The roles of plasmids in phytopathogenic bacteria: mobile arsenals?

Alan Vivian1, Jesús Murillo1 and Robert W. Jacksona,1

Centre for Research in Plant Science, Faculty of Applied Sciences, UWE-Bristol, Coldharbour Lane, Bristol BS16 1QY, UK1
Laboratorio de Patología Vegetal, Departamento de Producción Agraria, Universidad Pública de Navarra, 31006 Pamplona, Spain2

Author for correspondence: Alan Vivian. Tel: +44 117 344 2470. Fax: +44 117 344 2904. e-mail: alan.vivian{at}uwe.ac.uk

Keywords: virulence, type III secretion, Erwinia, Pseudomonas, Xanthomonas

a Present address: Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK.


   Overview
TOP
Overview
Background
Roles of plasmids
The pPT23A family of...
Concluding remarks
REFERENCES
 
Plasmids are extrachromosomal elements of finite size, usually stably inherited within a bacterial cell line and potentially capable of transfer between strains, species or genera. The most widely used systems of grouping for plasmids rely on incompatibility between closely related replicons. This review surveys our current knowledge of plasmids among members of six Gram-negative genera of phytopathogenic bacteria (Burkholderia, Erwinia, Pantoea, Pseudomonas, Ralstonia, Xanthomonas), but excludes Agrobacterium. A major aspect concerns the growing interest in the role of plasmids in pathogenicity and host specificity and the possible advantages of plasmid-borne locations for the genes involved. A range of plasmid-borne phenotypes, including toxin and hormone production, and resistance to bactericides, are reviewed. The role of mobile elements and their association with pathogenicity islands on plasmids and in the bacterial chromosome provide indicators of possible evolutionary routes to the acquisition of disease-inducing capabilities. The paucity of knowledge concerning almost all aspects of plasmid biology among phytopathogenic bacteria is implicit: we argue the need for more work in this somewhat neglected area, to provide a clearer understanding of the molecular strategies adopted by bacteria that enable them to cause disease and evade host defences among a wide range of important crop plants.


   Background
TOP
Overview
Background
Roles of plasmids
The pPT23A family of...
Concluding remarks
REFERENCES
 
Bacterial groups and scope of this review
It is over 10 years since Coplin (1989) published his comprehensive survey of plasmids in phytopathogenic bacteria. While this review remains an important source for earlier work, it appears timely to revisit this relatively neglected area of microbiology. The development of PCR and the advent of relatively inexpensive DNA sequencing have contributed to a revival in interest about the precise contribution of plasmid-borne genes to the host–pathogen relationship. This review will focus on plasmids from bacteria among six genera (Burkholderia, Erwinia, Pantoea, Pseudomonas, Ralstonia and Xanthomonas) that cause diseases in plants, but excludes Agrobacterium (see review by Kado, 1998 ). These pathogens cause a variety of symptoms in their plant hosts, ranging from soft rots through wilts, necroses and blights to knots and cankers, and some examples are shown in Table 1. Some genera contain species with complex subdivisions based largely on their host specificity, such as Pseudomonas syringae, which comprises some 51 pathovars (Young et al., 1996 ) and the genus Xanthomonas, which comprises 20 groups identified by DNA–DNA hybridization (Vauterin et al., 1995 ). Many recent nomenclatural changes have created a taxonomic minefield for those unfamiliar with this field of bacteriology; for example, the wilt pathogen Ralstonia solanacearum was previously Pseudomonas and Burkholderia, before assuming its current name (Yabuuchi et al., 1995 ).


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Table 1. Symptoms associated with phytopathogenic bacterial groups

 
Plant-pathogenic bacteria were for many years regarded as essentially opportunists, entering plants via wounds and natural openings such as stomata, and multiplying in the intercellular spaces within plant tissues. Resistance in plants was associated with a form of localized, programmed host cell death at the site of infection termed the hypersensitive reaction (HR), which restricted the ability of the pathogen to survive and multiply (Richberg et al., 1998 ). However, recent experimental results have indicated that the relationships between host and pathogen are much more intimate and subtle (Alfano & Collmer, 1997 ; Anderson et al., 1999 ).

This review explains briefly how bacterial plasmids are classified into groups and considers the relevance of these systems for plasmids from plant-pathogenic bacteria. The roles of plasmids found in plant-pathogenic bacteria are considered in terms of the phenotypes they confer on their host cells. These include pathogenicity and host specificity, toxin and hormone production, and resistance to bactericides such as copper and antibiotics, and to UV irradiation. We briefly review mobile elements associated with plasmids and the implications of these for evolving host–pathogen relationships. Attention is devoted to the pPT23A family of plasmids, which constitute the largest and best-studied type of plasmid found among these phytopathogenic bacteria. The review concludes by listing a number of areas where our knowledge concerning plasmids remains strictly limited and in need of renewed scientific support and effort.

