1 Laboratory of Plant Virology, Faculty of Agriculture, Saga University, Saga 840-8502, Japan
2 School of Botany and Zoology, Australian National University, Canberra, ACT 0200, Australia
3 Departamento de Biotecnologia, INIA, Autopista A-6 km 7, 28040 Madrid, Spain
Correspondence
Kazusato Ohshima
ohshimak{at}cc.saga-u.ac.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences used in this paper are available as supplementary material in JGV Online.
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INTRODUCTION |
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Genome evolution has been shown to allow plant and animal viruses and bacteriophages to adapt to their susceptible hosts or host cells (Bull et al., 1997, 2003
; Valli & Goudsmit, 1998
; Wichman et al., 1999
; Crill et al., 2000
; Moya et al., 2000
; Brown et al., 2001
; García-Arenal et al., 2001
; Schneider & Roossinck, 2001
; Liang et al., 2002
; Novella, 2004
). Such evolution has been found to be either random or selected, depending on the virus and host species involved (Domingo, 2000
; Elena & Lenski, 2003
). Plant viruses differ in the numbers of host species that they infect. Some, such as Turnip mosaic virus (TuMV) and Cucumber mosaic virus (CMV), infect several species, whereas others, such as Wheat streak mosaic virus (WSMV) and Cowpea chlorotic mottle virus (CCMV), are more host-specific and infect only a few host species. The host range and virulence of a virus are usually among its most malleable characters. All isolates of a single viral species may infect the same range of host species, but individual isolates may be mostly confined to different members of that set; different viral species usually have different host ranges (Gibbs et al., 1995
). The molecular evolutionary changes that accompany changes in host ranges of plant viruses have been studied by using susceptible hosts; viral populations were transferred serially in single or different host(s), as reported for Tobacco mosaic virus (TMV), CMV, CCMV, WSMV and Hibiscus chlorotic ringspot virus (Kurath & Palukaitis, 1989
; Kurath & Dodds, 1995
; Kearney et al., 1999
; Schneider & Roossinck, 2000
, 2001
; Hall et al., 2001
; Liang et al., 2002
). Similar studies of serially transferred bacteriophage Microvirus
X174 (Bull et al., 1997
; Wichman et al., 1999
) and of orthomyxovirus A (Brown et al., 2001
) identified, in the genomic sequences of adapted variants, two sorts of convergent changes: some sites in independently passaged isolates had identical mutations, whereas others had different mutations. These were distinguished as resulting from parallel evolution and directional evolution.
TuMV infects a wide range of plant species, most from the family Brassicaceae. It is probably the most widespread and important virus that infects both crop and ornamental species of this family and occurs throughout the world, including the temperate and tropical regions of Africa, Asia, Europe, Oceania and North and South America (Provvidenti, 1996; Ohshima et al., 2002
). TuMV was ranked second only to CMV as the most important virus to infect field-grown vegetables in a survey of virus disease in 28 countries and regions (Tomlinson, 1987
; Walsh & Jenner, 2002
). TuMV belongs to the genus Potyvirus. This is the largest genus of the largest family of plant viruses, the Potyviridae, which itself belongs to the picorna-like supergroup of viruses of animals and plants (Shukla et al., 1994
; van Regenmortel et al., 2000
). TuMV, like other potyviruses, is transmitted by aphids in a non-persistent manner (Hamlyn, 1953
). Potyviruses have flexuous, filamentous particles that are 700750 nm long; each of these contains a single copy of the genome, which is a single-stranded, positive-sense RNA molecule of about 10 000 nt. The genomes of potyviruses have terminal untranslated regions and, between them, a single ORF that is translated into a single large polyprotein, which is hydrolysed after translation into at least ten proteins by virus-encoded proteinases that are part of the polyprotein (Riechmann et al., 1992
; Urcuqui-Inchima et al., 2001
).
