Graduate School of Agricultural and Life Sciences, The University of Tokyo, Kashiwa, Chiba 277-8562, Japan1
Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-8562, Japan2
Author for correspondence: Shigetou Namba. Tel: +81 4 7136 3700. Fax: +81 4 7136 3701. e-mail: snamba{at}k.u-tokyo.ac.jp
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ABSTRACT |
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Keywords: phytoplasma, extrachromosomal DNA, rolling circle replication, recombination
Abbreviations: EC-DNA, extrachromosomal DNA; OY, onion yellows; OY-M, mild-symptom line of onion yellows phytoplasma; OY-W, wild-type onion yellows phytoplasma
b The GenBank accession numbers for EcOYW and EcOYM are AB076262 and AB076263, respectively.
a Present address: Graduate School of Agriculture, Ibaraki University, Ami, Inashiki-gun, Ibaraki 300-0393, Japan.
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INTRODUCTION |
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The wild-type line (OY-W) of onion yellows (OY) phytoplasma is the causal agent of onion yellows disease, which produces a wide variety of symptoms in its plant host, including virescence (excessive greening of floral tissue), yellowing, phyllody (leaf-like petals and sepals), stunting, proliferation and witches broom (excessive formation of shoots) (Shiomi et al., 1996 ). OY-W has been maintained in a greenhouse using a leafhopper vector, Macrosteles striifrons, and the host plant garland chrysanthemum, Chrysanthemum coronarium. In our previous work, a mild-symptom line (OY-M) was isolated from OY-W that had been maintained by insect and plant hosts (Oshima et al., 2001b
). Plants infected with OY-M still produce many lateral shoots, but show mild leaf yellowing and almost no stunting. The host range and incubation period of OY-M are not significantly different from those of OY-W.
The genome size of phytoplasmas ranges from 530 to 1350 kb, representing the smallest genome in the class Mollicutes and of all known living organisms (Whitley & Finch, 1990 ; Neimark & Kirkpatrick, 1993
; Carle & Neimark, 1995
; Gibb et al., 1995
; Zreik et al., 1995
; Marcone et al., 1999
). Moreover, this variability in genome size is strikingly large (Pyle et al., 1990
; Ladefoged & Christiansen, 1992
; Marcone et al., 1999
). It has been observed that dynamic variation in the genome size occurs after maintenance of a phytoplasma isolate for a certain period of time (Oshima et al., 2001b
). However, little is known about the dynamics of extrachromosomal DNA (EC-DNA) species of phytoplasmas.
Genes encoded in EC-DNAs, such as plasmids, are known to play important roles in the pathogenicity and virulence of many plant-pathogenic bacteria (Panopoulos & Peet, 1985 ; Vivian et al., 2001
). The isolation and characterization of EC-DNAs in some phytoplasmas have been described (Denes & Sinha, 1991
; Kuske et al., 1991
; Schneider et al., 1992
; Goodwin et al., 1994
; Nakashima & Hayashi, 1997
; Kuboyama et al., 1998
; Oshima et al., 2001a
; H. Nishigawa and others, unpublished). The western aster yellows phytoplasma contains at least four different species of EC-DNA (Kuske & Kirkpatrick, 1990
). However, isolation of the complete set of EC-DNAs from one phytoplasma isolate has not yet been reported. In our previous work, we isolated and characterized two different classes of EC-DNAs from OY phytoplasma. One class of EC-DNA, which is 7 kbp in length and has a rep gene similar to that of the geminiviruses, has been shown to exist only in the OY-W line (EcOYW1) and not in the OY-M line (Nishigawa et al., 2001
). Another class of EC-DNA, which is 4 kbp in size and has a rep gene similar to that of pLS1-like plasmids, has been isolated from the OY-W (pOYW) (Kuboyama et al., 1998
; Oshima et al., 2001a
) and OY-M (pOYM) (H. Nishigawa and others, unpublished) lines. The rep gene of pOYW was found to encode a unique protein, pOY plasmid Rep. The N-terminal region of pOY plasmid Rep is similar to the Rep of the pLS1 family of plasmids, but, unlike the Rep of other plasmids, it has a C-terminal extension unexpectedly similar to the helicase domain of Rep of eukaryotic viruses, especially the circoviruses (single-stranded-DNA viruses of vertebrates). It was suggested that an ancestral phytoplasma plasmid, pOYW, may have acquired a helicase domain from host phytoplasmal DNA, entered the surrounding eukaryotic cytoplasm, and subsequently evolved into an ancestral eukaryotic single-stranded-DNA virus (Oshima et al., 2001a
).
