1 Instituto de Agrobiotecnología y Recursos Naturales, CSIC-UPNA, and Laboratorio de Patología Vegetal, Departamento de Producción Agraria, Universidad Pública de Navarra, 31006 Pamplona, Spain
2 UMR de Pathologie Végétale INRA-INH-Université, Beaucouzé, 49071 France
3 Centre for Research in Plant Science, University of the West of England, Coldharbour Lane, Bristol BS16 1QY, UK
Correspondence
Jesús Murillo
jesus{at}unavarra.es
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ABSTRACT |
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Deceased (d. 16 December 2002); this paper is dedicated to his memory.
The EMBL accession numbers for the sequences reported in this paper are AJ568000 (IS50, 734 bp), AJ568001 (IS50, 295 bp), AJ568002 (ERIC, 1289 bp), AJ550186 (EEL-Pph1), AJ550187 (EEL-Pph2) and AJ550188 (EEL-Pseudomonas syringae pv. glycinea).
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INTRODUCTION |
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P. syringae pv. phaseolicola can readily be distinguished from other pathovars of P. syringae pathogenic to beans, such as pathovars syringae and glycinea, by nutritional characteristics and because only P. syringae pv. phaseolicola isolates produce water-soaked lesions on bean pods (Palleroni, 1984; Völksch & Weingart, 1997
; Marques et al., 2000
). In general, P. syringae pv. phaseolicola appears to be a more or less homogeneous pathovar, although it displays a degree of genetic and phenotypic variation that overlaps with isolates from P. syringae pv. glycinea (Marques et al., 2000
). On the basis of phenotypic characteristics and ERIC-PCR-generated profiles, strains of P. syringae pv. glycinea, P. syringae pv. phaseolicola isolated from bean and P. syringae pv. phaseolicola isolated from kudzu (Pueraria lobata), can be divided into three distinct groups (Völksch & Weingart, 1997
). Additionally, intrapathovar variation in P. syringae pv. phaseolicola can be linked, in some cases, to the host plant species of isolation (Marques et al., 2000
). Isolates that produce natural infections on kudzu vine are distinguished, among other characters, for carrying a plasmid-borne efe gene (Nagahama et al., 1994
) and, similar to isolates from Vigna radiata, by their REP-PCR profile with ERIC primers (Völksch & Weingart, 1997
; Marques et al., 2000
).
Most isolates of P. syringae pv. phaseolicola are reported to be Tox+ and naturally occurring isolates unable to synthesize phaseolotoxin (Tox- isolates), which usually possess the corresponding argK-tox gene cluster region, are rare (Rudolph, 1995; Schaad et al., 1995
). We reported recently, however, that over 60 % of the Spanish field isolates of P. syringae pv. phaseolicola were Tox- and did not produce the expected PCR amplification using a primer pair specific for ORF6 (Rico et al., 2003
), which is essential for phaseolotoxin biosynthesis and is routinely used as a target for the detection of this pathogen (Schaad et al., 1995
). Additionally, Tox- isolates did not show hybridization to an ORF6-specific DNA probe (Rico et al., 2003
), suggesting the absence of part or of the entire argK-tox gene cluster. This raised the possibility that the Spanish Tox- isolates were genetically separable from the more common isolates that synthesize phaseolotoxin. In this study, we analyse the genetic variability within the Spanish P. syringae pv. phaseolicola population, in comparison with P. syringae pv. phaseolicola and P. syringae pv. glycinea isolates from international collections. Collectively, our results allowed the differentiation of two genetic lineages in P. syringae pv. phaseolicola and suggest the separate evolution of their pathogenicity gene complement.
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METHODS |
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PCR analysis.
