Departments of Plant Pathology and 2Plant Biology, University of Minnesota, St Paul, MN 55108, USA
Author for correspondence: Ben Lockhart. Fax +1 612 625 9728. e-mail plpa{at}puccini.crl.umn.edu
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Abstract |
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Introduction |
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Methods |
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Purification, characterization and serology of TVCV.
Virus-like particles were extracted from fresh or frozen symptomatic N. edwardsonii leaf tissue by the method described for cauliflower mosaic virus (CaMV) (Hull & Shepherd, 1976 ). Further purification was done by isopycnic density-gradient centrifugation in Cs2SO4 in 100 mM sodium/potassium phosphate, pH 7·0. Gradients were fractionated from the bottom using an ISCO density-gradient fractionator and Fluorinert FC-40 (Sigma) as chase liquid. Gradient fractions (500 µl) were examined by EM. Fractions containing virus-like particles and no host plant material were pooled. The pooled fractions were diluted fourfold with 100 mM sodium/potassium phosphate, pH 7·0, and centrifuged at 148000 gmax for 70 min. The resulting pellet was resuspended in dH2O and the suspension used for particle characterization and antiserum production. Extraction and electrophoretic analysis of particle-associated nucleic acid and protein were done as described (Lockhart, 1990
). Polyclonal antibodies were prepared in rabbits by an initial intramuscular injection of purified TVCV adjusted to 150 mM NaCl and emulsified in an equal volume of Hunters TiterMax adjuvant (Sigma). Six weeks later a second immunization was done by multiple-site subcutaneous injection of purified antigen emulsified in Freunds incomplete adjuvant. Blood samples were collected at weekly intervals starting at 2 weeks after the second immunization. Serological tests were done by DAS-ELISA (Clarke & Adams, 1977
) and by immunosorbent electron microscopy (ISEM) as described previously (Ahlawat et al., 1996
). Antisera to cauliflower mosaic (CaMV), carnation etched ring (CERV) and dahlia mosaic (DaMV) caulimoviruses were obtained from T. Guilfoyle, University of Missouri, St Louis, MO, USA, the ATCC and A. A. Brunt, Horticulture Research International, Wellesbourne, UK, respectively. An isolate of PVCV was obtained from petunia cv. Fantasy Pink (Lockhart & Lesemann, 1998
).
Detection of TVCV.
Particles of TVCV were detected by EM examination of partially purified extracts (minipreps) as described for badnaviruses (Ahlawat et al., 1996 ). After an antiserum against TVCV became available, the presence and identity of TVCV were determined by DAS-ELISA using sap extracts, and by ISEM using minipreps.
Mechanical, graft and aphid transmission tests.
Mechanical transmission tests were done using both crude and partially purified extracts of symptomatic N. edwardsonii leaf tissue in which the presence of TVCV had been confirmed by ISEM. Crude or partially purified extracts in 25 mM potassium phosphate, 0·2% (v/v) 2-mercaptoethanol, pH 7·5, were used to rub-inoculate Carborundum-dusted leaves of five healthy plants each of N. benthamiana, N. clevelandii, N. debneyi, N. glutinosa, N. occidentalis, N. rustica and N. tabacum Xanthi. In graft-transmission tests symptomatic shoots of N. edwardsonii were side-grafted onto N. benthamiana, N. glutinosa and N. tabacum Xanthi. Aphid transmission tests were done using late-instar and adult apterae of Myzus persicae raised on Chinese cabbage (Brassica oleraceae var. pekinensis) grown from seed. Aphids were given a 24 h acquisition access feed on symptomatic N. edwardsonii leaves and then transferred in groups of ten to each of four healthy test plants of N. benthamiana, N. clevelandii and N. glutinosa. After an inoculation access period of 24 h aphids were killed by insecticide. Test plants inoculated mechanically, by graft or by aphids were maintained in the greenhouse for up to 1 year post-inoculation. The plants were observed for development of vein-clearing symptoms and were indexed by DAS-ELISA and ISEM for the presence of TVCV.
Seed-transmission tests.
Seeds of N. edwardsonii from the three sources listed above were germinated in insect-proof cages and maintained in an insect-proof greenhouse after transplanting. Plants were grown one per pot, and were monitored for 1618 months after transplanting. The plants were inspected visually for development of vein-clearing symptoms, and were indexed by DAS-ELISA and ISEM for presence of TVCV.
