Laboratory of Bioresource Technology, Graduate School of Frontier Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan1
Laboratory of Plant Breeding and Genetics, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan2
National Agricultural Research Center, 3-1-1 Kannondai, Tsukuba, Ibaraki 305-8666, Japan3
Koibuchi College of Agriculture, 5965 Koibuchi, Uchihara-cho, Higashi-ibaraki, Ibaraki 319-0323, Japan4
Author for correspondence: Shigetou Namba. Tel: +81 424 69 3125. Fax: +81 424 69 8786. e-mail: snamba{at}ims.u-tokyo.ac.jp
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ABSTRACT |
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Keywords: phytoplasma, extrachromosomal DNA, Rep protein, geminivirus
Abbreviations: AY, aster yellows; OY, onion yellows; OY-M, mild symptom line of onion yellows; OY-W, wild-type onion yellows phytoplasma; SCWL, sugar cane white leaf; TGMV, tomato golden mosaic virus; Vaccinium witches broom
The GenBank accession number for EcOYW1 is AB010426.
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INTRODUCTION |
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Extrachromosomal DNA, including ss- and dsDNA, associated with some spiroplasmas, mycoplasmas and acholeplasmas, has been described (Maniloff, 1988 ; Razin, 1985
; Renaudin & Bove, 1994
). Extrachromosomal DNA was also found in phytoplasmas (Davis et al., 1988
; Sears et al., 1989
; Bertaccini et al., 1990
; Harrison et al., 1991
; Goodwin et al., 1994
). The nucleotide sequences of some extrachromosomal DNA from the AY group of phytoplasmas (partial sequence; approx. 200 bp), Vaccinium witches broom (VAC) phytoplasma (approx. 1·5 kbp) and sugarcane white leaf (SCWL) phytoplasma (approx. 2·7 kbp) have been reported (Rekab et al., 1999
; Kuske & Kirkpatrick, 1990
; Nakashima & Hayashi, 1997
). Previously, we reported a 3·6 kbp DNA fragment of the dsDNA, pOYW1, from OY-W phytoplasma and suggested that pOYW1 is a plasmid replicating by a rolling-circle replication mechanism (Kuboyama et al., 1998
).
In other plant-pathogenic bacteria, it is well known that plasmid-encoded genes play important roles in the pathogenicity and virulence (Panopoulos & Peet, 1985 ). We previously reported that the copy number of pOYW1 was higher in OY-W phytoplasma than OY-M and suggested that this difference might have some relationship to the differences in pathogenicity. However, nucleotide sequence information on pOYW1 alone is not sufficient to deduce its biological significance in the pathogenicity of phytoplasmas.
In this paper, we report another extrachromosomal DNA from OY-W. This replicon was revealed to encode a homologue of geminivirus replication protein Rep; it was not found in OY-M, suggesting that it might also be related to the pathogenicity of OY-W.
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METHODS |
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Extraction of DNA from infected plants.
The phytoplasma-enriched fraction of infected garland chrysanthemums was prepared as reported previously (Lee & Davis, 1983 ). Total DNA was extracted from this fraction following the method of a previous report (Kuboyama et al., 1998
).
Cloning and sequencing the extrachromosomal DNA.
The total DNA of the phytoplasma-enriched fraction of infected garland chrysanthemum plants was electrophoresed and stained with ethidium bromide. As shown in Fig. 1, several DNA bands were observed from OY-W and OY-M DNA. In addition to the chromosomal DNA band (indicated by an asterisk), at least four major extrachromosomal DNA bands were observed in lane W, whilst only two bands were observed in lane M.
