Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK1
Author for correspondence: Lesley Torrance.Fax +44 1382 526426. e-mail ltorra{at}scri.sari.ac.uk
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Attempts to obtain large (>2·7 kbp) cDNA fragments of PMTV RNA 2 by an RTPCR method consistently produced bands of smaller than expected size. This paper reports investigations to establish the nature of the smaller bands, and shows that a spontaneous deletion occurs in PMTV RNA 2 molecules.
Total RNA extracts of PMTV-T-infected Nicotiana benthamiana leaves at 4, 9 and 14 days post-inoculation (p.i.) were prepared by a modification of the method of Verwoerd et al. (1989) as described by Barker et al. (1993)
. RNA was extracted from leaf discs (0·51·0 g), either from systemically infected leaves, or inoculated leaves on day 4 p.i. In the first experiments, RTPCR was done using three primer sets (Fig. 1a).
1. Primers F1 [5' CGCTCGAGTTTAGGTGACACTATAGGTATTTCAACTCTACCTAG3', representing the 5'-terminal 19 nucleotides of RNA 2 with attached nucleotides (underlined) to create a XhoI site and an SP6 RNA polymerase promoter sequence] and R2702 (5' CCGAATTCCTGTAAGCACTAACAC 3', the complement of RNA 2 nucleotides 26792702, with the underlined nucleotide changed from G to T to create an EcoRI site) amplify >91% of RNA 2 including all of the open reading frames. 2. Primers F1 and R2007 (5' AACTGGCAAGAACAATTGGGAG 3', the complement of RNA 2 nucleotides 19862007) amplify the 5' 1·9 kb region of RNA 2. 3. Primers F1482 (5' CATTGTGTTCTCTGAAAGCACTCG 3', representing nucleotides 14821504 of RNA 2) and R2702 amplify the 1·2 kb 3' terminal region of RNA 2. The RTPCR was done essentially as described by Arif et al. (1994)
using primer R2702 or R2007 to prepare cDNA. The PCR was done in a Perkin Elmer PE9600 instrument using the following conditions: 95 °C for 5 min, then 30 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 60 s, followed by 70 °C for 10 min. Only faint bands of ~2·7 kbp were obtained using primers F1 and R2702 (Fig. 2a
; lanes 1, 2 and 3),
and bands of about 600 bp (
1) and 1100 bp (
2) were visible on gel electrophoresis of the products from the samples extracted 9 and 14 days p.i. (Fig. 2a
; lanes 2 and 3). However, the 5' and 3' segments could be amplified from these samples using primer combinations F1/R2007 and F1482/R2707, which indicates that full-length RNA 2 molecules were present (Fig. 2a
; lanes 610). The nucleotide sequence of the
1 product was obtained and analysis revealed that the fragment contained sequence that was identical to the 5' and 3' termini of PMTV RNA 2, and that a deletion occurred removing nucleotides 4802592 inclusive, or 4792591 inclusive depending on the assignment of the U residues at the junction of the deletion. The sequence of the
2 product was found to be identical to the 5' portion of the RNA 2 up to nucleotides 11401150, with no apparent deletion, and may have been the result of mispriming of primer R2702 at nucleotide 1175. To try to eliminate mispriming, the RTPCR was repeated using F1 and either of two different downstream primers, R2715 (5' CACTAACACTTAACGTAGTGTC 3', the complement of RNA 2 nucleotides 26942715), or R2736 (5' CTCCAATGGTTCGATTTAAGTG 3', the complement of nucleotides 27152736), and an increased annealing temperature of 60 °C. The
1 product only was obtained with both downstream primers, and Fig. 2(b)
shows the result obtained with combination F1/R2736.
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To exclude the possibility that the deletion occurred during cDNA synthesis, RTPCR was performed on transcripts of PMTV RNA 2 derived from a full-length cDNA clone of RNA 2 cloned downstream of the SP6 RNA polymerase promoter (B. Reavy, unpublished results). Transcripts were made in vitro following the manufacturer's instructions (MEGAscript, Ambion), and then the template DNA was removed by treatment with DNase I. The cDNA was prepared using primer R2715, and the PCR was done with the increased annealing temperature of 60 °C to minimize mispriming, with primer combinations F1/R2702 or F1/R2715. Only one band of approx. 2·7 kbp was obtained using either primer set (Fig. 3a).
