p22 gene, as found consistently in three experiments. As Tug2 was available only in cv. Tanzania co-infected with SPFMV, and m2-47 was obtained as cDNA from Peru for molecular analysis, these isolates were not included in the experiments. Co-infection of the p22-encoding isolates Ug or Tug2 with SPFMV caused a severe disease, which was eventually lethal to plants of I. setosa at 20 days post-inoculation. In contrast, co-infection with SPFMV and the other SPCSV isolates caused severe mosaic and leaf malformation but no necrosis in I. setosa. Infection of I. setosa with SPFMV or SPCSV alone caused only mild symptoms of vein clearing and chlorosis in leaves, respectively. Therefore, all tested isolates of SPCSV acted synergistically with SPFMV in I. setosa, as indicated by markedly increased symptom severity, but the symptoms were more severe with the p22-encoding isolates. Differences in virus accumulation were not tested because extensive necrosis in some virus combinations in contrast to others was expected to make comparisons unreliable.
All isolates of SPCSV tested also act synergistically with SPFMV in sweetpotato plants (cv. Tanzania). Severe symptoms of leaf malformation (Fig. 4) and stunting characteristic of SPVD developed following co-infection with SPCSV and SPFMV. In contrast, plants infected with SPFMV alone showed no symptoms, and those infected with SPCSV alone displayed only mild chlorosis of the upper leaves and some purpling of the lower leaves. Differences in accumulation of SPFMV were estimated by DAS-ELISA and quantitative (real-time) PCR in symptomatic leaves of the same developmental stage of sweetpotato plants 3–4 weeks post-inoculation. ELISA indicated highly increased titres of the SPFMV antigen in doubly infected plants compared with the plants infected with SPFMV only in which the virus was barely detectable (Fig. 5). Real-time PCR revealed that SPFMV accumulated at up to 500-fold higher titres in SPVD-affected plants compared with plants infected with SPFMV alone (Table 2). Hence, isolates of SPCSV were able to act synergistically with SPFMV in sweetpotato plants, regardless of the presence or absence of the p22 gene.
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100-fold) in sweetpotato plants co-infected with SPCSV-Ug compared with the 500-fold elevation in plants co-infected with the SPCSV isolates Unj2, Mis1 or Is (Table 2). Real-time PCR data indicated that the titres of isolate Ug were greatly decreased (33-fold) in plants co-infected with SPFMV, whilst the titres of isolates Unj2 and Mis1 were not significantly affected (Table 2). In sweetpotato plants infected with the isolates Unj2, Ug and Mis1 alone, all isolates accumulated at similar titres (Table 2). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) although no sequence data are available for direct comparison. The Kenyan isolate was reported to contain only an ORF for a putative 30 kDa protein at the end of the RNA1 sequence (Hoyer et al., 1996a). An ORF for a putative 30 kDa protein would also be predicted for RNA1 of isolates Ug, Tug2, Unj2, Mis1 and m2-47 (but not Is) due to an AUG translation initiation codon at nt 7481 (according to the sequence of SPCSV-Ug; Kreuze et al., 2002). However, this codon is upstream from the determined 5' end of the sgRNA for the 27 kDa RNase3 (Kreuze et al., 2002) and most likely is not used. Hence, whilst it cannot be verified further due to unavailable sequence data, it seems that the Kenyan isolate (Hoyer et al., 1996a) encodes RNase3 but no p22. Taken together, characterization of the 3'-proximal sequences of SPCSV isolates provides the first example of intraspecific variability in gene content in viruses of the family Closteroviridae.
The results of this study show that isolates of a virus species can vary with regard to the presence of genes encoding RSS proteins. Previous studies using chimeric viruses have shown that, whilst some viral RSS proteins are important for efficient RNA accumulation in certain hosts, they are dispensable for virus replication and systemic movement (Peremyslov et al., 1998; Qu & Morris, 2002; Silhavy et al., 2002; Stenger et al., 2005; Scholthof, 2006). Closteroviruses such as citrus tristeza virus encode several RSS proteins (Lu et al., 2004), which may also explain how SPCSV isolates still show high levels of virulence as well as synergism with SPFMV in the absence of the p22 RSS protein. Indeed, p22 was not essential for the ability of SPCSV to act synergistically with SPFMV and for the consequent increases in SPFMV accumulation by several hundred fold and the development of the severe symptoms of SPVD. These results were consistent with previous studies on isolates Is (Milgram et al., 1996) and m2-47 (Gutiérrez et al., 2003; Untiveros et al., 2007), which cause SPVD in plants co-infected with various isolates of SPFMV. Isolate Tug2 is maintained in a sweetpotato plant also infected with SPFMV and affected by SPVD (unpublished data). Hence, isolates of SPCSV act synergistically with SPFMV in sweetpotato plants, regardless of the presence or absence of the p22 gene. This is an important finding as it means that any effort to engineer pathogen-derived resistance to SPCSV and SPVD in sweetpotato should not rely on p22 as the transgene and the target sequence.