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To obtain the remaining part of the possibly circular extrachromosomal DNA, we utilized the sequence information of p2 . 5 to design inverse PCR primers in outward orientations. A pair of inverse PCR primers, P1 (5'-TCT CTT TCT TGT CAA AGC CCC TA-3') and P2 (5'-TCT TCG GGT TCT TGT TTT TCA GTT-3'), and OY-W DNA as a template, were used for inverse PCR. Amplification was performed in a thermal cycler (Perkin Elmer 9700) for 30 cycles, under the following conditions: denaturation for 15 s at 94 °C, annealing for 30 s at 55 °C, and extension for 3 min at 60 °C. The amplified 4·5 kbp fragment was cloned (p4 . 5) and sequenced. Complete sequencing of p2 . 5 and p4 . 5 suggested that the original DNA was a circular 7·0 kbp DNA possessing a single HindIII restriction site. Subsequently, from a shotgun library of HindIII-digested total DNA of an OY-W phytoplasma-enriched fraction, a single 7·0 kbp clone (p7 . 0), which hybridized to both p2 . 5 and p4 . 5, was obtained. Sequence analysis of p7 . 0 was performed as described above.
PCR amplification to detect the circular extrachromosomal DNA.
To detect EcOYW1 from OY-W or OY-M infected plants, each PCR primer set for the 2·5 kbp or the 4·5 kbp DNA fragment of EcOYW1 was used. The primers P1 and P2 were used for the 4·5 kbp DNA fragment amplification. The primers P3 (5'-AAC CCT GAA ATC TCA TTT GA-3') and P4 (5'-AAC TTT AAC CAC AGG TGC A-3') were synthesized and used to amplify the 2·5 kbp DNA fragment.
Southern blot hybridization.
Total DNA extracted from phytoplasma-enriched fractions of OY-W and OY-M infected plants was digested with restriction endonucleases, and Southern hybridization analysis was performed. Transblotting was done using the method described by Sambrook et al. (1989) . Detection was done with a digoxigenin-labelled probe and a DIG DNA labelling and detection kit (Roche Diagnostics) following the manufacturers guidelines. Fragments of 306 bp and 354 bp, corresponding to nt 50335338 (probe A, Fig. 2
) and 65836936 (probe B, Fig. 2
), respectively, of EcOYW1 were used as probes.
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SDS-PAGE and Western blot analysis.
Total protein was extracted from healthy and OY-W infected garland chrysanthemum plants. The phytoplasma-enriched fraction, prepared as described above, was suspended in 1 ml sonication buffer (20 mM Tris/HCl, pH 7·5, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM 2-mercaptoethanol) (Sambrook et al., 1989 ) and sonicated for 5 min, before separation into soluble and insoluble fractions by centrifugation (20000 g, 15 min); the insoluble fraction was suspended in 1 ml sonication buffer. These samples were electrophoresed into SDS-PAGE gels and analysed using Western immunoblotting (Sambrook et al., 1989
).
The fractionated proteins were transferred onto PVDF membranes. These membranes were first reacted with rabbit anti-ORF1 protein IgG, and then with a mouse anti-rabbit Ig conjugated with alkaline phosphatase. Signals were detected using the ECF Western Blotting Reagent Pack (Amersham Pharmacia Biotech) and a Fluoro Imager (Molecular Dynamics). Pre-immune IgG was used as a control. Prestained SDS-PAGE Standards (Bio-Rad) were used as molecular mass markers.
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RESULTS |
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EcOYW1 was not found in OY-M
EcOYW1 was cloned from OY-W DNA. DNA extracted from OY-M (Fig. 1, lane M) suggested the absence of this molecule in OY-M. Therefore, the presence of EcOYW1 was examined by PCR using two primer sets specific for p4 . 5 and p2 . 5. As shown in Fig. 3(a)
, neither a 4·5 kbp nor a 2·5 kbp fragment was amplified from healthy plant DNA or OY-M infected plant DNA extracted from the phytoplasma-enriched fraction. Southern blot hybridization was then performed against total DNA of the phytoplasma-enriched fraction using 306 bp and 354 bp DNA fragments of EcOYW1 (regions A and B in Fig. 2
) as probes that specifically hybridize 2·5 kbp and 4·5 kbp fragments, respectively. As shown in Fig. 3(b)
, when OY-W DNA was used, a 7·0 kbp HindIII fragment was detected with both probes, and 2·5 kbp and 4·5 kbp EcoRV fragments were detected with 2·5 kbp- and 4·5 kbp-fragment-specific probes, respectively. However, when OY-M DNA was used, no signal was detected. These data suggest that EcOYW1 does not exist in OY-M infected tissues.
