Characterization and genomic analysis of tobacco vein clearing virus, a plant pararetrovirus that is transmitted vertically and related to sequences integrated in the host genome

B. E. Lockhart1, J. Menke1, G. Dahal1 and N. E. Olszewski2

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


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
A previously undescribed caulimo-like virus was identified in the hybrid tobacco species Nicotiana edwardsonii, and was named tobacco vein clearing virus (TVCV) after the symptoms associated with its occurrence in this plant. The virions of TVCV are 50 nm in diameter and are composed of a 45 kDa capsid protein and a 7767 bp dsDNA genome. Each strand of the genome is interrupted by a site-specific discontinuity. In genome sequence and arrangement of ORFs TVCV was most similar to cassava vein mosaic virus, indicating that TVCV is a pararetrovirus. No serological relationship was detected between TVCV and any other caulimoviruses, including petunia vein clearing virus, which has similar biological properties. In N. edwardsonii TVCV was seed-transmitted to 100% of progeny plants, but was not transmitted by mechanical inoculation, grafting or Myzus persicae to any of seven other Nicotiana spp. Genomic DNA of TVCV hybridized to genomic DNA of N. edwardsonii and of N. glutinosa, its male parent, but not to genomic DNA of N. clevelandii, the female parent. TVCV has 78% sequence identity with pararetrovirus-like sequences that are present in high copy number in the N. tabacum genome, and TVCV genomic DNA hybridized to genomic DNA of N. tabacum and N. rustica. These observations suggest that the episomal form of TVCV may arise from integrated pararetroviral elements present in N. edwardsonii, that these integrants were inherited from the male parent N. glutinosa, and that these elements are related but not identical to pararetroviral elements occurring in other Nicotiana spp.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Nicotiana edwardsonii is a hybrid tobacco species resulting from a cross between N. clevelandii (female parent) and N. glutinosa (male parent) (Christie & Hall, 1979 ). All three species are widely used as indicator plants in biological assays of plant viruses (Christie, 1969 ). Uninoculated plants of N. edwardsonii grown from seed in an insect-proof greenhouse at the University of Minnesota all developed virus-like vein-clearing symptoms. Virus-like particles 50 nm in diameter and similar in morphology to virions of caulimoviruses (Hull, 1984 ) were observed by electron microscopy (EM) in partially purified extracts of symptomatic N. edwardsonii leaf tissue. No other virus-like particles were observed in these partially purified extracts. These observations suggested that the caulimovirus-like particles associated with vein-clearing symptoms in N. edwardsonii were seed-transmitted in 100% of progeny plants. No caulimoviruses are known to occur naturally in Nicotiana spp. (Hull, 1984 ), and there have been no previous reports of seed-transmission of caulimo-like viruses in Nicotiana (tobacco). However, petunia vein clearing virus (PVCV) is known to be transmitted through both pollen and seed in petunia, another member of the Solanaceae (Richert-Pöggeler & Shepherd, 1997 ). Recent reports have presented evidence that genomic sequences of PVCV (Richert-Pöggeler & Shepherd, 1997 ) and banana streak badnavirus (BSV) (Ndowora et al., 1999 ; Harper et al., 1999 ), two plant pararetroviruses, are integrated into the host genome, and that integrated viral sequences can give rise to episomal viral infection (Ndowora et al., 1999 ). The results presented below describe the characterization and genomic analysis of TVCV and the results of initial studies on the possible link between vertical transmission of TVCV and the presence of viral sequences integrated in the host genome.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Seed sources and plant culture.
Three seed lots of N. edwardsonii were used in this study. One was produced in the greenhouse at the University of Minnesota from an original source obtained in 1982 from D. E. Purcifull, University of Florida, Gainesville, FL, USA. Two other seed lots were obtained in 1997 from D. E. Purcifull and from S. M. Garnsey, USDA, Orlando, FL, USA. Seeds from all three lots were germinated in insect-proof cages and grown in an insect-proof greenhouse after transplanting. Plants were grown in a steam-pasteurized potting mix at 24–28 °C under medium shade, and were kept for 12–18 months after transplanting.

