Instituto de Agrobiotecnología y Recursos Naturales, CSIC-UPNA, and Laboratorio de Patología Vegetal, Departamento de Producción Agraria, Universidad Pública de Navarra, 31006 Pamplona, Spain1
Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843-2132, USA2
Author for correspondence: Jesús Murillo. Tel: +34 948 169133. Fax: +34 948 169732. e-mail: jesus{at}unavarra.es
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ABSTRACT |
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Keywords: pathogenicity and virulence plasmids, avirulence gene avrD, repeated DNA, rulAB, ultraviolet light resistance genes
Abbreviations: AP-PCR, arbitrarily primed PCR; ERIC, extragenic repetitive consensus
The EMBL accession numbers for the sequences reported in this paper are AJ276998AJ277021.
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INTRODUCTION |
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Little is known about the specific genetic determinants that affect host range in P. syringae or other necro genic bacteria, although it is becoming clear that host specificity is defined by the coordinated action of positive factors and the antagonistic action of negative factors. The negative factors, or avirulence (avr) genes, are the best known and act by restricting the plant cultivars or species that can be infected by a given isolate (Vivian et al., 1997 ). The positive factors would include determinants that govern the ability to produce disease or increase virulence in a given plant host. In P. syringae, phytotoxins and growth regulators generally increase the aggressiveness of the bacterium towards a plant host. However, there is growing evidence to suggest that avr genes could also be the main positive factors determining virulence and/or pathogenicity (Kearney & Staskawicz, 1990
; Swarup et al., 1991
; Lorang et al., 1994
; Ritter & Dangl, 1995
; Yang et al., 1996
; Jackson et al., 1999
). For example, the ability of P. syringae pv. phaseolicola 1449B to infect beans and soybean is conferred by a plasmid-encoded pathogenicity island of about 30 kb which contains three avr genes and four other ORFs with the characteristics of avr genes (Jackson et al., 1999
). One of the ORFs, designated virPphA, confers the ability to infect beans and to cause a hypersensitive response in certain soybean cultivars (Jackson et al., 1999
). Avirulence gene avrPphF, also included within this pathogenicity island, determines the capacity to infect soybean and causes a specific gene-for-gene hypersensitive response on the bean cultivar Red Mexican (Tsiamis et al., 2000
).
Many of the determinants involved in virulence and pathogenicity of P. syringae, including several that clearly influence host range, are encoded on native plasmids. Outstanding examples are the genes involved in the biosynthesis of the phytotoxin coronatine (Sato, 1988 ; Bender et al., 1999
), auxins (Comai & Kosuge, 1980
; Glickmann et al., 1998
) and avr genes (Vivian et al., 1997
). In some cases, related genes or gene clusters are conserved in unrelated pathovars; examples of this include the coronatine biosynthetic cluster which is present in five pathovars (Mitchell, 1982
; Wiebe & Campbell, 1993
; Cuppels & Ainsworth, 1995
) and avrD sequences which have been detected within a wide pathovar range (Yucel et al., 1994
). However, the relationships among different native plasmids from P. syringae have not been studied extensively and authors have reported both similarity and dissimilarity among plasmids from a given pathovar (Curiale & Mills, 1983
; Denny, 1988
; King, 1989
; Sundin et al., 1994
). The possibility of plasmid transfer within natural populations via conjugation must also be considered in terms of the introduction of novel genes affecting host interactions into unrelated P. syringae pathovar strains.