EcOYW1 encodes a homologue of geminivirus replication protein Rep
We found seven ORFs in the complete nucleotide sequence of EcOYW1 (Fig. 2, Table 1
). A search for similar sequences using BLAST showed that the deduced protein encoded by ORF1 shares a high level of similarity with Rep protein, a multifunctional replication protein found in plant viruses belonging to the family Geminiviridae. An overall similarity between a protein encoded by ORF1 and a Rep protein of Tomato golden mosaic virus (TGMV), a geminivirus, is 66%. Homologous deduced proteins were also reported for the extrachromosomal DNA from SCWL and VAC phytoplasmas (Rekab et al., 1999
; Nakashima & Hayashi, 1997
). Some of these proteins are aligned in Fig. 4
. It was reported that motifs 13 and helices 1 and 2 in the N-terminal domain of the Rep protein of TGMV were very important for replication of geminivirus genomic DNA (Orozco & Hanley-Bowdoin, 1998
). These domains were highly conserved in ORF1 of EcOYW1. Specifically, motifs 1 and 3 showed higher similarities of 80% and 83%, respectively. The Tyr residue in motif 3 (indicated by an arrowhead in Fig. 4
), which is considered necessary for DNA cleavage, was conserved. In addition, the nucleotide-binding site called the P-loop in the C-terminal domain was also highly conserved with 100% similarity. Therefore, we suggest that the protein encoded by ORF1 functions as a replication-initiation protein.
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DISCUSSION |
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Rekab et al. (1999) reported geminivirus-related extrachromosomal DNA from an X-clade phytoplasma. Although they only reported a partial sequence, their ORFB was highly homologous to the C-terminal region of geminivirus replication proteins. Our results imply that both X-clade and AY-clade phytoplasmas (containing OY-W) possess extrachromosomal DNA, which encodes a Rep gene homologue. Furthermore, we confirmed the expression of this Rep homologue protein. This suggests that ORFB of the X-clade-specific extrachromosomal DNA might also be expressed.
It has been suggested that the geminiviruses originated from a prokaryotic plasmid containing the rolling circle DNA replication initiation protein (Koonin & Ilyina, 1992 ). In addition, the Nicotiana tabacum nuclear genome was reported to carry multiple direct repeats of a geminivirus-related DNA containing the Rep gene region of TGMV (Kenton et al., 1995
). These sequences are thought to have arisen during the evolution of Nicotiana, from illegitimate recombination following geminivirus infection. Therefore, it appears that Rep homologous sequences have been widely distributed in viruses, mollicutes and plants during evolution. Further experiments are now in progress using antibodies against Rep to clarify whether an ORF1 protein exists both in phytoplasma-infected plants and in insects.
We previously reported that the copy number of a plasmid pOYW1 from OY-W was 4·2 times greater than that from OY-M (Kuboyama et al., 1998 ). In addition, here we showed that EcOYW1 was not found in OY-M. Thus, our results confirm differences in the extrachromosomal DNA between OY-W and OY-M. However, the relationship between the milder symptoms of OY-M and the absence of the EcOYW1 is still unknown. It could be possible that the EcOYW1 ORFs with no similarity to any known function encode unknown pathogenicity genes. Further investigation of the extrachromosomal DNA should enable the elucidation of the mechanism for these intriguing differences in symptoms, and the pathogenicity of phytoplasmas.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 13 June 2000;
revised 4 October 2000;
accepted 3 November 2000.