Typing systems for plasmids
Plasmids were first discovered in enteric bacteria and from the late 1950s onwards were increasingly recorded in relation to antibiotic resistance (Datta & Hughes, 1983 ; Hughes & Datta, 1983 ). Typing systems were developed for plasmids, based on the observation that replicons sharing common replication functions were unable to stably co-exist, as independent plasmids, in the same cell: in general, when selection for the acquisition of a second plasmid was applied to the culture, the related replicon was either displaced or recombined with the incoming plasmid to form a cointegrate. In this way, systems which assigned plasmids to different incompatibility groups, were developed for Escherichia coli (groups assigned alphabetical letters; Hedges, 1974 ; Bukhari et al., 1977 ) and Pseudomonas aeruginosa (P1–P23; Jacoby, 1977 ). These systems have proved to be functionally robust for medically related groups of bacteria, but there are indications that they may be less appropriate for plant-related bacteria. For example, among Ps. syringae, for which the most evidence is available, the commonest replicon type appears to be the recently described pPT23A family (Gibbon et al., 1999 ), which show similarities to the Es. coli ColE2-type plasmids. Related members of this famiFCly stably co-exist in the same strain and thus require direct identification of the replicon type by sequence analysis or hybridization. Currently, very little is known about the range and types of replicon in phytopathogenic bacteria, including the groups covered by this review.

In the case of Erwinia, since members of this genus are closely related to other enteric bacteria there is the potential to base replicon typing on the current systems for Es. coli (Couturier et al., 1988 ). However, the cryptic plasmid (pCPP60) identified in Erwinia amylovora strain Ea322, while showing strong hybridization to IncFII and IncFIV replicons, and producing pili for F-plasmid-specific phages, did not show incompatibility with any IncF replicons tested (Steinberger et al., 1990 ).

A small cryptic replicon pPS10 from Pseudomonas savastanoi pv. savastanoi (previously known as Ps. syringae pv. savastanoi; Young et al., 1996 ) has been intensively used to study replication using a minimal replicon of 1267 bp, derived from the parent plasmid (Nieto et al., 1990 ), but little use seems to have been made of this in plant-related studies of the original host bacterium.

Plasmids that have been identified from the six plant-related genera are shown in Table 2.


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Table 2. Plasmids found in Gram-negative phytopathogenic bacteria

 

   Roles of plasmids
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Overview
Background
Roles of plasmids
The pPT23A family of...
Concluding remarks
REFERENCES
 
Pathogenicity and host specificity
The genes involved in pathogenicity and host specificity comprise two main groups, those termed avirulence (avr) and virulence (vir) genes (Vivian & Gibbon, 1997 ; Vivian et al., 1997 ; Vivian & Arnold, 2000 ), and those involved with a type III protein secretion system, the ‘harp’ (hrc/hrp) genes (Bogdanove et al., 1996 ; Lee, 1997 ; Galan & Collmer, 1999 ). The latter group of genes, with two notable exceptions, which will be described later, are chromosomally encoded (Lindgren, 1997 ; Nizan et al., 1997 ). It appears that the type III secretion system, also present in many animal pathogens, determines the production of a pilus-like structure, which is thought to deliver certain protein products, including Avr proteins, inside plant cells (Roine et al., 1997 ; Taira et al., 1999 ). In contrast, those avr genes that have been described are evenly divided between plasmid and chromosomal locations (Vivian & Gibbon, 1997 ). Avirulence genes were identified as conferring the ability to induce an HR in plant hosts that carry a matching gene for resistance (R), the so-called gene-for-gene theory, based on the original work of Flor (1971) .

Distinct races, which differ in their abilities to cause disease in cultivated varieties (termed cultivars) of crop plants, have been identified for certain pathovars of Ps. syringae and Xanthomonas campestris. For example, nine races of Ps. savastanoi pv. phaseolicola, the cause of bacterial halo blight of bean (Phaseolus spp.), can be distinguished on the basis of their differential interactions with eight bean cultivars. From the pattern of susceptible and resistant reactions, it has been deduced that six pairs of matching avr and R genes are responsible (Taylor et al., 1996 ). As long as one pair of avr/R genes matches in pathogen and host, an HR results, while in the absence of this matching, disease is the outcome.

A substantial number of genes involved in pathogenicity and host specificity that have been assigned to plasmids in particular strains are shown in Table 3; some of these are also found as homologues in the chromosome of other strains. These include avr genes involved in the determination of gene-for-gene host specificity. Some of them may also be involved, together with hrp and vir genes and genes for extracellular enzyme production, in pathogenicity. Here we will briefly consider some examples, which strengthen the view that plasmids, with their potential for inter-strain mobility, play an important role in relation to the bacterial interaction with plants.