Characterization of the TVCV genome.
Nucleic acid was isolated from purified TVCV as described previously (Lockhart, 1990 ). This procedure included pre-treatment with DNase and RNase to eliminate contaminating (i.e. non-encapsidated) host nucleic acids. Electrophoretic analysis to determine the type, strandedness and mobility of the extracted nucleic acid was done as described (Lockhart, 1990
). After it was established (see Results below) that TVCV contained a dsDNA similar in properties to the genomic DNAs of caulimoviruses and badnaviruses (Hull, 1984
; Lockhart, 1990
), this DNA was linearized by digestion with PstI, ligated into Bluescript SK(+) (Stratagene) and the resulting genomic clone, pTVCV, was propagated in E. coli. The full-length clone was sequenced in both directions by primer-walking using an ABI automated sequencer (DNA Sequencing Facility, Iowa State University, Ames, IA, USA); the sequence has been deposited in GenBank (acc. no. AF190123). Computer analyses of the DNA sequence and the deduced amino acid sequence of proteins encoded by the TVCV ORFs were performed using the Wisconsin Package version 10.0, Genetics Computer Group (GCG), Madison, WI, USA. The TVCV genome was analysed for the presence of site-specific discontinuities as described by Bouhida et al. (1993)
.
Southern blotting.
Total genomic DNA was isolated from freeze-dried leaf tissue of N. edwardsonii, N. glutinosa, N. clevelandii, N. tabacum cv. NC 2326 and N. rustica (Gawel & Jarret, 1991 ). DNA samples (1520 µg each) were electrophoresed in 0·8% agarose gels either untreated or after digestion with EcoRI or HindIII. After overnight capillary transfer to nylon membranes (Micron Separations) the DNA was UV cross-linked with a Bio-Rad GS Gene Linker and the membranes baked at 80 °C for 2 h. Membranes were probed in QuikHyb solution (Stratagene) with the full-length clone of TVCV labelled with 32P using a Promega Prime-a-Gene kit. Membranes were washed for 30 min at 60 °C in 0·1x SSC, 0·1% SDS and exposed to X-ray film for 14 days at -80 °C.
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Results |
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No vein-clearing symptoms were observed in any of six other Nicotiana spp. (N. benthamiana, N. clevelandii, N. debneyi, N. glutinosa, N. occidentalis and N. rustica) grown alongside N. edwardsonii for up to 12 months after transplanting. TVCV was not detected by ISEM or DAS-ELISA in these other Nicotiana spp.
Characterization and serological and biological properties of TVCV
Preparations of TVCV purified by isopycnic banding in Cs2SO4 were shown by EM examination to be free of contaminating host plant material (Fig. 1b) and had an A260/A280 absorbance ratio of 1·401·45. The virions had a buoyant density of 1·348 gm/cm3 in Cs2SO4 and measured 4850 nm in diameter when stained negatively in 2% sodium phosphotungstate, pH 7·0 (Fig. 1b
). A major 45 kDa polypeptide species was detected by Coomassie blue staining (Fig. 2
). Minor polypeptides of 56, 50, 41 and 39 kDa were also visualized by Coomassie blue staining (Fig. 2
). Secondary polypeptides are also associated with virions of other caulimoviruses (Hull, 1984
) and badnaviruses (Lockhart, 1990
), and are possible polyprotein processing intermediates and capsid degradation products (Hull, 1984
; Lockhart et al., 1995
). Nucleic acid extracted from the purified preparations (Fig. 3b
) was shown by RNase and S1 nuclease digestion experiments to consist of dsDNA (data not shown). TVCV genomic DNA migrated on agarose gels as a series of conformational forms (Fig. 3b
) similar to those described for caulimoviruses [Hull (1984)
and references therein] and badnaviruses (Lockhart, 1990
). These conformational species represent linear, circular and knotted forms of genomic virion DNA (Hull, 1984
; Lockhart, 1990
). In addition to full-length genomic DNA, TVCV virions contained an 850 bp moiety (Fig. 3b
) that hybridized to the full-length TVCV clone and was digested by S1 nuclease (data not shown). This DNA species may be analogous to encapsidated single-stranded subgenomic caulimovirus DNAs which have been described previously (Covey et al., 1983
; Marsh et al., 1985
).