This paper reports the isolation and characterization of another class of EC-DNA species, EcOYW2 and EcOYM, which are approximately 5 kbp in size, from OY-W and OY-M, respectively. Southern blot analysis indicated that no more EC-DNAs exist in either OY-W or OY-M, which means that the complete set of EC-DNAs has been cloned from OY phytoplasma. Interestingly, both EcOYW2 and EcOYM have chimeric structures of two other classes of EC-DNAs: EcOYW1 and pOYW. We postulate that the new class of EC-DNAs originated from intermolecular recombination between different classes of EC-DNAs. To our knowledge, such an event has never been reported for phytoplasmal EC-DNAs. The roles of recombination in the evolution of phytoplasmal EC-DNAs and in increasing the biological diversity of phytoplasma are discussed.
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METHODS |
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Southern hybridization.
Southern blot analysis was performed with an AlkPhos alkaline phosphatase labelling kit (Amersham Pharmacia) and by chemiluminescent detection with CDP-Star (Amersham Pharmacia) according to the manufacturers instructions. DNA fragments containing ORF1 (rep) from EcOYW1 were amplified by primers Rep-N and Rep-C (Nishigawa et al., 2001 ) and used as a probe. A DNA fragment containing the rep gene from pOYW was amplified by primers 105Rep1 (5'-TATTT ATCAA AATGA TAAAG AGGCT C-3') and 105Rep2 (5'-CAACG ACGTT TTAAT TGAGT AATAC-3') and also used as a probe (Oshima et al., 2001a
). Total DNA, extracted from either OY-W- or OY-M-infected plants, was digested with EcoRI. For hybridizations, this digested DNA was electrophoresed on 0·7% agarose gels, transferred to nylon membranes (Roche Diagnostics) and detected by chemiluminescence on X-ray films (Fuji Medical X-ray Film RX-U; Fuji Photo Film).
Cloning of EC-DNA.
EC-DNA species were extracted, by gel purification from OY-W and OY-M lines, and digested with EcoRI. The DNA was ligated into pUC18 cloning vectors and introduced into a host Escherichia coli strain, JM109. The ends of the cloned fragments were amplified by PCR using primers EW1 (5'-CATAA TTAGG TTCTA ATGAT TCAGC-3') and EW2 (5'-TATTC TCATT ACTCC TTTAC TGCC-3') for OY-W, and EM1 (5'-CAATT CTTTA ATATC AGATT CAAAG AT-3') and EM2 (5'-TGCGT ATATG TAGGG TCATT TTTAT-3') for OY-M. Amplification of these fragments was performed in a thermal cycler (Perkin Elmer 9700) for 30 cycles, under the following conditions: denaturation for 15 s at 94 °C, and annealing and extension for 3 min at 60 °C.
Sequence analysis.
The EC-DNA species were sequenced by the dideoxynucleotide chain termination method using a PRISM 377 DNA Sequencer (Applied Biosystems). Sequence analysis was performed with DNASIS version 3.7 (Hitachi Software Engineering). Amino acid sequence similarity analysis by BLAST-P and PSI-BLAST (Altschul et al., 1990 ) was performed with the server of the National Center for Biotechnology Information, USA.
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RESULTS |
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Sequence analysis
Sequence analysis of the cloned full-length EC-DNAs revealed that EcOYW2 was 5560 bp and EcOYM 5025 bp in length. The physical map of these EC-DNAs and the locations of putative ORFs encoding more than 100 amino acids are shown (Fig. 2). Seven ORFs were found in EcOYW2, and six were found in EcOYM. ORFs 1, 2, 3, 4 and 5 of EcOYW2 and EcOYM were similar to each other, suggesting that EcOYW2 and EcOYM share the same ancestry.
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Southern blot analysis
To confirm that the OY phytoplasma has no more EC-DNA species, Southern blot analysis was performed. When a virus-type rep gene was used as a probe (Fig. 1), no DNA bands were detected other than EcOYW1, EcOYW2 and EcOYM. Therefore, we concluded that there were no other virus-like EC-DNAs present in OY-W or OY-M. When a plasmid-like rep gene of pOYW was used as a probe (Fig. 4
), two bands from OY-W and one band from OY-M were detected. We previously reported that pOYW was composed of heterogeneous molecules; a newly cloned full-length clone of pOYW (Oshima et al., 2001a
) contains two EcoRI sites, while a newly cloned almost full-length clone of pOYW (Kuboyama et al., 1998
) did not possess one of the EcoRI sites found in the full-length clone. We presume that the 2·2 kbp band represents the pOYW molecule with two EcoRI sites, and that the 3·9 kbp band represents that with only one EcoRI site. The 3·9 kbp band from OY-M should be pOYM. Therefore, we conclude that there are no other plasmid-type EC-DNAs present in OY-W or OY-M.