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Table 3. Plasmid-borne effector genes

 
As already mentioned in the Background, phytopathogenic bacteria have a specialized secretion system, involved in delivery of proteins to plant cells, termed the type III system. The cluster of genes involved in this pathway is chromosomally located in Ps. syringae, X. campestris and Er. amylovora: however, in Erwinia herbicola pv. gypsophilae the hrp genes are on a 150 kb plasmid, pPATH (Lichter et al., 1995a ; Nizan et al., 1997 ) and in R. solanacearum these genes are on a megaplasmid, pVir (previously designated pWi; Boucher et al., 1986 ). It is worth noting that there are two phylogenetically distinct sets of hrp genes, one found in R. solanacearum and X. campestris, and the other found in Ps. syringae and Er. amylovora; the plasmid-borne counterparts in R. solanacearum and Er. herbicola pv. gypsophilae are similar to their respective chromosomal homologues.

The type III systems serve to deliver effector proteins into plant cells and these include both avr and vir gene products. Until recently no clearly defined virulence role had been assigned to avr gene products in plant pathogens and the only ‘virulence’ genes identified were for those specifying extracellular enzymes, plant hormones and toxins (see below). Attempts to identify vir genes by mutagenesis of the pathogen, followed by screening for loss of pathogenicity in a compatible host plant, almost invariably resulted in the isolation of hrp mutants (Boucher et al., 1985 ; Niepold et al., 1985 ; Lindgren et al., 1986 ; Malik et al., 1987 ). A number of avr genes were shown to confer varying degrees of fitness in their interaction with the plant, but none showed clear loss of pathogenicity when mutated (Lorang et al., 1994 ; Ritter & Dangl, 1995 ). Some early indications that the failure to obtain single gene mutations affecting pathogenicity alone, as opposed to hrp genes, were provided by Yang et al. (1996) , who showed that, in the cotton (Gossypium spp.) pathogen X. campestris pv. malvacearum, a number of plasmid-borne avr genes together redundantly encoded the ability to cause watersoaking in the host plant.

An early report of plasmid involvement in host-specific pathogenicity genes in Ps. syringae pv. pisi by Mazarei & Kerr (1991) was based on superficial evidence and has not yet been confirmed among the type races. In contrast, there is convincing evidence available from the halo-blight pathogen of bean, Ps. savastanoi pv. phaseolicola, which comprises nine races, eight of which harbour large plasmids of about 150 kb. Curing of one of these plasmids, pAV511, from a race 7 strain resulted in the loss of virulence toward bean cultivars. The cured strains, however, continued to elicit an HR in bean, implying that the chromosomal type III system remained functional and was delivering some signal to the plant that resulted in the elicitation of a defence reaction. Characterization of the region on pAV511 responsible for virulence led to the identification of a low G+C, 30 kb pathogenicity island (PAI), comprising several previously identified avr genes, together with a number of potential vir genes and sequences resembling mobile elements (Jackson et al., 1999 ).

One of the potential vir genes, designated virPphA, when introduced into the cured race 7 strain RW60 was shown to restore virulence to bean, while conferring avirulence to certain soybean (Glycine max) cultivars. This, together with the common regulatory features shown by virPphA and avr genes in Ps. syringae, implies that this gene can be regarded as a virulence gene for bean and that the soybean is capable of recognizing its product. Recently, a gene showing dual avr/vir gene behaviour was isolated from the pPATH plasmid of Er. herbicola pv. gypsophilae (Ezra et al., 2000 ).

A similar duality of function has been shown for a second gene (avrPphF) from the PAI on pAV511: AvrPphF acts as a virulence factor for bean cv. Tendergreen, an avirulence determinant for bean cv. Red Mexican (which carries the matching R1 resistance gene), but enhances the HR seen in the cured race 7 strain on the bean cv. Canadian Wonder. A third gene from pAV511, avrPphC, which controls avirulence toward soybean, blocks the activity of avrPphF in cv. Canadian Wonder, but not that in cv. Red Mexican (Tsiamis et al., 2000 ). Thus, the fortuitous curing of a single large native plasmid in the bean pathogen has provided significant insights into the complexity of interactions between pathogen genes and a range of host plants.

Curing of an approximately 82 kb native plasmid by heat shock (32 °C) from a strain of Ps. syringae pv. eriobotryae, the causal agent of stem canker of loquat (Eriobotrya japonica) led to loss of pathogenicity on the host plant (Kamiunten, 1995 ). A single gene, psvA, was capable of restoring pathogenicity. The gene, with a low % G+C and potential hrpL promoter sequence, showed no similarity to other genes in the databases, except for a small portion of the N terminus of the putative protein product, with similarity to AvrA (Kamiunten, 1999 ). Like virPphA, psvA is flanked by a gene with similarity to transposases and comparison of the DNA flanking psvA shows there to be further similarity in the non-coding regions (J. Murillo, R. W. Jackson & A. Vivian, unpublished). An important question regarding the evolution of host specificity is whether there are mechanisms that facilitate the acquisition and/or exchange of vir genes at common target sites in the bacterial genome; this topic is addressed later in the section on mobility of DNA sequences.