There have, to our knowledge, been four reports of studies of the phylodemography of TuMV (Ohshima et al., 2002; Tomimura et al., 2003
, 2004
; Tan et al., 2004
). These studies showed that TuMV isolates fell into four well-supported lineages: basal-B, basal-BR, Asian-BR and world-B. These groupings correlated with differences in pathogenicity and origin; the sister group to all others was Eurasian Brassica (B)-host type (strain) isolates from non-brassicas, which probably represents the ancestral TuMV population, and the most recently emerged branch of the population was probably that of the Brassica/Raphanus (BR)-host type isolates, which are found only in east Asia. Our previous studies indicated that the original TuMV population was probably of the B-host type, but the BR-host type isolates have evolved from the B-host type on several occasions. These may indicate that the basal-B group isolates, which are optimally adapted to crops of brassicas, spread worldwide in the footsteps of modern agriculture more readily than those adapted to other species, although it could also indicate that the older populations of TuMV are more variable and hence contain more variants that are able to infect non-brassica species. Therefore, the basal-BR, Asian-BR and world-B group isolates seem to be more adapted to their host plants, such as Brassica and Raphanus, than those in the ancestral basal-B group (Ohshima et al., 2002
; Tomimura et al., 2003
). Most, but not all, of the isolates belonging to the world-B group are of the B-host type; for instance, one isolate, UK1, is of the B-host type and belongs to the world-B group and, although many Brassica plants are susceptible to it, it rarely infects Raphanus sativus systemically and then only asymptomatically (Walsh, 1989
; Tomimura et al., 2003
).
Despite growing interest in the molecular evolution of potyviruses (Bousalem et al., 2000; Bateson et al., 2002
; Ohshima et al., 2002
; Tomimura et al., 2003
; Moreno et al., 2004
; Tan et al., 2004
), there have been few attempts to study adaptive evolution, although this could provide crucial information about the nature of the interaction between host and virus. As phylodemographic studies have shown that the TuMV B-host type has evolved into the BR-host type on several occasions, we decided to investigate whether this evolutionary step could be simulated experimentally. Here, we present a model system for studying plant virus adaptation to a novel host. Using it, we have explored the molecular and evolutionary basis of host-specific adaptation of TuMV by adapting several lineages that were obtained from a single clone of the virus to R. sativus. Changes in the virulence of parallel populations during passaging of these lineages were assessed and the genomes in successive samples were sequenced and compared.
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METHODS |
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Serial passages.
First, 2 µg p35Tunos DNA was inoculated into each of two N. benthamiana plants. Both of the inoculated plants showed clear mosaic symptoms within 10 days of inoculation (d.p.i.). These two plants provided the parental virus stocks, designated N1 and N2. They were kept in a greenhouse for 60 d.p.i. and sap from them was used to inoculate the first of a series of successively infected R. sativus plants, to which the virus adapted. Systemically infected upper leaves of each N. benthamiana plant were collected and 1 g was homogenized separately in 1·5 ml potassium phosphate buffer (PPB), pH 7·0. Each sap extract was applied to the primary leaves of three young B. rapa plants and, respectively, to the primary leaves of 108 and 90 young R. sativus plants. Clear mosaic symptoms appeared on all six B. rapa plants by 10 d.p.i., whereas none of the R. sativus plants showed visible symptoms, even after 90 d.p.i. The uninoculated upper leaves of each of the 198 R. sativus plants were collected at 90 d.p.i. and indexed for the presence of TuMV by double antibody-sandwich ELISA (DAS-ELISA) and also by RT-PCR (see below). Virus samples from the upper leaves of the plants that gave positive reactions in both DAS-ELISA and RT-PCR were designated variants A and B and these were used for further adaptation by R. sativusR. sativus passaging; at each transfer, one or more of the samples (variants) with the greatest DAS-ELISA A405 value was chosen as the source of inoculum for infecting the next R. sativus plant (Fig. 1). These were kept for a further period of 90 days to enable the virus to adapt further.
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RNA was extracted from the uninoculated upper leaves of inoculated plants by using ISOGEN (Nippon Gene) as instructed by the manufacturer. UK1 RNA in the extracts was reverse-transcribed into cDNA by using the minus-sense primer Tu3T9M (5'-GGGGCGGCCGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3'), which hybridizes with the 3'-terminal poly(A) region of the viral genome. The cDNA was then amplified into double-stranded DNA by using primer Tu3T9M together with TuNIB15P. The latter has the sequence 5'-TTGA(C/T)AA(G/A)GAACCAGCTCAAG-3' and hybridizes with the 3' end of the nuclear inclusion b (NIb) gene of UK1. Reverse transcription was done by using ReverTra Ace reverse transcriptase (Toyobo) at 42 °C for 1 h. PCR was done for 35 thermocycles: denaturation at 94 °C for 15 s, annealing at 40 °C for 30 s and polymerization at 68 °C for 60 s, with high-fidelity Platinum Pfx DNA polymerase (Invitrogen). The homogeneity and concentration of amplified cDNA were checked visually after electrophoresis in agarose gels. Infectivities of the variant samples were estimated from the numbers of plants that gave positive reactions in both DAS-ELISA and RT-PCR.