{blacksquare} 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 Hunter’s TiterMax adjuvant (Sigma). Six weeks later a second immunization was done by multiple-site subcutaneous injection of purified antigen emulsified in Freund’s 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 ).

{blacksquare} 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.

{blacksquare} 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.

{blacksquare} 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 16–18 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.

{blacksquare} 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) .

{blacksquare} 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 (15–20 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 1–4 days at -80 °C.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Association of TVCV with vein-clearing symptoms in N. edwardsonii
Under winter greenhouse conditions in Minnesota (December–March), vein-clearing symptoms (Fig. 1a) were first observed in seedlings of N. edwardsonii 5–6 weeks after transplanting, and had appeared in approximately 25% of the plants at 8–12 weeks. The percentage of plants showing symptoms increased over the next 2 months, until all plants developed various degrees of vein-clearing symptoms. All symptomatic plants contained TVCV identified by ISEM, and TVCV-associated antigen detected by DAS-ELISA. No other virus-like particles were observed in either purified or partially purified preparations from either symptomatic or asymptomatic N. edwardsonii leaf tissue.



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Fig. 1. Vein-clearing symptoms and virions of TVCV that occur in N. edwardsonii. (a) Vein-clearing symptoms (right) that occur typically in early winter. Asymptomatic leaves at left. (b) Virions of TVCV purified by isopycnic density-gradient centrifugation in Cs2SO4 and negatively stained with 2% sodium phosphotungstate, pH 7·0. Scale bar represents 100 nm.

 
Initial vein-clearing symptoms disappeared in N. edwardsonii plants at 10–20 weeks after transplanting. Thereafter, plants produced asymptomatic leaves containing low but detectable levels of TVCV and TVCV-associated antigen. Vein-clearing symptoms were absent during the summer, and reappeared at the beginning of winter (November–December). There was a direct correlation between symptom expression and the concentration of TVCV detectable by ISEM and DAS-ELISA, respectively. A similar pattern was repeated in a second growth cycle, when symptoms disappeared towards the end of winter, and there was a corresponding decline in the concentration of TVCV.

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 1·40–1·45. The virions had a buoyant density of 1·348 gm/cm3 in Cs2SO4 and measured 48–50 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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Fig. 2. Analysis of TVCV virion components by SDS–PAGE and agarose gel electrophoresis. TVCV capsid polypeptides visualized by Coomassie blue Staining following SDS–PAGE. Left lane: protein size markers with molecular masses indicated in kDa. Right lane: TVCV. Major 45 kDa capsid polypeptide species indicated by arrowhead.

 


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Fig. 3. Each strand of the TVCV genome is interrupted by a discontinuity. (a) Circular map of TVCV. The tRNAMet binding site and minus-strand discontinuity (cloverleaf), approximate position of the plus-strand discontinuity (arrow), and EcoRI and PstI sites are indicated. The arcs indicate the positions of the ORFs. (b) Ethidium bromide-stained gel containing undigested TVCV virion DNA (lane 1), PstI-digested TVCV virion DNA (lane 2), PstI-digested pTVCV DNA (lane 3) and markers (lane 4). (c) DNA samples were denatured and subjected to electrophoresis on a 0·8% agarose gel. Ethidium bromide-stained gel containing TVCV genomic DNA either undigested (lane 1) or digested with EcoRI (lane 2) or PstI (lane 3). Lane 4 contains pTVCV that has been digested with PstI and lane 5 contains markers. The open squares to the right of the lanes indicate the location of fragments that hybridized with pTVCV and in the case of lanes 2–4 are derived from digested DNA. Lanes 2 and 3 contain a DNA fragment with the same mobility as the full-length denatured TVCV genome, which hybridized to pTVCV; the nature of this DNA is unclear but we believe that it arises from DNA that was not digested by the restriction enzyme. The full-length TVCV insert of pTVCV runs as two species (upper two bands in lane 4); this is probably because the two strands have different secondary structure under the electrophoresis conditions employed.