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DISCUSSION |
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Most interestingly, sequence analysis strongly suggests that EcOYW2 and EcOYM might be the products of DNA recombination between EcOYW1 and pOYW. We know of no reports of intermolecular recombination among EC-DNAs in phytoplasmas. The rolling-circle-replication plasmids of Bacillus subtilis are notoriously unstable (Ehrlich et al., 1986 ; Ehrlich, 1989
), probably because their replication generates intermediates that are susceptible to rearrangement, and they show high levels of recombination (Niaudet et al., 1984
; Jannière & Ehrlich, 1987
). Since both the geminivirus-like rep gene and the pLS1-plasmid-like rep gene mediate rolling-circle replication, it may be possible that the EC-DNAs of phytoplasmas can also undergo recombination (Saunders & Stanley, 1999
).
Recombination of EC-DNA plays a major evolutionary role by creating genetic diversity, and provides the potential for rapid adaptation to new environmental conditions (Riley, 1993 ). A cluster of genes encoding bactericidal colicins and related proteins of E. coli resides on plasmids, and its diversity results from plasmid mobilization and subsequent recombination within and between the clusters (Riley, 1993
). In addition, a type I restriction/modification system in Lactococcus lactis was reported to acquire novel specificities via the recombination of two plasmids, each having different hsdS genes encoding a specificity-determining subunit. The recombination of two plasmids at the hsdS loci generates a large plasmid with two chimeric hsdS genes with novel specificities (OSullivan et al., 2000
). Recombination also plays a major evolutionary role by creating genetic diversity within prokaryotic and eukaryotic virus populations (Domingo & Holland, 1997
; Saunders & Stanley, 1999
). Interspecific recombination has been well documented in many virus families (Roossinck, 1997
), for example, between RNA viruses (White et al., 1995
), between DNA viruses (Zhou et al., 1997
), and between RNA and DNA viruses (Morse et al., 1992
). Indeed, some instances of recombination between different viral rep genes have been reported (Gibbs & Weiller, 1999
; Saunders & Stanley, 1999
).
The mechanism of EC-DNA recombination in phytoplasmas is not clear. It should be noted, however, that the non-coding region between ORF4 (ssb) and ORF5 (rep) of EcOYW2 has similarity to sequences upstream from ORF1 (rep) of EcOYW1 and to sequences downstream from ssb of pOYW (Fig. 5), indicating that this common region between EcOYW1 and pOYW may be the recombination site for generating EcOYW2. No other regions showed as clear junctions as this region. It should also be noted that the homologous, non-coding region b shown in Fig. 2
is located differently between EcOYW2 and EcOYM. This could suggest that these chimeric EC-DNAs are not a result of a simple single recombination event of EcOYW1 (or related EC DNAs) and pOYW (or related ones), but further deletions, insertions or recombination events should have taken place.
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Another speculation is that in the OY-W line there may have been a considerable number of variant EC-DNAs consisting of EcOYW1, pOYW and recombinant EcOYW2, each of which may have been heterogeneous itself. The phytoplasmal cells of the OY-W line may contain several different combinations of the three replicons. In the serial maintenance of the OY-W line, some may have lost EcOYW1, resulting in an OY-M line that may have contained only ancestral sets of EcOYM and pOYM.
Comparison of the gene organization of EC-DNAs of OY phytoplasma (Fig. 2) revealed that the EC-DNAs differ in OY-W and OY-M. Since genes encoded in EC-DNAs have been reported to play important roles in the pathogenicity and virulence of plant-pathogenic bacteria (Panopoulos & Peet, 1985
; Vivian et al., 2001
), it is possible that the difference in the genes of these EC-DNAs is responsible for the difference in the pathogenicity of the OY lines. This is the first evidence of intermolecular recombination between extrachromosomal DNAs in phytoplasma.
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ACKNOWLEDGEMENTS |
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Received 22 October 2001;
revised 15 January 2002;
accepted 28 January 2002.