Race change is observed when a plant pathogen extends its host range as a result of the loss or inactivation of an avirulence determinant. Race change due to loss of a plasmid carrying avrPpiB was detected in Ps. syringae pv. pisi (Bavage et al., 1991 ), while a similar plasmid loss from race 1 strains of X. campestris pv. vesicatoria involving avrBs3 resulted in extension of host range (Kousik & Ritchie, 1996 ). A more recent report of race change in Ps. savastanoi pv. phaseolicola found a >40kb region of DNA, containing avrPphB (Jenner et al., 1991 ), that appeared to have inserted and then excised from a tRNA-lysine gene (Jackson et al., 2000 ). Analysis of the left junction revealed homology with plasmid rep and par genes and an unknown plasmid gene from the human periodontal pathogen Actinobacillus actinomycetemcomitans. Kiewitz et al. (2000) simultaneously reported an almost identical phenomenon in clinical isolates of Ps. aeruginosa, involving a chromosomal insertion/excision of a 106 kb plasmid. Further analysis of the Ps. savastanoi pv. phaseolicola deletion should help to elucidate whether the region containing avrPphB also has the potential to exist as an independent plasmid.

Many plant pathogens, particularly the soft-rot pathogen Erwinia carotovora and some pathovars of X. campestris, secrete extracellular enzymes, which are specified by chromosomal genes. However, in the pathogen Burkholderia cepacia, particular strains appear to be adapted to different environments: some (BCP) causing bulb-rot of onion (Allium cepa), others (BCC) opportunistic human infections and others (BCS) colonizing soil. Production of an endopolygalacturonase in a BCP strain was shown to be due to a gene, pehA, located on a 200 kb plasmid. Mobilization of this plasmid to other BCP strains conferred enhanced maceration of onion, while transfer to a BCC strain conferred some slight ability to macerate onion (González et al., 1997 ).

Toxins
Phytopathogenic bacteria produce a variety of toxins that affect the host plant, often causing chlorosis and stunting. The genetic determinants for one of these, coronatine, are generally located on plasmids, as shown in Table 4. However, among a majority of strains of Ps. syringae pv. maculicola examined, the genes were chromosomal (Cuppels & Ainsworth, 1995 ). The toxin is a polyketide, coronafacic acid, coupled by an amide bond to a cyclopropyl amino acid, coronamic acid. Production is thermoregulated, in a manner consistent with its expression during plant infection, which is optimal at 18 °C (Palmer & Bender, 1993 ; Ullrich & Bender, 1994 ).


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Table 4. Plasmid-borne coronatine genes in Ps. syringae and Ps. savastanoi

 
In most strains of Ps. savastanoi pv. glycinea and Ps. syringae pv. tomato, coronatine production is specified by a 30 kb cor gene cluster located on large 90–100 kb plasmids (Bender et al., 1996 ). Interestingly, plasmid-borne cor genes in Ps. syringae were always associated with strains capable of utilizing sorbitol (Cuppels & Ainsworth, 1995 ). Coronatine is a virulence factor, enabling producer strains to form larger lesions and develop higher in planta populations; in this way it appears to confer a selective advantage in the natural habitat of these bacteria (Bender et al., 1987 ; Mittal & Davis, 1995 ). It was recently shown that the coronafacic acid gene cluster (located on plasmid pPT23A) in Ps. syringae pv. tomato PT23 was required for the expression of unknown virulence determinants on the closely related plasmid pPT23B, offering a possible explanation for the maintenance of this gene cluster prior to its incorporation into the coronatine gene cluster (Sesma et al., 2001 ).

Hormones
It is perhaps not surprising that phytopathogenic bacteria have acquired the ability to produce certain plant hormones and examples are listed in Table 5. Indoleacetic acid (IAA) is a plant growth regulator that affects cell proliferation. In bacteria it is synthesized via indoleacetamide and the genes involved are found on the T-DNA of Agrobacterium tumefaciens, the cause of crown gall disease in many dicotyledonous plants. In this case, the bacteria make use of the plant host to manufacture this compound as a nutritional source for the invading bacteria: transfer of the T-DNA to the plant cells permits bacterial gene expression in planta, a mechanism that does not appear to occur with other plant pathogens (Patten & Glick, 1996 ).