The majority of the native plasmids identified in P. syringae belong to the recently described pPT23A-like family; these plasmids share replication sequences and, in most cases, additional areas of homology (Murillo & Keen, 1994 ; Sundin & Bender, 1996
; Glickmann et al., 1998
; Sesma et al., 1998
; Gibbon et al., 1999
). Furthermore, many P. syringae strains contain two to six coexisting pPT23A-like plasmids (Murillo & Keen, 1994
; Sesma et al., 1998
), suggesting their potential role in the bacterial life cycle and underlying their capacity to overcome incompatibility. In the best characterized case, that of pPT23A and pPT23B, both from P. syringae pv. tomato PT23, approximately 74% of these plasmids consist of repeated sequences (Murillo & Keen, 1994
). We are interested in the evolution of the pPT23A-like plasmid family, in particular the molecular evolution of the plasmids and of the sequences they encode with emphasis on the host-selective forces resulting in the compartmentalization of specific plasmid genes within a limited pathovar range. The replication regions from two pPT23A-like plasmids contain a determinant, repA, that is highly homologous to the major replication gene of ColE2 replicons, a plasmid group widespread in Escherichia coli and found in other members of the
-Proteobacteria, and to a plasmid from Thiobacillus intermedius (Gibbon et al., 1999
). In this study, we report an analysis of repA sequences as a strategy to examine the phylogeny of native P. syringae plasmids; the repA determinant is essential for replication of the pPT23A-like plasmids and thus is an appropriate locus for comparative study.
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METHODS |
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Fragments to be used as probes were either amplified by PCR or separately cloned in pBluescript (Stratagene) or pK184 (Jobling & Holmes, 1990 ); in this case, they were excised from the vector and separated in low-melting-point agarose before labelling. Labelling of DNA with digoxigenin and hybridization of uncut plasmid DNA separated by electrophoresis and transferred to nylon membranes (Hybond N+; Amersham) were performed following the manufacturers instructions (Boehringer Mannheim).
Cloning of origins of replication.
The possession of pPT23A-like plasmids among the strains included in this study either was already reported (Murillo & Keen, 1994 ; Sundin & Bender, 1996
; Sesma et al., 1998
; Sundin & Murillo, 1999
) or was examined by hybridization using the 0·8 kb EcoRI fragment from the replication region of pPT23A (Gibbon et al., 1999
) as probe. repA genes were amplified using primers 532 (5'-GAACGGTGGACTTATGG-3') and 1588 (5'-CTCCAGCTTGCGGCCCC-3') which flank a fragment of 1399 bp containing 1279 bp of the repA coding region plus 120 bp upstream of the putative start codon (Gibbon et al., 1999
). Total plasmid DNA was used as template for strains containing a single native plasmid and for P. syringae pv. tomato strain OK-1. The origins of replication from pPT23A, pR6C and pAV505 (see Table 1
for details) were cloned in plasmids pAKC (Murillo & Keen, 1994
), pORI601 (Sesma et al., 1998
) and pPPY50 (Gibbon et al., 1999
), respectively, which were also used as templates for amplification. Amplifications were carried out in a total volume of 25 µl using 1 µl DNA as template under the following conditions: 4 mM MgCl2, 0·75 mM each dNTPs, 1 pmol each primer µl-1 and 1·0 U Taq polymerase. PCR was performed in a Perkin Elmer 480 thermocycler with one cycle of 94 °C for 5 min, 55 °C for 5 min and 72 °C for 90 s, followed by 32 cycles at 94 °C for 1 min, 55 °C for 1 min, 72 °C for 90 s and a final extension step of 10 min at 72 °C. Ten microlitres of the PCR mix was loaded on a 1% agarose gel and bands of the correct size were purified using a gel extraction kit (Qiaquick; Qiagen) and ligated to vector pCR2.1 (Invitrogen). The repA gene from pAV505 was not cloned because its complete sequence was already available (accession no. AJ222648; Gibbon et al., 1999
). The amplicon cloned from total plasmid DNA from strain OK-1 was confirmed to be derived from plasmid pOK1B, by comparison of the restriction profile of repA amplicons from purified DNA of each of the two native plasmids of OK-1 with that of the cloned amplicon. For restriction analysis, DNA was amplified with primers 532 and 1588 as above and PCR products digested with HaeIII or Sau3AI were separated on 2·5% high-resolution agarose (MS8; Hispanlab) gels. As templates, we used purified plasmid DNA for the cloned amplicons and for P. syringae strains containing a single native plasmid. For strains containing two or more pPT23A-like plasmids, these were separated by electrophoresis as indicated above and template DNA was purified from appropriate bands excised from the gels using 0·2 µm Nanosep MF columns (Pall Filtron) as described previously (Sesma et al., 1998
).
Analysis of nucleotide sequences.