Genetic variability among P. syringae strains was examined by PCR fingerprinting of repetitive DNA sequences using primers for extragenic repetitive consensus (ERIC), repetitive extragenic palindromic (REP) and the arbitrarily primed PCR (AP-PCR) techniques. For ERIC and REP analyses, primers and reaction conditions were as described by McManus & Jones (1995). AP-PCR was carried out using the universal M13 reverse primer (5'-AGCGGATAACAATTTCACAGG-3') or a single 20 bp oligonucleotide primer (5'-GGTTCCGTTCAGGACGCTAC-3') complementary to the IS50 portion of Tn5, as described by Sundin & Murillo (1999)
. For the amplification of phaseolotoxin biosynthetic genes, we assayed two different primer pairs which are specific for DNA regions separated in the genome that are essential for phaseolotoxin biosynthesis. Primers PHA19 and PHA95 amplify a 480 bp internal fragment from the amidinotransferase gene amtA (Marques et al., 2000
; Hernández-Guzmán & Alvarez-Morales, 2001
) and primers OCTF-03 and OCT-R amplify a 632 bp DNA fragment of the ornithine carbamoyltransferase gene argK (Sawada et al., 2002
), which confers resistance to phaseolotoxin. Amplification of genes included in the pathogenicity island was performed with primers DL-04523 (5'-GTAATCGAGTCGCCGCTCTG-3') and DR-05216 (5'-GAAAGTGAAGCGAACGCAAG-3') for avrD, and primers CL-19541 (5'-GATCGTAAGAACGGGCGATT-3') and CR-20852 (5'-CGTGCATGGTAGCATGTATGAA-3') for avrPphC. The exchangeable effector locus (EEL) region of the hrp pathogenicity island (Alfano et al., 2000
) was amplified using primers avrPphE-FOR (Stevens et al., 1998
) and queA-2 (5'-AATCAGGGAATCGGGGAGTT-3') within the coding regions of the hrpK and queA genes, respectively. A 627 bp fragment from the insertion sequence element IS801 (Romantschuk et al., 1991
) was amplified from P. syringae pv. phaseolicola strain 1449B using primers IS801F (5'-AGTCCTGCCTACACACCTCGA-3') and IS801R1 (5'-GCCTCTTTGTGGAACGACAG-3'). The occurrence of a chromosomal insertion of IS801 in P. syringae pv. phaseolicola was tested by amplification with primers RP-1 and RP-2 (González et al., 1998
). For amplifications, bacterial cell suspensions of isolates grown on KMB were prepared in 500 µl sterile distilled water and subjected to freezethaw lysis. Standard PCR reactions were performed in a final volume of 25 µl containing as template 50 ng total genomic DNA or 5 µl bacterial lysates, using either Taq DNA polymerase (Biotaq; Bioline) or Ready To Go PCR Beads (Amersham Pharmacia Biotech).
General molecular techniques.
Total DNA was extracted using a Puregene DNA isolation kit (Gentra Systems), according to the manufacturer's instructions. Plasmids were isolated by a modified alkaline lysis procedure (Zhou et al., 1990) and intact native plasmids were separated by electrophoresis on 0·6 % agarose gels in 1x TAE as described previously (Murillo et al., 1994
). PCR products were purified using the GFX PCR DNA purification kit (Amersham Pharmacia Biotech). DNA sequencing was performed by MWG-Biotech AG. Nucleotide sequences were aligned using CLUSTALW (Thompson et al., 1997
) and database comparisons were made via the BLASTN, BLASTP and TBLASTX algorithms (Altschul et al., 1997
). Preliminary sequence data from P. syringae pv. tomato DC3000 and pv. syringae B728a genome projects were obtained from The Institute for Genomic Research (http://www.tigr.org) and the DOE Joint Genome Institute (http://www.jgi.doe.gov) websites, respectively.
For Southern blots, chromosomal DNA was routinely digested with appropriate restriction enzymes, and DNA fragments separated by electrophoresis in 1 % agarose gels were transferred to a nylon membrane (Roche Diagnostics). For the preparation of DNA probes, specific DNA fragments were gel-extracted and cloned into the pGEM-T Easy vector (Promega). After restriction digestion, the inserts were separated by electrophoresis, excised from the gels and used as probes. Preparation of labelled probes with digoxigenin, Southern hybridization and detection of the hybridized DNA were carried out with the DIG DNA labelling and detection kit (Roche Diagnostics).
Heteroduplex mobility assay (HMA).
The sequence polymorphism of the internal transcribed spacer (ITS) region between 16S and 23S rRNA genes was analysed using a DNA HMA (Delwart et al., 1993). The ITS region was amplified using primers D21 and D22 (Manceau & Horvais, 1997
) and PCR products were migrated in 5 % polyacrylamide gels (Delwart et al., 1993
).
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RESULTS |
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PCR amplification using primers internal to amtA (Fig. 1a) and argK (not shown) yielded the expected 480 and 632 bp amplification products, respectively, for all the Tox+ isolates tested, as well as for the Tox- isolates Hb-1b and M2/1. Conversely, no strong specific amplicons were observed for any of the Spanish Tox- isolates or for P. syringae pv. glycinea strains PG4180 and 49a/90 (Fig. 1a
). We determined that the published sequence of primer PHA19 (Marques et al., 2000
) showed two mismatches in its 5' end with the sequence of the amtA gene deposited in the databases (accession no. AF186235; Hernández-Guzmán & Alvarez-Morales, 2001
). Although the argK gene was shown to be highly conserved (Sawada et al., 1999
), these results suggest that the observed lack of amplification observed for some of the Tox- isolates might be due to possible sequence variations in their argK-tox gene cluster with respect to the primers used. We therefore examined the conservation of this cluster by DNA hybridization.