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Table 5. Plasmid-borne genes for plant hormone production

 
However, a number of phytopathogenic bacteria cause outgrowths on their plant hosts, termed knots or galls, and here we will consider two examples. The first is that of Ps. savastanoi pv. savastanoi, strains of which infect olive (Olea europea), oleander (Nerium oleander) and ash (Fraxinus excelsior), causing knots and resulting in economic losses for olive in particular (Wilson, 1935 ; Smidt & Kosuge, 1978 ). The strains appear to be essentially host-specific, and while the IAA genes occur on plasmids in oleander strains, they are chromosomally located in ash and most olive strains (Caponero et al., 1995 ). There is no evidence that the genes for IAA production are transferred to the plant and it is probable that the ability to infect these hosts does not reside solely in their ability to produce IAA; however, the basis of their host specificity remains unknown. Olive and oleander strains, but not ash strains, produce a further hormone, cytokinin, specified by the ipt (ptz) gene in Ps. savastanoi pv. savastanoi (MacDonald et al., 1986 ; Powell & Morris, 1986 ).

IAA production is also specified on a large 150 kb plasmid, pPATH, in Er. herbicola pv. gypsophilae, which causes galls on gypsophila (Gypsophila sp.) (Manulis et al., 1991 ; Clark et al., 1993 ). On the same plasmid, the etz gene specifies the production of a second hormone, a cytokinin, which affects the size of galls (Lichter et al., 1995a , b ). Adjacent to the etz gene, an ‘insertion-like’ element, IS1327, could have been involved in the horizontal acquisition of the phytohormone gene cluster (Lichter et al., 1996 ). The pPATH plasmid also harbours a cluster of hrp genes, as described above (Nizan et al., 1997 ).

Recently, Glickmann et al. (1998) investigated the production of IAA among 57 strains representing Ps. syringae pathovars from eight of the nine genomospecies groups identified by Gardan et al. (1999) . Most produced IAA, particularly in the presence of tryptophan. In general, high-level production of IAA, which was limited to a small number of strains, correlated with the presence of homologues of the genes iaaM and iaaH from Ps. savastanoi pv. savastanoi; in pathovars myricae and photiniae these genes were located on plasmids. In contrast, many strains hybridized with a probe for the iaaL gene for IAA-lysine synthase, which was often plasmid-borne. The authors concluded that the majority of strains appeared to produce IAA by an alternative pathway, possibly involving indolepyruvate as an intermediate (Glickmann et al., 1998 ).

Among certain strains of the bean pathogen Ps. savastanoi pv. phaseolicola which infect a tropical weed called kudzu (Pueraria lobata) (Goto & Hyodo, 1987 ), a plasmid, pPSP1, has been shown to carry a gene, efe, for the production of ethylene, a hormone involved in the ripening of fruit (Fukuda et al., 1992 ). The efe gene was also found on plasmids in Ps. syringae pv. cannabina and Ps. savastanoi pv. glycinea (Sato et al., 1997 ; Watanabe et al., 1998 ; Table 5).

The production of hormones is frequently host-specific and the location of such genes may simply reflect their precise mode of acquisition – whether by the stable maintenance of an incoming plasmid or by some transposition or recombination event involving the transient acquisition of a plasmid.

Copper and antibiotics
Bordeaux mixture, primarily a fungicide based on copper plus lime, whose accidental discovery is recounted by Schumann (1991) , has been used to control plant disease in crops for over 100 years. Plasmid-borne resistance to copper has been found in several phytopathogenic bacteria, including X. campestris pv. vesicatoria pathogenic on pepper (Capsicum annuum), in Ps. syringae pv. syringae pathogenic to ornamental fruit trees [mainly pear (Pyrus calleriana)] and Ps. syringae pv. tomato, a pathogen of crucifers and tomato (Lycopersicon esculentum) (Stall et al., 1986 ; Bender & Cooksey, 1986 , 1987 ; Mellano & Cooksey, 1988 ; Kidambi et al., 1995 ). The genes specifying resistance to copper appear to be widely conserved among the two genera (Voloudakis et al., 1993 ) and are generally located on large plasmids, with the exception of a walnut (Juglans regia) pathogen, X. campestris pv. juglandis, in which they are chromosomal (Lee et al., 1994 ). Table 6 lists examples of plasmid-borne resistance to bactericides used in agriculture.


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Table 6. Plasmid-borne bactericide resistance genes

 
Resistance to streptomycin, which has been used since the late 1950s to control disease in fruit orchards, was detected in the pathogen Ps. syringae pv. papulans and a number of other Gram-negative bacteria present in apple (Malus sp.) orchards in the USA (Norelli et al., 1991 ). Common fluorescent epiphytic bacteria, which were not associated with disease, were found to transfer streptomycin resistance efficiently to the pathogen, Ps. syringae pv. papulans, in the laboratory, suggesting that these bacteria may provide a reservoir for streptomycin resistance (Huang & Burr, 1999 ).