 
The TVCV genome contains single EcoRI and PstI sites (Fig. 3a, b). After denaturation, both EcoRI- and PstI-digested virion DNA migrated as four fragments on an agarose gel (Fig. 3c). Denatured but undigested virion DNA migrated as a single species with a mobility similar to that of denatured full-length cloned TVCV DNA (Fig. 3c). These results are consistent with each strand of virion DNA being interrupted by a site-specific discontinuity (Fig. 3a).

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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Fig. 4. Comparisons of genomic features of TVCV, TPV and CsVMV. The thin line indicates the genomic DNA starting at nucleotide 1 and extending to the last nucleotide of the sequence. The thick lines indicate the locations of the ORFs. The dotted lines extending between the ORFs of CsVMV (GenBank acc. no. U59751) and those of TVCV (AF190123) and TPV (AJ238747) indicate the corresponding sequence identities. The predicted functions of the proteins encoded by the ORFs of TVCV are indicated. Coat, virion capsid protein; Movement, cell-to-cell movement protein; Pr/RT/RH, a polyprotein composed of aspartic protease, reverse transcriptase and RNase H; and Transactivator, a protein with similarity to the caulimovirus inclusion body/transactivator protein. {clubsuit}, tRNAMet binding site; , zinc finger-like RNA binding domain of the capsid protein; , active site of the aspartic protease; {diamondsuit}, active site of reverse transcriptase; , RNase H consensus.

 

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Table 1. Identify between TVCV, TPV and CsVMV proteins

 
Southern blotting
Total DNA isolated from N. edwardsonii seedlings prior to the appearance of symptoms or the occurrence of detectable episomal virus contained sequences that hybridized with TVCV. Hybridization occurred with both undigested and restriction endonuclease-digested N. edwardsonii genomic DNA (Fig. 5). The hybridization signal from undigested DNA occurred at the location of the N. edwardsonii genomic DNA rather than at that (~8 kb) expected for TVCV virion or minichromosome DNA. Similar hybridization was observed with total genomic DNA of N. glutinosa. TVCV DNA also hybridized to N. tabacum and N. rustica genomic DNA but the signal was weaker and the pattern of hybridization was different. No hybridization with genomic DNA of N. clevelandii was detected. In parallel experiments no hybridization was detected with genomic DNA from other Solanaceae (eggplant, potato, tomato, petunia, Physalis) or from unrelated taxa (corn, soybean, wheat, turnip) (data not shown).



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Fig. 5. Sequences that cross-hybridize with TVCV are present in the genomes of several Nicotiana spp. (a) Blot of total genomic DNA that had been digested with EcoRI (odd-numbered lanes) or HindIII (even-numbered lanes) from healthy N. edwardsonii (lanes 1 and 2), N. glutinosa (lanes 3 and 4), N. clevelandii (lanes 5 and 6), N. tabacum cv. NC 2326 (lanes 7 and 8) and N. rustica (lanes 9 and 10). (b) Blot of undigested total genomic DNA from healthy N. edwardsonii (lane 1), N. glutinosa (lane 2), N. clevelandii (lane 3), N. tabacum cv. NC 2326 (lane 4) and N. rustica (lane 5). Both blots were probed with the TVCV insert of pTVCV.

 

   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
On the basis of the data presented above, we have concluded that TVCV is a previously undescribed plant pararetrovirus known to occur only in N. edwardsonii and apparently transmitted only vertically. Because TVCV was not transmitted between host plants, it could not be proven that it is the causal agent of the vein-clearing syndrome in N. edwardsonii. However, the consistent association of TVCV virions with symptom appearance, and the failure to detect any other virus-like particles in purified preparations from symptomatic plants, support the hypothesis that TVCV infection induces vein-clearing symptoms in N. edwardsonii.

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 7–8 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.


   Footnotes
 
The sequence of TVCV has been deposited in GenBank, accession no. AF190123.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 8 November 1999; accepted 20 February 2000.