Plasmid DNA was purified using Qiagen columns. Nucleotide sequencing was done using the Big Dye kit (ABI) following the instructions of the manufacturer; sequence reactions were run at the Genetic Technologies Center, Texas A&M University. Both ends of the cloned amplicons were sequenced using universal and custom-synthesized primers based on the sequence of the minimal replication regions of plasmids pPT23A and pAV505 from P. syringae (Gibbon et al., 1999 ). Sequence comparison, alignment and construction of phylogenetic trees using the neighbour-joining method was done using the programs CLUSTAL W and MULTALIN at EBI, Cambridge, UK, or IBCP, Lyon, France. The strength of the tree topology was assessed by the bootstrap method using the CLUSTALX software package (Thompson et al., 1997
). Phylogenetic trees were viewed using the NJPLOT software (Saitou & Nei, 1987
).
ERIC, arbitrarily primed PCR and data analysis.
The genetic relationships among P. syringae strains was examined by PCR using primers for extragenic repetitive consensus (ERIC) and the arbitrarily primed PCR (AP-PCR) techniques. For ERIC analysis, primers ERIC1R and ERIC2 were used for amplification as described by McManus & Jones (1995) . AP-PCR was carried out with a single 20 bp oligonucleotide primer complementary to the IS50 portion of Tn5 as described previously (Sundin & Murillo, 1999
).
Amplification bands or bands resulting from digestion of amplification products from the origins of replication were scored as 1 (present) or 0 (absent), and a similarity matrix was computed using Dices coefficient with the program NTSYS-PC (Applied Biostatistics) as described previously (Sundin & Murillo, 1999 ).
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RESULTS |
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The minimal fragment able to support the replication of pPT23A was defined as an approximately 1·6 kb fragment that spans gene repA (Fig. 1) (Gibbon et al., 1999
), which encodes a 437 aa putative replicase that is essential for plasmid replication. A 1399 bp fragment containing 1279 bp of the 1311 bp repA coding region plus 120 bp upstream of the putative start codon (Fig. 1
; Gibbon et al., 1999
) was amplified from the pPT23A-like plasmids and separately cloned in vector pCR2.1 which, in our hands, did not replicate in P. syringae. All the cloned amplicons, except those originating from p5D425A and pB76A, were able to replicate in the plasmidless strain P. syringae pv. syringae FF5 (data not shown), suggesting that they were functional in their parental plasmids. Since in all cases the repA gene was in the opposite orientation with respect to the vector lac promoter, as determined by nucleotide sequencing (data not shown), this suggests that the 120 bp preceding the putative start codon could be sufficient to initiate transcription and for supporting autonomous replication in P. syringae.
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The DNA fingerprint of the strains belonging to pv. syringae and pv. tomato was determined by using ERIC-PCR and AP-PCR. The position of the strains in the resulting dendrograms and their genetic relatedness (Fig. 6) agreed in general with results obtained by other authors using combinations of these strains (Hendson et al., 1992
; Legard et al., 1993
; Sundin & Murillo, 1999
). In many cases, the relatedness between two strains did not reflect the relatedness between their native plasmids (Figs 5
and 6
). For instance, strains 8B48 and B76 showed a low relative genetic similarity, although the plasmids they harboured, pPSR11 and pB76A, respectively, were 98% identical, which is the highest identity value among the native plasmids if we exclude pB8617A and pBBS325A (100% identical).
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DISCUSSION |
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Knowledge of individual plasmid genotypes may provide important clues in the evolution of the pPT23A-like plasmid family. A determinant such as rulAB may have been acquired early in the evolution of this plasmid family and may have been fixed within the plasmid genome because of its importance to phyllosphere fitness, a trait which is universally important to the success of P. syringae strains (Beattie & Lindow, 1995 ). The acquisition of loci such as avr genes might have stimulated the emergence of new pathovars and, concomitant with that, the opportunity for continued plasmid evolution within the context of a new hostpathogen interaction. Further analysis will determine if certain plasmid lineages are defined by the presence of additional determinants of importance to hostpathogen dynamics. Likewise, the limited distribution of determinants of importance to plasmid maintenance (IncC and stbCBAD) could be illustrative of the requirement of such features only in certain host backgrounds, or may reflect a possibly large diversity of maintenance systems among pPT23A-like plasmids. The distribution of IS elements such as IS801 and IS1240 may also be interesting in terms of the timing of evolutionary acquisition. IS801-like sequences, and those of other transposable elements, have been found to flank avr sequences and sequence regions suspected to be pathogenicity islands in several P. syringae pathovars (Kim et al., 1998
; Jackson et al., 1999
). These observations imply that the mobility of these types of sequences among strains is important, possibly from the standpoint of a pathogen adding to its host range or combating a new plant resistance gene. It should be noted that IS801 and avr genes were not detected in pv. syringae in this study or a previous study (Sundin & Bender, 1996
), indicating that the mobility of these sequences may be limited by factors which are currently unknown.