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Isolates containing or lacking the argK-tox gene cluster can be differentiated into two groups by REP-PCR
The phaseolotoxin biosynthetic cluster appears to have been acquired by horizontal gene transfer (Sawada et al., 1997, 1999
) and, as a consequence it is possible that the P. syringae pv. phaseolicola isolates containing this DNA and those lacking it might represent distinct genetic lineages. We used PCR fingerprinting of repetitive DNA sequences (REP-PCR) to assess the genetic diversity among the above 21 Pph1 and 24 Pph2 isolates. We also analysed two strains of P. syringae pv. glycinea, because strains of this pathovar also lack the argK-tox gene cluster and are closely related phylogenetically to P. syringae pv. phaseolicola (Gardan et al., 1999
; Marques et al., 2000
; Yamamoto et al., 2000
).
The REP-PCR amplification profiles were similar among all isolates examined (Fig. 2), although strains of P. syringae pv. phaseolicola showed several strong differential bands that allowed their distinction from the P. syringae pv. glycinea isolates. One of these was a 1700 bp band present in the ERIC profile (Fig. 2
). Additionally, strains belonging to Pph1 and Pph2 could be distinguished on the basis of significant differences in their REP-PCR banding profiles (Fig. 2
). Besides several minor differential bands, a strong 734 bp band was present in the IS50 profile of all the Pph1 strains (Fig. 2
), independently of their place of isolation. Hybridization experiments showed that the 45 P. syringae pv. phaseolicola isolates examined contained several fragments with homology to the sequences included in the 734 bp fragment (not shown). However, the pattern of hybridization to the probe showed significant differences between Pph1 and Pph2 isolates (not shown), indicating the existence of more dissimilarities than those revealed by REP-PCR. The analysis of the nucleotide sequence of the 734 bp band, obtained in this work, indicated that it is a mosaic (Table 1
) that probably resulted from a reorganization event. Comparison with the databases showed that parts of this sequence are also repeated and scattered in different positions of the P. syringae pv. tomato DC3000 genome and plasmid pDC3000A (Table 1
).
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Conservation of the exchangeable effector loci
The hrp cluster encodes a type III secretion system that injects specialized proteins, or effectors, into the plant host cell; these effectors appear to be the main host range determinants, promoting pathogenicity or defence reactions of the plant. In P. syringae, the hrp cluster is bordered by two DNA regions containing diverse effector genes (Alfano et al., 2000). One of them, the exchangeable effector locus (EEL), begins 3 nt downstream of the stop codon of the hrp gene hrpK and ends near tRNALeu, queA and tgt sequences, which are highly conserved among different Pseudomonas species. The size and gene sequence of the EEL are highly diverse among different isolates of P. syringae (Charity et al., 2003
; Deng et al., 2003
).
The EEL region from different isolates belonging to both groups of P. syringae pv. phaseolicola and from P. syringae pv. glycinea strains PG4180 and 49a/90 was amplified by PCR using primers located within the coding regions of genes hrpK and queA. Identical 2·4 kb PCR amplification products were observed for all the isolates examined (not shown), suggesting that the EEL region is conserved among Pph1, Pph2 and P. syringae pv. glycinea. The EEL sequence (1083 bp) between gene queA and the effector gene avrPphE, located immediately downstream of hrpK, was determined for one representative isolate each of Pph1 (strain 1449B), Pph2 (strain CYL325) and P. syringae pv. glycinea (strain 49a/90). Pairwise comparison showed from one to a maximum of three nucleotide differences, indicating a high degree of conservation. The analysis of the 1083 bp EEL sequence showed the presence of an ORF homologous (85 % identity) to ORF3 (eelF1) located in the EEL region of P. syringae pv. tomato DC3000 (Alfano et al., 2000; Charity et al., 2003
).