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Rabbit polyclonal antibodies raised against TVCV reacted in DAS-ELISA and ISEM with homologous antigen. No serological relationship to PVCV, CaMV, CERV or DaMV was detected by ISEM, nor to PVCV, CaMV or DaMV by DAS-ELISA.
TVCV was not transmitted to other Nicotiana spp. by mechanical inoculation using either sap extracts or partially purified preparations. No vein-clearing symptoms were observed in inoculated plants of N. benthamiana, N. clevelandii, N. debneyi, N. glutinosa, N. occidentalis or N. rustica that were observed for up to 6 months post-inoculation and TVCV was not detected in these plants by ISEM or DAS-ELISA. Similarly, TVCV was not detected in plants of N. benthamiana, N. clevelandii and N. glutinosa inoculated by M. persicae, nor in graft-inoculated N. benthamiana and N. tabacum that were indexed at 3 month intervals over a period of 9 months.
Cloning and sequencing of TVCV
The sequence of a cloned TVCV genome was determined (GenBank acc. no. AF190123). The genome is 7767 bp and contains four ORFs. Based on both sequence identity and the arrangement of the ORFs, TVCV is most similar to integrated pararetrovirus-like sequences that are present in high copy number in the N. tabacum genome (Jakowitsch et al., 1999 ; TPVL) and to cassava vein mosaic virus (CsVMV; de Kochko et al., 1998
) (Fig. 4
and Table 1
). While the order of protein functions is conserved, ORF1 of CsVMV is split into two ORFs in TVCV and the putative viral genome assembled from the integrated TPVL sequences (TPV) and ORF2 of CsVMV is not present in TVCV and TPV (Fig. 4
). In addition, ORF1 of CsVMV has an N-terminal extension of 200 amino acids relative to TVCV and TPV. The TVCV genome and putative proteins contain all of the features expected to be present in a pararetrovirus (Fig. 4
).
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Discussion |
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TVCV resembles caulimoviruses (Hull, 1984 ) in virion properties and genome type, but is distinct from caulimoviruses in biological properties and genome organization. There are obvious similarities between TVCV and PVCV. Both are highly seed-transmitted, are not transmitted by mechanical inoculation or by aphids, and are related to sequences integrated in the host genome. However, TVCV and PVCV are serologically unrelated, do not cross-hybridize, and differ in genome organization. In nucleotide sequence and genome organization, TVCV most closely resembles CsVMV, which has been proposed as the type member of a subgroup of plant pararetroviruses intermediate in genome organization between caulimoviruses and badnaviruses (de Kochko et al., 1998
), but CsVMV is transmitted horizontally (Calvert et al., 1995
) and has not been reported to be either seed-transmitted or related to sequences present in the host genome.
It has recently been reported (Ndowora et al., 1999 ; Harper et al., 1999
) that episomal banana streak badnavirus (BSV) infection in banana and plantain (Musa spp.) might arise from integrated pararetroviral sequences, and a similar mechanism has been proposed for PVCV (Richert-Pöggeler et al., 1996
; Richert-Pöggeler & Shepherd, 1997
). Results of genomic Southern blotting experiments reported above (Fig. 5
) show that TVCV-related sequences are associated with high molecular mass genomic DNA of N. edwardsonii, and preliminary results from screening of an N. edwardsonii genomic library have revealed the presence of TVCV genomic sequences contiguous to presumed host plant genomic sequences (data not shown). These data suggest that N. edwardsonii may contain integrated plant pararetroviral sequences capable of producing episomal viral infection similar to that proposed for BSV (Ndowora et al., 1999
; Harper et al., 1999
). Interestingly, a recent report (Jakowitsch et al., 1999
) has described the occurrence of pararetrovirus-like sequences (TPVL) that occur in high copy number in the genome of N. tabacum. From a composite of TPVL integrants the complete genomic sequence of a putative tobacco pararetrovirus (TPV; GenBank acc. no. AJ238747) was assembled. There is high similarity between TVCV and TPV both in nucleotide sequence and genome organization (Fig. 4
). The nucleotide sequence of the protein coding region of TPV is 81% identical to the corresponding region of TVCV, while the intergenic regions are only 60% identical (Table 1
). The differences in the respective extent of identity between the intergenic and protein coding regions of TVCV and TPV suggest either that the TPVL sequences are not orthologous to the N. edwardsonii integrants, which are postulated to give rise to the episomal form of TVCV, or that the protein-coding regions of the integrated sequences are subject to selective pressures that slow the rate of divergence relative to the intergenic region.