We utilized the repA gene as our starting point for large-scale phylogenetic analysis of the pPT23A-like plasmid family. repA was appropriate because of its requirement for plasmid replication in P. syringae and because it is the only gene currently known to be distributed among all pPT23A-like plasmids. Dendrograms generated from analyses of restriction digest patterns or 5' and 3' sequence data of repA lacked congruence. This could indicate that the restriction digest data were not substantial enough to resolve relationships among the plasmids, or that additional diversity was present within portions of repA that were not sequenced. Nevertheless, similar sequencing strategies have been utilized to examine the diversity present among replication genes of closely related plasmids (Burgos et al., 1996 ; Turner et al., 1996
) and imply that the relationships generated from our analysis of the 5' and 3' sequence data (Fig. 5
) would be the most valid. These results indicate that the repA sequences from plasmids isolated from different P. syringae pathovars were not always clearly distinguished. From these observations, we can infer that ecological factors such as plasmid transfer or similar selection pressures faced by different pathovars could result in the current distribution of repA sequences among P. syringae pathovars. There have been several examinations of plasmid transfer in planta (Bender & Cooksey, 1986
; Burr et al., 1988
; Sundin et al., 1989
; Björklöf et al., 1995
). However, although these studies indicate that plasmid transfer can occur, they do not provide evidence for the natural occurrence of plasmid transfer or if plasmids are mobile among more distantly related strains. In one retrospective analysis of plasmid and host genotypes examining a population of P. syringae pv. syringae under bactericide (copper and streptomycin) selection pressure, recent plasmid transfer events were inferred, but limited to closely related host strains (Sundin et al., 1994
). The issue of transfer of pPT23A-like plasmids into P. syringae strains that already contain an indigenous pPT23A-like plasmid(s) must also be addressed. In one study utilizing the P. syringae pv. syringae strains 4A39 and FF3, each of which contained a single pPT23A-like plasmid, conjugation experiments were done resulting in the transfer of the copper resistance (CuR) plasmid pPSR4 into strain FF3 which contained the streptomycin resistance (SmR) transposon Tn5393 on plasmid pPSR5 (Sundin & Bender, 1996
). Further experiments showed that, in the absence of selection, the plasmids were incompatible; however, selection for the CuR and SmR markers enabled the FF3 strain to harbour both pPSR4 and pPSR5 for 32±8 generations, after which pPSR5 was lost, but Tn5393 transposed into pPSR4 (Sundin & Bender, 1996
). This experiment showed that surface exclusion or restriction modification systems did not preclude the transfer of a pPT23A-like plasmid into a strain harbouring an incompatible plasmid, at least for these P. syringae pv. syringae hosts. Plasmid incompatibility may prevent the establishment of a plasmid in a new P. syringae background; however, the extensive homology present on pPT23A-like plasmids may contribute to genetic rearrangements resulting in the acquisition of new sequences by these plasmids. Whether such barriers to plasmid transfer exist in other P. syringae pathovars is currently unknown. The results of other studies of Agrobacterium and Rhizobium spp. have also indicated the long-term stability of plasmid and host genotypes (Young & Wexler, 1988
; Otten et al., 1996
; Wernegreen et al., 1997
). Our results suggest that most of the PT23A-like plasmids studied have been maintained for long periods within their respective hosts. Our current analysis of strains HS191, 7B12 and B120, however, does not preclude the possible occurrence of plasmid transfer in nature.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 23 March 2000;
revised 30 June 2000;
accepted 11 July 2000.