The data showed that the antagonistic effect of SPFMV, resulting in a decrease in SPCSV accumulation (Mukasa et al., 2006), was most pronounced towards SPCSV-Ug, which encodes p22. The titre of this isolate was decreased 33-fold in full-grown sweetpotato leaves co-infected with SPFMV, whereas the titres of the two other EA strain isolates Mis1 and Unj2 lacking the p22 gene remained unaffected. In I. setosa, the p22-encoding isolates Ug and Tug2 caused necrotic symptoms and eventually lethal necrosis following co-infection with SPFMV, which made assessment of SPCSV titres unreliable. Necrosis caused by Ug and Tug2 may be due to a higher level of accumulation in infected tissues of I. setosa compared with other isolates, or perhaps the activities of p22 in tissues infected with heterologous viruses. For example, chimeric potato virus X (genus Potexvirus) expressing SPCSV p22 causes lethal necrosis in N. benthamiana plants, whereas the same vector virus causes only mosaic symptoms in the absence of p22 (Kreuze et al., 2005). The mechanisms behind the different interactions of the p22-containing SPCSV isolates compared with others in host plants co-infected with SPFMV require further study.
Recombination whereby functional units from homologous or non-homologous sources are brought together lies behind the processes that lead to gene gain (duplication and extensive gene divergence; gene shuffling and horizontal gene transfer) in plant and animal viruses (Lai, 1992; Worobey & Holmes, 1999). Viruses of the family Closteroviridae belonging to three different genera share homologous conserved genes and genome structures, but also show a high level of variability in gene content at the 3' end of the genomic RNA (closteroviruses and ampeloviruses) and RNA1 (criniviruses) (Aguilar et al., 2003; Dolja et al., 2006). This is illustrated in Fig. 6 for five different criniviruses, which contain a diversity of genes with low or no detectable sequence homology among each other in the 3'-proximal region of RNA1. All of these genes are expressed from sgRNAs, which in turn have been implicated in the diversification of the closteroviruses through recombination and gene gain (Bar-Joseph et al., 1997).
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). Its full sequence was reported in 2002 (Kreuze et al., 2002) and the sequence of the 3'-proximal region of RNA1 was reconfirmed in this study. The virus has been maintained in living plants of sweetpotato and I. setosa since its isolation, but no mutants lacking p22 have been observed. An independent isolate, Tug2, was obtained from a sweetpotato plant in Uganda in 2005 and, as reported here, carries the p22 gene. The characterized RNA1 sequences of isolates Ug and Tug2 are highly similar but not identical. Notably, the high sequence conservation of the two Ugandan EA strain isolates in the p22 and flanking regions, an identical size and position of the unique sequence compared with other EA and WA strain isolates, and the distinct geographical location of the p22-encoding isolates suggest recent incorporation of this coding region into the SPCSV genome. An alternative scenario would be that p22 was deleted from an early genetic line of SPCSV before diversification of the EA and WA strains. Spontaneous natural deletions of viral sequences in plant viruses have been attributed to errors in RNA replication, such as a ‘copy choice’ mechanism where the RNA polymerase dissociates and primes elsewhere on the template during RNA synthesis (Simon & Bujarski, 1994). Partial or complete deletion of ORFs has been reported, e.g. for potato mop-top virus (genus Pomovirus) (Torrance et al., 1999; Sandgren et al., 2001) and tobacco rattle virus (genus Tobravirus) (Hernandez et al., 1996), especially upon serial passage by mechanical inoculation under experimental conditions where the genomic regions involved in transmission of the virus by vectors become redundant and can be deleted. The loss of functionally redundant sequences is also observed with viral genomes engineered to become gene vectors and from which the inserted heterologous sequences are often deleted during an extended period of virus replication and spread in the host (see, for example, Chung et al., 2007). However, the p22 gene and its flanking sequences seem to be stable in SPCSV-Ug, and p22 is probably not redundant. The two isolates encoding the p22 RSS protein accumulated in higher amounts than the other isolates in young systemically infected leaves but did not induce more severe symptoms in I. setosa plants infected with SPCSV alone. In the field, higher titres of SPCSV might indirectly enhance virus transmission via better virus acquisition by the whitefly vectors, which would provide a selective advantage to the p22-encoding isolates. In conclusion, it is less likely that the p22-containing region was independently and identically deleted from SPCSV isolates of two genetically distinct strains and isolates that are geographically widely distributed in East Africa, Israel and Peru. Therefore, the evidence best fits the theory of recent acquisition of p22 by some EA isolates of SPCSV, in line with recombination-mediated gene gain, which is a frequent phenomenon in the evolution of members of the family Closteroviridae (Dolja et al., 2006).
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ACKNOWLEDGEMENTS |
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Received 22 September 2007;
accepted 24 October 2007.
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