The 150 kb virulence plasmid of Pph1 is not present in Pph2
Strains of P. syringae pv. phaseolicola usually contain a large native plasmid of around 150 kb that, in the race 7 strain 1449B, was shown to carry the PAI (Jackson et al., 1999). We therefore decided to evaluate the conservation and physical location of the PAI between groups Pph1 and Pph2 by examination of the plasmid profiles and by Southern hybridization with probes specific for effector genes avrD and avrPphC, which are located in the leftmost border and in the centre of the PAI, respectively (Yucel et al., 1994
; Jackson et al., 1999
). avrD is widely distributed in P. syringae and restricts infection on certain soybean cultivars by triggering a defence response, as does avrPphC. Additionally, avrPphC also behaves as a virulence gene on bean cultivar Canadian Wonder (Tsiamis et al., 2000
).
The profiles of Pph1 isolates showed diverse native plasmids and all of them contained a large plasmid similar to the 150 kb virulence plasmid present in strain 1449B (Fig. 3a). In contrast, the Pph2 isolates contained one or two native plasmids of 3050 kb, with absence of the typical 150 kb plasmid present in Pph1 (Fig. 3a
). DNA probes specific for genes avrD and avrPphC showed hybridization with the large plasmid present in strain 1449B and in all the other Pph1 isolates (Fig. 3b
), indicating that the physical location of the PAI is conserved in Pph1. Conversely, avrD did not show hybridization with any of the plasmids of the Pph2 isolates (Fig. 3b
), although it hybridized to a 5·6 kb HindIII fragment when digested total genomic DNA was used instead of intact native plasmids (not shown). The avrPphC probe, however, hybridized to a single plasmid of 4050 kb in each Pph2 isolate (Fig. 3b
). These results suggest a different organization of the pathogenicity genes included in the PAI among Pph1 and Pph2 isolates.
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Pph1 and Pph2 can be differentiated by HMA analysis of the ITS sequences
The ITS is a non-coding sequence located between the 16S and 23S rRNA genes that is frequently used for taxonomic studies (Gurtler & Stanisich, 1996). HMA is a PCR-based technique (Delwart et al., 1993
) that facilitates the analysis of even minor sequence differences among the five rDNA operons present in P. syringae and it has been successfully used for the establishment of phylogenetic relationships among P. syringae pathovars and other species of bacteria (Sutra et al., 2001
; Catara et al., 2002
).
Different electrophoretic HMA profiles obtained by direct migration of PCR-amplified ITS, indicated a clear diversity between P. syringae pv. glycinea and P. syringae pv. phaseolicola and allowed the differentiation of groups Pph1 and Pph2 (Fig. 5). The HMA profiles of all the Pph1 isolates presented a unique homoduplex band, indicating that the ITS copies in the different rDNA operons were identical within each strain. Conversely, the Pph2 isolates showed a homoduplex band that co-migrated with that observed for Pph1 isolates, but also showed two supplementary bands with reduced mobility that correspond to heteroduplexes, indicating sequence differences between the ITS copies in the different rDNA operons within each strain. For the three P. syringae pv. glycinea strains analysed, the ITS sequences were identical within each strain and shorter than the ITS sequences of P. syringae pv. phaseolicola (Fig. 5
).
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DISCUSSION |
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The hybridization patterns of genomic DNA to IS801 clearly distinguished Pph1 and Pph2, but the phylogenetic significance of this observation is uncertain because most of the hybridizing bands corresponded to plasmid DNA. However, a chromosomal insertion of IS801 that was present in all Pph1 strains, but absent from Pph2 and strains of other P. syringae pathovars, could be a reliable marker for identification. This is because IS801 belongs to a family of insertion elements that follow a replicative rolling-circle transposition (Mendiola et al., 1994; Richter et al., 1998
), making it likely that IS801 insertions would be permanent. Also, the relatively relaxed target specificity of IS801 (Richter et al., 1998
) makes the independent occurrence of two insertions in the fragment amplified by RP-1 and RP-2 rather improbable, even more so if we take into account the limited occurrence of IS801 chromosomal insertions in P. syringae pv. phaseolicola. In contrast to a previous report (González et al., 1998
), our results show that this IS801 insertion is not race-specific.
Additional evidence for the separation of groups Pph1 and Pph2 is provided by the different HMA patterns of the ITS sequences, indicating the existence of sequence differences among the ITS copies only in the Pph2 genomes. This is significant because sequence differences in the ITS among pathovars of P. syringae are strongly correlated with significant genomic differences (Manceau & Horvais, 1997; Sawada et al., 1997
). By using DNA hybridization, several restriction fragment length polymorphisms have been described among different P. syringae pv. phaseolicola strains (González et al., 2000
), suggesting further variation associated to the rDNA operons of this bacterium. We do not know, however, if there are similar restriction site variations that could distinguish Pph1 and Pph2.