Sequencing of the entire genomes of the variants.
The RNA of variant viruses was extracted from UK1-infected R. sativus and B. rapa leaves by using ISOGEN (Nippon Gene). The RNA was reverse-transcribed and amplified by 25 cycles of PCR using high-fidelity Platinum Pfx DNA polymerase (Invitrogen) and the conditions given above. The amplified cDNA was electrophoresed in agarose gels; bands were excised and then separated from the gel by using a QIAquick gel extraction kit (Qiagen). The nucleotide sequences of the complete genomes of the variants were obtained by primer walking along the genome in both directions by using a BigDye Terminator v3.0 cycle sequencing ready reaction kit (Applied Biosystems) and an Applied Biosystems genetic analyser DNA model 310; about 11 overlapping genomic fragments were amplified independently at least twice by using 22 UK1-specific plus- and minus-sense primers. The 35 nt sequences that were used as primers to amplify the 5' ends of the genomes were not sequenced. Sequences were assembled by using BioEdit version 5.0.9 (Hall, 1999).
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RESULTS |
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Molecular analyses of passaging variants
In order to identify the mutations that adapted TuMV to R. sativus, we determined the complete nucleotide sequences of the genomes of each of the 16 variants' (i.e. passage samples). In total, 39 substitutions, involving eight of the ten viral protein genes, were detected (Table 2, Fig. 2
). No mutations were detected in the 6 kDa 1 (6K1) protein gene, the NIb gene or the 5' and 3' non-coding regions. Half the changes were synonymous and half were non-synonymous; the latter resulted in changes in seven of the ten virally encoded proteins (Fig. 2
). Two-thirds of them were in the protein 3 (P3), cylindrical inclusion protein (CI) and genome-linked viral protein (VPg) genes; six, three and three coding changes were in the P3, CI and VPg genes, respectively. There were also one or two amino acid substitutions in the protein 1 (P1) and helper component proteinase (HC-Pro) genes or 6 kDa 2 (6K2) and coat protein (CP) genes, but there were no non-synonymous changes in the nuclear inclusion a proteinase (NIa-Pro) gene. Mutations appeared in the P1, HC-Pro, P3, CI and 6K2 genes after the first passage, and then in other genes. Most, but not all, mutations found in one passage variant were also in variants that were subsequently derived from it. Thirty-two transitions and eight transversions were found and, of the transitions, 17 were C/U changes and 15 were G/A changes. Seven mixed sites were found in variants A1, A111, B, B11 and B2211. There were four parallel-evolution sites among the genomes of the adapted variants, and all were non-synonymous: (i) variant A1 had C at nt 722 in the P1 gene and the independently selected variants A11 and A12 had U, a non-synonymous change; (ii) parental isolate N1 had G at nt 3430 in the P3 gene, whereas variants A and B had A, a non-synonymous change; (iii) variants B1 and B2 had A at nt 4674 in the CI gene and the independently selected variants B11 and B22 had C, a non-synonymous change; and (iv) parental isolate N1 had G and A at nt 6350 and 6351 in the VPg gene, whereas variants A11 and B22 had A and C, respectively, resulting in different amino acid changes (from Asp to Asn or Ala at an identical position of aa 2074, non-synonymous changes).