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We performed two further experiments to determine whether the production of a deletion in RNA 2 was peculiar to the laboratory isolate PMTV-T which has been maintained by manual inoculation for more than 30 years. In the first experiment N. benthamiana plants were infected with a more recently acquired isolate (PMTV-S; Arif et al., 1994 ) that had been maintained by manual inoculation. In the second experiment, tomato cv. Kondine Red plants were inoculated with PMTV by exposure of roots to virus-carrying S. subterranea spore balls. Total RNA extracts were made and assayed by RTPCR as above. A product of approx. 600 bp was obtained with primers F1 and R2702 from cDNA prepared from both the PMTV-S-infected N. benthamiana leaves, and the naturally infected tomato leaves (data not shown). Nucleotide sequence analysis of the 600 bp product from the infected tomato plants again revealed the same RNA 2 deletion (nucleotides 4802592).
RTPCR was done on purified virus particles to investigate whether the deleted forms are encapsidated. PMTV particles were obtained from infected N. benthamiana leaves essentially as described by Torrance et al. (1993) except that after treatment with chloroform the particles were precipitated by 8% (w/v) polyethylene glycol and 0·2 M NaCl followed by one cycle of differential centrifugation over a 3 ml 25% w/v sucrose cushion. The virus preparation was stored at -20 °C before use. RTPCR was done as described above with primers F388 and R2715 either directly on the preparation, or after immunocapture of particles by incubation of the preparation for 1·5 h at 4 °C in PCR tubes (Greiner) pre-coated with anti-PMTV antibodies. A 200 bp product of the size expected for
1 was obtained in both immunocapture and direct RTPCR (Fig. 3b
) experiments, and sequence analysis revealed the same RNA 2 deletion (nt 4802592).
Our experiments show that a contiguous segment of PMTV RNA 2 sequence has been deleted rather than several short fragments, giving a discrete band in the RTPCR which comprises 5' and 3' sequence moieties. Sequence deletions have been found previously in several RNA plant viruses (reviewed in Simon & Bujarski, 1994 ) and 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 (King et al., 1987
). Mechanisms requiring either double-stranded intermolecular duplexes as occurs in Brome mosaic virus or sequence motifs resembling the 5' ends of genomic or subgenomic RNAs in the case of Turnip crinkle virus have been described (Simon & Bujarski, 1994
). Examination of the PMTV RNA 2 sequences flanking the deletion did not reveal any extensive areas of sequence identity between them. However, a model can be proposed where the deletion occurs during synthesis of plus-strand RNA 2 from minus-strand RNA templates. The complement of nucleotides 25632592 and of nucleotides 480507 in the minus-strand can base-pair to form a stem 29 nucleotides long with five mismatches and one unpaired nucleotide. This would bring nucleotides 480 and 2592 into direct proximity at the base of the stem and the deletion would be produced by template switching from nucleotide 479 to 2593 (Fig. 1b
).
Some deleted molecules have been shown to interfere with replication of genomic RNA and have been called DI RNAs (Simon & Bujarski, 1994 ; Zaccomer et al., 1995
). Among the fungus-transmitted rod-shaped viruses, deletions are commonly found in the readthrough domain of the capsid protein, and such deletions have been correlated with lack of fungus transmissibility (Tamada et al., 1996
). Artificial DI RNAs derived from BNYVV RNA have been produced, and the RNA-2-derived molecules were shown to inhibit replication of genomic RNA 1 and 2 (Hehn et al., 1994
). It is not known whether the
1 deleted form of RNA 2 identified here is a true DI in that it inhibits genomic replication. Although the deletion occurs spontaneously, it was present in cultures obtained by fungal inoculation as well as in a laboratory culture maintained by manual inoculation for many years, and is encapsidated.
Deletion of nucleotides 4802592 in RNA 2 removes the entire TGB sequence (apart from the 5' 110 nucleotides of ORF 1) but results in an in-frame fusion of the 5' end of ORF 1 with the 3' end of ORF 4. The TGB, by analogy with other viruses, is thought to encode proteins responsible for virus movement. PMTV is known to be erratically distributed in plants and can be found in some stems but not others from the same plant (Torrance et al., 1992 ). Furthermore, PMTV is gradually self-eliminating from potato stocks if sources of re-infection are removed (Calvert, 1968
; Cooper et al., 1976
). Deletion of the TGB sequence from the virus culture may explain this behaviour.
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Acknowledgments |
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Footnotes |
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References |
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Received 6 April 1999;
accepted 17 May 1999.