It is highly unlikely that episomal TVCV infection in N. edwardsonii could have arisen from external sources, because TVCV was not transmitted by mechanical, aphid or graft inoculation. Since TVCV was seed-transmitted to 100% of progeny plants, episomal virus infection could only have arisen from either integrated pararetroviral sequences as has been postulated for BSV (Ndowora et al., 1999 ; Harper et al., 1999
), or from subliminal infection in the form of virions or supercoiled virion DNA (Covey et al., 1997
). The latter possibility seems unlikely, because there has been no history of pararetrovirus infection in N. edwardsonii, in the parental genotypes N. clevelandii and N. glutinosa or in any other Nicotiana spp. (Jakowitsch et al., 1999
), and in Southern blot analysis of total genomic DNA of N. edwardsonii (Fig. 5
) no hybridizing sequences corresponding to 78 kb episomal virion or supercoiled virion DNA were detected. The available evidence, therefore, suggests that the episomal form of TVCV arises from integrated pararetroviral sequences present in the N. edwardsonii genome and inherited from the male parent, N. glutinosa. Work in progress, including characterization of TVCV-related sequences identified in an N. edwardsonii genomic library, is expected to provide information on the structure of the integrated pararetroviral sequences and clues to possible molecular mechanisms by which the integrants could yield a replication-competent episomal viral genome. Neither an intact integrated nor an episomal form of TPV has been detected in N. tabacum (Jakowitsch et al., 1999
) and TVCV is therefore the first pararetrovirus described from Nicotiana spp. Evidence presented above and previously (Jakowitsch et al., 1999
) suggests that pararetroviral sequences are integrated in the genomes of other Nicotiana spp. and of other Solanaceae. It would be interesting to see if other instances of episomal pararetrovirus infection are discovered in these solanaceous species.
This report raises two interesting questions. The first is why was TVCV not discovered previously in an indicator plant that has been widely used for over 20 years. The second is why do symptoms and virions occur in N. edwardsonii but not in the parental genotype N. glutinosa, nor other Nicotiana spp. that contain related integrated viral sequences. One plausible answer to the first question is that vein-clearing symptoms and virions occur only in older plants growing under certain conditions. In normal virus-transmission experiments plants are inoculated at a young stage and kept for only a few weeks before being harvested or discarded, i.e. prior to the time when vein-clearing symptoms first appear. In addition, vein-clearing symptoms seem to develop mainly during late fall and early winter under Northern Hemisphere conditions and were generally not observed at other periods of the year. This suggests that in areas where seasonal changes are less pronounced (e.g. Florida), vein-clearing symptoms may not appear even in plants kept for long periods. The discovery of TVCV may therefore have been due to a fortuitous accident of geography.
The answer to the second question, i.e. the reason for the possible episomal expression of integrated viral sequences in N. edwardsonii but not in N. glutinosa or other Nicotiana spp., is more speculative. One simple hypothesis is that N. clevelandii and N. glutinosa contain incomplete or replication-deficient viral integrants, and that interspecific hybridization brought together complementary viral genomic sequences. However, the failure to detect TVCV sequences in N. clevelandii (Fig. 5) does not support this hypothesis. An alternative hypothesis is based on the process of synthesis of N. edwardsonii, which is an amphihexaploid generated by colchicine-induced chromosome doubling (Warmke & Blakeslee, 1939
; Christie, 1969
). Increased chromosome breakage and rearrangements associated with this process may have led to rearrangement of viral integrants to produce a new replication-competent viral genome capable of giving rise to episomal virus. A further possibility is that interactions between the parental genomes activate the integrated virus. We have noted the occasional presence of what appear to be clonal sectors on leaves (not shown), suggesting that genome rearrangement or epigenetic events occur during plant growth. When grown under short days the growth habit of N. edwardsonii is similar to N. clevelandii while under long days the habit is that of N. glutinosa suggesting that the dominant genome changes with daylength.
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Footnotes |
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References |
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Received 8 November 1999;
accepted 20 February 2000.