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Some of the sequence changes also correlated with changes in virulence (Fig. 1). The clear increase in virulence between variants A1 and A11 correlated with changes from C to U at nt 722, from G to A at nt 6350 and from A to G at nt 6496, indicating that the two parallel changes at nt 722 and 6350 may be responsible for the increase in virulence. Similarly, the increase in virulence between variant B111 and B1111 is correlated with a C to U change at nt 6223. Likewise, the B2 to B22 increase in virulence correlates with an A to G change at nt 2929 and an A to C change at nt 6351, and the B22 to B221 increase in virulence seems to correlate with four changes: C to U at nt 1094, U to C at nt 1656, A to G at nt 5668 and C to U at nt 8935. Interestingly, none of these changes reverted in either variant B2211 or B2212, even though B2212 is in the same virulence group as B221, whereas B2211 seemed to have decreased in virulence compared with its parent. B2212 differed from both B221 and B2211 in having U rather than C at nt 3499 and A rather than C at nt 9614, whereas B2211 differed from both B221 and B2212 in having U rather than C at nt 3281, A rather than C at nt 5755 and C/A rather than U at nt 5944. This may indicate that combinations of particular nucleotides at particular sites determine the virulence of the virus. Half of these virulence-associated changes were non-synonymous. The clear increases in virulence between variants A1 and A11 and between variants B2 and B22 correlated with changes from G to A at nt 6350 and from A to C at nt 6351; both nucleotide changes resulted in amino acid changes from Asp to Asn and Ala at the identical position of aa 2074.
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DISCUSSION |
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There is, of course, the possibility that the mutations that we detected were an artefact of PCR sequencing, as this has been shown to occur when low-fidelity polymerase is used. In the present study, Platinum Pfx DNA polymerase, a high-fidelity polymerase, was used for amplifying cDNA. The fidelity of this polymerase was checked by amplifying and then sequencing 11 000 bp, in total, of PCR products from cloned TuMV DNA of known sequence. We found no mismatch between the sequences of PCR products and those of original DNA clones. Therefore, we conclude that the variant nucleotides we found are not artefacts.
Plant viruses are convenient for studying viral evolution, as infectious viral RNAs generated in vitro or in vivo can be used to infect inbred or even cloned hosts. Isolates of plant viruses with RNA genomes are usually mixed populations of genomes, even when cloned repeatedly, as lesions do not result from infections by single genomes. By contrast, a cloned cDNA plasmid encoding an infectious full-length transcript is a single molecular species that can provide a defined genome for studying adaptation by repeated passaging (Kurath & Dodds, 1995; Kearney et al., 1999
; Schneider & Roossinck, 2001
). However, the method by which the genome encoded in the plasmid is transcribed into RNA seems to be important. Schneider & Roossinck (2000)
transcribed an encoded genome in vitro by using a T7 promoter and PCR (15 cycles with Pfu polymerase) and found a composite mutation frequency of 4·5x105 nt1. By contrast, infections using a single molecular species were obtained from viral cDNA clones with CaMV 35S promoters that were used directly to inoculate host plants by particle guns or by rubbing (Sánchez et al., 1998
; Jenner et al., 2002
, 2003
; Suehiro et al., 2004
). Therefore, in our study, we used an infectious clone, p35Tunos, that has a promoter rendering it directly infectious to N. benthamiana plants.
As initial experiments showed that R. sativus plants were not susceptible to infection with p35Tunos, N. benthamiana plants, which are fully susceptible to TuMV, were employed to establish the parental populations of viruses for passaging. These were kept for 60 d.p.i. before use, to enable a stable and, hopefully, diverse viral population to form in them. However, no sequence differences were found between the region of p35Tunos that encoded the viral genome and the genomes in the parental isolates N1 and N2. This indicates that any variant genomes in the parental isolates represent a small component of each population (Domingo et al., 1978).
As most of the R. sativus plants that were infected during adaptation showed no symptoms, it is likely that selection for virulence and the ability to infect R. sativus systemically does not also select for variants that produce clear and persistent symptoms. The latter, a visual effect, may possibly be coselected when, under natural conditions, transmission by flying aphids is the selection mechanism.
In total, 39 mutations were detected in the genomes of the 16 R. sativus-adapted variants that we sequenced. Some of these may have adapted the virus populations to R. sativus plants, as anticipated, by increasing their virulence to that species; however, others may be mere evolutionary noise. A strong indicator of adaptive change at the molecular level is convergent selection, characterized by repeated and independent occurrence of the same or similar mutations in adapted variants; the occurrence of the same nucleotide substitution at the same site occurring in two or more lineages is termed parallel evolution, whereas non-identical mutations at the same site are termed directional evolution (Bull et al., 1997; Wichman et al., 1999
; Brown et al., 2001
). Four of the changes that we found appeared in parallel lineages of successively infected plants, suggesting that they were involved in adaptation to R. sativus. Their frequency also suggests that they are not the product of random processes as, if the mutations had occurred at random in genomes of 10 000 nt, one would expect to find around one parallel mutation in 250 genomes, not four in 16. There were, however, no sites that changed multiple times with different outcomes (i.e. directional evolution), nor could our experiments indicate whether different combinations of the other 35 independent mutations were adaptive in different lineages.