Our results concerning genes involved in pathogenicity also suggest the separate evolution of at least part of the pathogenicity gene complement for Pph1 and Pph2. The differential capacity to synthesize phaseolotoxin, which is a putative virulence factor, is accompanied by differences in the genomic organization of the effector genes avrD and avrPphC. However, the EEL sequences adjacent to the hrp cluster are highly conserved among Pph1, Pph2 and P. syringae pv. glycinea, indicating that the genes responsible for host range have a different genomic location.
Among many other plant-pathogenic bacteria, including several pathovars of P. syringae, only strains of P. syringae pv. phaseolicola and P. syringae pv. actinidiae, as well as a single isolate of P. syringae pv. syringae, were found to produce phaseolotoxin and contain DNA specific for this biosynthetic gene cluster (Tourte & Manceau, 1995; Sawada et al., 1997
; Tamura et al., 2002
). The complete conservation of the argK coding sequence, as compared to the phylogeny of the chromosomal genes gyrB and rpoD, and the pathogenicity-related genes hrpL and hrpS, suggests that the argK-tox gene cluster was horizontally transferred after the divergence of the ancestor of P. syringae into the modern pathovars (Sawada et al., 1999
). In support of this, we showed that the internal organization of the argK-tox gene cluster was highly conserved among diverse Pph1 strains. Moreover, all the Pph2 isolates appear to lack the entire argK-tox gene cluster, because they failed to hybridize to two specific probes that correspond to well separated genes (amtA and argK) within this cluster. Therefore, it seems likely that the capacity to infect beans was acquired by P. syringae pv. phaseolicola earlier than the capacity to synthesize phaseolotoxin. The role of this toxin in pathogenicity is not clear, although there is some evidence that it might increase the virulence of the infection (de la Fuente-Martínez et al., 1992
) or allow it to become systemic on bean plants (Patil et al., 1974
). However, the production of phaseolotoxin is considered a defining characteristic of P. syringae pv. phaseolicola and strains unable to synthesize it are only rarely reported (Rudolph, 1995
; Schaad et al., 1995
; Völksch & Weingart, 1998
; Marques et al., 2000
), suggesting that the production of phaseolotoxin, or the activity of other gene(s) that might have been co-transferred with the argK-tox gene cluster, could confer an important selective advantage.
The PAI in P. syringae pv. phaseolicola strain 1449B spans around 30 kb of contiguous DNA located in the 150 kb native plasmid and includes several effector genes, some of which are involved in pathogenicity and virulence (Jackson et al., 1999; Tsiamis et al., 2000
). Other pathovars of P. syringae contain homologues of one or more of the genes included in this PAI, although the PAI itself is not conserved among them and the number of genes and their physical location (plasmid versus chromosome) is highly variable (Jackson et al., 2002
). However, the PAI would appear to be conserved among the Pph1 group of strains since all of them contained a large plasmid that hybridized to both avrD- and avrPphC-specific probes. By contrast, in all the Pph2 isolates the DNA homologous to avrD was located in the chromosome while a plasmid smaller than 50 kb contained the avrPphC homologue.
It was suggested that kudzu strains could represent a different group because they can also be differentiated from other P. syringae pv. phaseolicola strains by their REP- and ERIC-PCR fingerprints, esterase zymotypes, O-serogroup, capacity to utilize mannitol, ethylene production and infection of kudzu plants (Goto & Hyodo, 1987; Völksch & Weingart, 1997
; Marques et al., 2000
). In our opinion, it seems more likely that the kudzu strains could represent a subdivision of the Pph1 group, because they also harbour the argK-tox gene cluster and it is unlikely that this group of genes has been independently acquired by P. syringae pv. phaseolicola twice during evolution. In addition, strains isolated from Vigna spp., which also possess the argK-tox gene cluster, can also be differentiated by their ERIC-PCR pattern and their O-serogroup (Marques et al., 2000
). Nevertheless, given the existence of several independent characters that separate the currently delineated Pph1 and Pph2, there is a likelihood that other possible genomic groups may exist within P. syringae pv. phaseolicola, showing characteristics intermediate between the different groups. We have clearly demonstrated the existence of two P. syringae pv. phaseolicola genetic lineages and provided a basis for a clearer understanding of the mechanisms behind the acquisition of virulence genes and their clustering in pathogenicity islands.
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ACKNOWLEDGEMENTS |
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Received 10 July 2003;
revised 30 October 2003;
accepted 31 October 2003.
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