Copper resistance is often linked to streptomycin resistance and dual resistance to these bactericides was detected on conjugative plasmids in Ps. syringae pv. syringae, ranging in size from 68 to 220 kb (Sundin & Bender, 1993 ). One such determinant is the Smr transposon, Tn5393, first identified in Er. amylovora (Chiou & Jones, 1993 ) and subsequently identified as the basis of streptomycin resistance seen in Ps. syringae and X. campestris (Sundin & Bender, 1995 ). Insertion sequences, IS1133 in Er. amylovora and IS6100 in X. campestris, increase the expression of the resistance genes, leading to higher resistance among the bacteria (Chiou & Jones, 1993 ; Sundin & Bender, 1995 ).

In a very revealing study, Sundin & Bender (1996) examined the relationships between bacteria and their plasmids among strains of Ps. syringae pv. syringae isolated from nurseries for the propagation of ornamental pear (Pyrus calleriana cv. Aristocrat). They isolated nine strains, each harbouring a pOSU900 replicon [Mukhopadhyay et al., 1990 ; now known to belong to the pPT23A family (Gibbon et al., 1999 )] and determined that six of the plasmids carried only Cur, two carried Cur plus Smr and one carried only Smr. The bacterial genomes were compared by arbitrarily primed PCR (AP-PCR) amplification of total DNA and a similarity matrix based on the resulting banding patterns enabled the generation of a dendrogram reflecting the relatedness of the strains. Using restriction patterns to compare the plasmid replicons from the same strains, a further dendrogram was obtained that showed a remarkable similarity to the strain dendrogram, suggesting that there had been little or no recent plasmid transfer between these strains. This in turn implies that the spread of resistance to copper and streptomycin may owe more to the agency of transposable elements rather than to plasmid transfer per se (Sundin & Bender, 1996 ).

Resistance to UV irradiation
Many Ps. syringae pathogens exist as epiphytes on plant leaf surfaces, without causing visible disease symptoms. However, it is clear that this phase, in many cases, is critical for the spread of the pathogen and for the development of disease under favourable environmental conditions (Hirano & Upper, 2000 ). Recent studies have indicated that for growth under conditions of environmental stress, such as dry leaf surfaces, pathogenic epiphytes have a better growth fitness compared to non-pathogenic epiphytes, although this advantage disappeared under moist conditions (Wilson et al., 1999 ). One aspect of life on surfaces exposed to sunlight is the effect of UV light, which is classified by photobiologists into UV-A (320–400 nm) and UV-B (290–320 nm). Exposure to the longer wavelength UV-A causes indirect damage to DNA via the generation of chemical intermediates such as reactive oxygen species, whereas UV-B causes direct damage through the formation of DNA photoproducts (Eisenstark, 1989 ; Friedberg et al., 1995 ).

Plasmid-borne resistance determinants, rulAB, homologous to DNA repair operons umuDC in Es. coli, have been identified and characterized in Ps. syringae (Sundin et al., 1996 ). These genes confer resistance to UV-B and are widely distributed among pathovars and strains of Ps. syringae: in 49 strains with plasmid-borne rulAB-hybridizing sequences, 48 also hybridized to a pPT23A-specific probe, indicating a high correlation between this replicon type and carriage of these determinants (Sundin & Murillo, 1999 ). However, caution is needed in the interpretation of these data since it is clear from the work presented below (Arnold et al., 2000 , 2001b ) that these genes may no longer be functional. In consequence, the question arises of the precise role of the rulAB genes among Ps. syringae strains, whether in protection against UV or as mobile regions of potential homology for integration of virulence genes.

Mobility of DNA sequences
A number of insertion sequences (IS) found on plasmids in plant pathogens are shown in Table 7. Some were detected because they appear to specifically inactivate certain genes, such as IS51 and IS52, which inactivate the iaaM gene in Ps. savastanoi pv. savastanoi, leading to loss of the ability to induce gall formation (Comai & Kosuge, 1983 ; Yamada et al., 1986 ), and IS476, which inactivates the avirulence gene avrBs1 in pXvCu1 in strains of the pepper pathogen X. campestris pv. vesicatoria (Kearney & Staskawicz, 1990 ). Others may serve as sites of potentially mobile homology, such as IS1327 in pPATH of Er. herbicola pv. gypsophilae and RSII in Ps. savastanoi pv. phaseolicola strain LR700, the latter being involved in integration and excision of the plasmid, pMMC7105 (Szabo & Mills, 1984a , b ).


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Table 7. Plasmid-borne insertion sequence elements in Gram-negative phytopathogenic bacteria

 
IS801 (previously RSI) from Ps. savastanoi pv. phaseolicola strain LR700 was reported to show a predilection for the target sequence TGAAC (Romantschuk et al., 1991 ). This element belongs to the IS91 family (Mendiola et al., 1992 ) and was recently found to be present in an ORF showing features of an avr/vir gene in race 2, but not in race 1 strains of Ps. savastanoi pv. phaseolicola. Attempts to show that the cultivar specificity of these two strains resulted from the insertion in the ORF were unsuccessful, but further experiments might reveal whether this gene functions as an avr gene towards non-host plants or as a vir gene towards bean. Interestingly, transpositions of this element are permanent and the target sequence was considered likely to be TGNAC, where N is any of the four nucleotides (González et al., 1998 ).