Many regions of the potyviral genome have been implicated as determinants of symptom induction (Rodríguez-Cerezo et al., 1991; Atreya et al., 1992
; Johansen et al., 1996
; Chu et al., 1997
; Simón-Buela et al., 1997
; Sáenz et al., 2000
; Ullah & Grumet, 2002
; Spetz & Valkonen, 2004
). Most of the mutations that we found in the adapted TuMV genomes were in the P3, CI and VPg genes (Fig. 2
). The P3 proteins, together with the P1 proteins, are among the most variable potyvirus proteins (Shukla et al., 1994
; Urcuqui-Inchima et al., 2001
) and are thought to be involved in virus replication (Merits et al., 1999
), accumulation (Klein et al., 1994
), symptomatology (Chu et al., 1997
; Sáenz et al., 2000
), resistance-breaking (Johansen et al., 2001
; Hjulsager et al., 2002
; Jenner et al., 2002
, 2003
) and cell-to-cell movement (Dallot et al., 2001
; Johansen et al., 2001
). Recently, Suehiro et al. (2004)
showed, by exchanging the genomic fragments between the two isolates, that the C terminus of TuMV P3 has an important role in systemic infection and/or symptomatology in cabbage (Brassica oleracea) and radish (R. sativus). In our study, one of four parallel substitutions, 3430G
A, which resulted in a 1100Met
Ile amino acid change in the C terminus of P3, seems likely to be important in the initial stages of adaptation to R. sativus, as it was found in the early passages of all lineages and was maintained in subsequent variants.
To check whether the other three parallel-evolution sites aided adaptation to R. sativus, we compared the nucleotide and aligned amino acid sequences of B- and BR-host type isolates (Tomimura et al., 2003); however, there was no clear difference between the B and BR isolate sequences and, thus, they do not seem to be involved in BR-host type adaptation or may merely be background sites. On the other hand, variant series B (Fig. 1
) had a mutation site, 3394G
A, that resulted in a 1088Met
Ile change (Table 2
) and, although this site was not one of the four parallel-evolution sites, it is also believed to be involved in systemic movement in R. sativus (Suehiro et al., 2004
). Hence, both positive selection and genetic drift seem to be involved in the adaptation of UK1 to R. sativus. Evidence of genetic drift resulting from population bottlenecks' during systemic movement of TMV was reported recently (Sacristán et al., 2003
) and could have been important in the work that we report here.
On the other hand, the parallel substitutions 6350GA and 6351A
C that resulted in a 2074Asp
Asn/Ala amino acid change in VPg may be important for the increase in virulence, although the former substitution reverted to the original nucleotide after subsequent passages. However, the same parallel substitutions did not occur in all three lineages passaged in R. sativus, despite the possibility that all these substitutions would have arisen many times during the course of these selections. This may suggest that stochastic features, such as the identity of the earliest change to sweep through the population, and the order in which substitutions arise may influence the pattern of adaptation, even in systems where parallelism of adaptation is the rule rather than the exception. Moreover, there is evidence from studies of Human immunodeficiency virus (Nijhuis et al., 1998
), bacteriophage Microvirus
X174 (Wichman et al., 1999
) and TMV (Sacristán et al., 2003
) that the effective population size of a virus may be much smaller than its apparent total population size and, if this is also true for the constrained TuMV populations in R. sativus, it may explain why different changes are found in different lineages.
This paper is, to our knowledge, the first report of experiments using a plant virus and an almost non-susceptible host to study the genetic basis of evolutionary adaptation, and has revealed that several nucleotide sites may be involved in adaptation to R. sativus. Our earlier studies of the phylodemography of TuMV (Ohshima et al., 2002; Tomimura et al., 2003
, 2004
; Tan et al., 2004
) showed that TuMV has adapted to R. sativus on several occasions and has formed major lineages, the Asian-BR and basal-BR groups, that are adapted to that host. The study reported here provides experimental insight into how global adaptation of a virus may occur and indicates that our attempt to simulate viral adaptation to a novel host plant is an idea that might be applied productively to other plant viruses.
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ACKNOWLEDGEMENTS |
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Received 20 August 2004;
accepted 12 October 2004.
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