An exciting and intriguing discovery of a ‘Russian Doll’ effect was found in the chromosome of the pea-blight pathogen Ps. syringae pv. pisi race 2, where an 8·5 kb fragment was detected, which was absent from a race 4B strain. This 8·5 kb fragment contained genes normally associated with plasmids, such as rulAB. The rulB gene was disrupted by a 4·3 kb fragment, including avrPpiA1 and three genes associated with transposons and phages. Flanking the 4·3 kb fragment were slightly imperfect, 12 bp inverted repeats and the 8·5 kb region was bounded by 7 bp slightly imperfect direct repeats (Arnold et al., 1999 , 2000 ). Homologues of avrPpiA1 and the 4·3 kb region are present on plasmids in Ps. syringae pv. pisi races 5 and 7 (Arnold et al., 2000 ). The DNA sequence clearly provides evidence of repeated insertions and topologically the 4·3 kb region containing avrPpiA appears similar to a transposable element. However, a functionally mobile element has yet to be demonstrated and these may represent relatively ancient events. The recent identification of a chromosomal PAI in Ps. savastanoi pv. phaseolicola race 4 strains provides further evidence of the mobility of genes involved in pathogenicity (Jackson et al., 2000 ).

Other plasmid-borne traits
Apart from those described above, remarkably few other phenotypes have been shown to be plasmid-determined among phytopathogens. There is an interesting report of thiamin prototrophy associated with pEa29 (previously described as a 28 kb plasmid; Chiou & Jones, 1993 ), suggesting that in this case the plasmid plays a role in the physiology or metabolism of Er. amylovora (McGhee & Jones, 2000 ).

The control of extracellular polysaccharide (EPS) production has been associated with plasmid pEa29 in Er. amylovora (Bereswill et al., 1992 ) and with pJTPS1 in R. solanacearum (Negishi et al., 1993 ). However, recent studies in R. solanacearum have shown that high-level transcription of eps requires the products of at least seven regulatory genes that are chromosomally located (Garg et al., 2000 ). In both cases, these traits are associated with full virulence of the pathogen.

Pantoea citrea causes a post-harvest disease of pineapple (Ananas comosus) called pink disease, which produces a pink coloration in canned pineapple due to the production of chromogenic 2,5-diketo gluconate from glucose (Pujol & Kado, 2000 ). Although the genes gdhA and gdhB are chromosomal, plasmid pUCD5000 was required for full expression of the pink colour (Pujol & Kado, 1998 ).


   The pPT23A family of replicons
TOP
Overview
Background
Roles of plasmids
The pPT23A family of...
Concluding remarks
REFERENCES
 
The first example of a pPT23A-like replicon was the cryptic 80 kb plasmid, pOSU900, isolated from Ps. syringae pv. syringae strain J900. Loss of this plasmid from the strain did not affect its pathogenicity toward bean. Two shuttle vectors for use with Ps. syringae and Es. coli were developed, but appear to have been little used (Mukhopadhyay et al., 1990 ). However, the pPT23A family is undoubtedly the best-characterized group of plasmids among phytopathogenic bacteria (Gibbon et al., 1999 ). Replicons of this type have so far only been identified in Ps. savastanoi, Ps. syringae and in a plasmid, pCPP519, from the yellow-pigmented, non-fluorescent epiphytic Pseudomonas sp. strain PyR19 (Huang & Burr, 1999 ). In pathovars of Ps. savastanoi and Ps. syringae, pPT23A-like plasmids often share large regions of homology at the DNA level (Murillo & Keen, 1994 ; Sundin & Bender, 1996 ; Glickmann et al., 1998 ; Sesma et al., 1998 ). This is surprising on two counts. Firstly, replicons sharing extensive regions of homology might be expected to undergo frequent recombination, leading to rearrangements, deletions and integration events, which might well result in significant instability of plasmids. Secondly, it was not clear how apparently related replicons stably co-exist, given the utility of the incompatibility typing systems used elsewhere. However, in general, plasmid profiles among strains of Ps. syringae appear to be remarkably stable (A. Vivian, unpublished observations over some 20 years). Indeed, attempts to obtain cured derivative strains were only successful with the advent of electroporation (Murillo & Keen, 1994 ).

Recent work has, however, provided some insights into the reasons for these apparent contradictions. A number of rep genes from pPT23A-like plasmids has shown them to be related to ColE2-type replicons (Hiraga et al., 1994 ; Gibbon et al., 1999 ), but to differ in being substantially larger, with an additional 100 residues at their C termini (Gibbon et al., 1999 ). Among members of the ColE2-type plasmids, two incompatibility determinants appear to function. The IncA specificity is due to the reliance on an antisense RNA, produced by the gene incA, that regulates the production of Rep protein, which is essential for replication. The RepA protein in ColE2-type plasmids functions in trans to activate replication by binding to a sequence at the replication origin in a plasmid-specific manner and synthesizing a primer RNA for the initiation of DNA synthesis by DNA polymerase. IncB incompatibility is due to competition for the Rep protein among origins of the same binding specificity. Five IncA and four IncB incompatibility specificities were identified and the specificity of interaction of the Rep proteins and origins appeared to be determined by insertion or deletion of single nucleotides and the substitution of several nucleotides at specific sites in the origins, with corresponding changes in the sequences found in the C-terminal regions of the Rep proteins (Hiraga et al., 1994 ).

Thus, plasmids closely related in terms of their DNA sequences have the ready potential to establish compatibility within a bacterial cell. Given this situation, homologous recombination between plasmids may occur with some frequency and might permit the maintenance of a level of heterogeneity in certain genes; however, no evidence for such mechanisms has appeared to date. Nevertheless, even relatively rare recombination events, particularly if these were (even indirectly) plant-inducible, could offer an explanation for the appearance of novel strains with changed host specificity or increased virulence.

To date, the precise mechanism of replication control is unknown for the pPT23A family and putative antisense RNA has not been detected, but it is not uncommon for strains of Ps. syringae to carry up to six plasmids sharing a pPT23A-like rep replicase gene (Sesma et al., 1998 ). It has been suggested that compatibility between closely related replicons could be due to a combination of sequence divergence within rep and ori, which would enable the maintenance of binding specificity and loss or modification of other Inc determinants, such as IncA and IncC (Sesma et al., 1998 ). The presence in one strain of closely related plasmids could indicate an ancestral duplication event and concomitant modification of a pre-existing plasmid.

Subsequently, the phylogenetic relationships, based on an analysis of the repA gene sequences, were examined among 14 pPT23A-like plasmids from 5 Ps. syringae pathovars. Two genes of this type have been completely sequenced and their predicted peptides show relatively high sequence conservation from the start to residue 373, but are poorly conserved from residue 374 to the end of the peptide at residue 437 (Gibbon et al., 1999 ). Consequently, regions at the 5' end (including 100–120 bp of upstream sequence) and 3' ends of the gene were sequenced and a combined phylogenetic tree based on sequence identity revealed four groups, with plasmids from the pathovars syringae and tomato being found in more than one group. Some repA genes had deletions in their upstream regions associated with a 9 bp direct repeat sequence (Sesma et al., 2000 ).

So do these plasmids represent mobile arsenals? Transfer of plasmid pCPP519 has been shown in vitro from a non-pathogenic Pseudomonas sp. (PyR19) to Ps. syringae pv. papulans and a number of similar plasmids were transmissible, both within Ps. syringae pv. papulans strains and to non-fluorescent, yellow Pseudomonas strains; however, failure to detect common R plasmids in the two types of strain suggests that plasmid transfer between these co-habiting epiphytes of apple orchards may rarely occur (Huang & Burr, 1999 ). This view is also supported by the phylogenetic studies mentioned above that provide evidence that chromosomes and plasmids of some Ps. syringae strains have co-evolved (Sundin & Bender, 1996 ). The use of bactericides and the subsequent spread of resistance to copper and streptomycin on a transposon clearly demonstrates the role of such elements in situations of strong genetic selection. Perhaps the plasmids act as ‘sponges’, soaking up useful genetic determinants carried into the strains by the agency of transposons. In this context, it will be interesting to see if more examples can be identified of the integrative disruption of rulB on plasmids: this gene appears to be a potential ‘hot-spot’ for such events involving the exchange of avr/vir genes (Arnold et al., 2001b ).


   Concluding remarks
TOP
Overview
Background
Roles of plasmids
The pPT23A family of...
Concluding remarks
REFERENCES
 
What we know of plasmids in plant-pathogenic bacteria is outlined in this article; what we do not know about plasmids in these groups is much more extensive. We would pose the following questions to address these unknowns. What other types of replicon might be found? What are the functions of the cryptic plasmids, given that their carriage must impose a load on their bacterial hosts? What is the nature of replication control in the pPT23A family and does this have implications for the evolution of the pathogen in relation to its plant host? What are the main agencies of gene mobility and what evolutionary timescales are involved? What factors are driving plasmid evolution among these pathogen groups?

In summary, this whole area provides inquisitive scientists with a range of opportunities to study many aspects of basic biology, such as replicon maintenance, recombination or apparent lack of it between homologous DNA sequences, pathogenicity islands and the significance of their distinctive properties in terms of function, that could have important consequences for the more effective and environmentally friendly control of plant disease.


   REFERENCES
TOP
Overview
Background
Roles of plasmids
The pPT23A family of...
Concluding remarks
REFERENCES
 
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