Centre for Research in Plant Science, Faculty of Applied Sciences, University of the West of England, Coldharbour Lane, Bristol BS16 1QY, UK1
Department of Biological Sciences, Imperial College, Wye, Ashford, Kent TN25 5AH, UK2
Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK3
Author for correspondence: Dawn L. Arnold. Tel: +44 117 3442526. Fax: +44 117 3442904. e-mail: dawn.arnold{at}uwe.ac.uk
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ABSTRACT |
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Keywords: Pseudomonas syringae, conserved DNA sequences, avr, plasmid, rulAB
Abbreviations: HR, hypersensitive reaction; PAI, pathogenicity island
The GenBank accession numbers for the sequences determined in this work are AJ277495 and AJ277496.
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INTRODUCTION |
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Avirulence genes appear to operate both in determining cultivar specificity among races of a single pathovar in P. syringae and Xanthomonas campestris and also at the pathovar/host plant species level in non-host recognition (Kobayashi et al., 1989 , 1990
; Fillingham et al., 1992
; Innes et al., 1993
; Wood et al., 1994
; Yucel et al., 1994
; Simonich & Innes, 1995
). The recent detection of a gene, virPphA, in P. syringae pv. phaseolicola, shown to be essential for full virulence towards the host plant bean, has provided some insight into the functional significance of avr genes. The same gene functions as an avr gene toward soybean, inducing a rapid cultivar-specific HR (Jackson et al., 1999
). Recently, Tsiamis et al. (2000)
showed that the gene avrPphF has multiple effector functions toward different plant hosts, supporting the notion that avr genes are probably virulence (vir) genes that are recognized by certain plants which do not act as hosts. There is also evidence that pathogenicity is redundantly encoded by vir genes (Yang et al., 1996
; Jackson et al., 1999
) and therefore the detection of potential vir genes is most readily accomplished through screening pathogen gene libraries for avr genes.
The genomic context of avr genes has attracted increasing attention since the earliest indications that genes avrB and avrC from P. syringae pv. glycinea were flanked by repeat DNA sequences (Staskawicz et al., 1987 ). A number of vir/avr genes have been associated with potentially mobile elements, such as insertion sequences, transposons and bacteriophage DNA, which might play a role in their horizontal transfer (Kim et al., 1998
). It is also clear that in some cases the distribution of avr genes is not random and that many are organized on pathogenicity islands (PAIs), which include DNA sequences indicative of gene mobility such as transposases, flanking direct repeats, or insertion sequences (Jackson et al., 1999
; Alfano et al., 2000
; Kjemtrup et al., 2000
).
In P. syringae pv. pisi, an 8·5 kb DNA fragment containing the avirulence gene avrPpiA1 is present in the chromosome of race 2, but is not present in race 4B. Sequence analysis of the 8·5 kb fragment revealed the presence of rulAB genes, which encode tolerance to UV radiation and are widely distributed on plasmids of the pPT23A family in P. syringae pathovars (Sesma et al., 1998 ; Sundin & Murillo, 1999
). They are homologues of the umuDC mutagenic DNA repair system identified in Escherichia coli (Smith & Walker, 1998
). Kim & Sundin (2000)
have recently demonstrated the likely significance of UV-B (290320 nm) for the function of rulAB in the survival of P. syringae in the phyllosphere habitat. In P. syringae pv. pisi race 2, the rulB gene has been disrupted by a 4·5 kb length of DNA, which includes avrPpiA1 and ORFs with similarity to bacteriophage and transposase genes (Arnold et al., 2000
).
In this study we investigated the sequences flanking two avr genes, previously isolated from P. syringae pv. pisi: avrPpiA1 from race 2 strain 203 matches the resistance gene R2 in certain pea cultivars (Vivian et al., 1989 ; Dangl et al., 1992
; Bevan et al., 1995
) and avrPpiB1 from race 3A strain 870A matches the gene R3 (Cournoyer et al., 1995
). Homologues of avrPpiA1 are present in races 5 and 7, whilst homologues of avrPpiB1 are present in races 1 and 7 (Cournoyer et al., 1995
; Gibbon et al., 1997
). The races of P. syringae pv. pisi can be divided into two genomic groups based on PCR amplification with two sets of specific oligonucleotide primers (Arnold et al., 1996
). Although avrPpiA and avrPpiB occur in races from both genomic groups, we show here that there is a high level of DNA sequence conservation in the regions flanking these avr genes and that these sequences are repeated elsewhere in the genome flanking previously undetected avr genes. The presence of conserved sequences therefore provides a route to the potential detection of novel avr/vir genes.
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METHODS |
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DNA manipulations.
DNA manipulations were performed as described by Sambrook et al. (1989) . Total bacterial DNA was extracted from an overnight broth using a Puregene DNA isolation kit (Flowgen). Plasmid DNA was extracted using the alkaline lysis method or using a Qiagen midi-prep kit. Restriction enzymes, obtained from Gibco Life Technologies, were used according to the manufacturers instructions. DNA fragments were ligated using T4 DNA ligase (Appligene) and transformed into E. coli strain DH5
. PCR products were cloned using the Original TA Cloning kit (Invitrogen). DNA sequencing was carried out by MWG-Biotech UK Ltd (Milton Keynes). The sequences obtained were analysed using programs at http://www.ncbi.nlm.nih.gov/ and using the University of Wisconsin GCG package, accessed through the MRC Human Genome Mapping Project, Hinxton, UK.
PCR.
The PCR reactions were as described by Arnold et al. (1996) using 30 cycles with an annealing temperature of 58 °C. PCR products (20 µl) were separated on 1·5% agarose gels (100 V); bands of interest were excised from the gel and the DNA purified using QIAEX II (Qiagen).
Hybridization.
DNA digests and PCR products for hybridization analysis were transferred from agarose gels to nylon membranes by vacuum blotting with a commercial apparatus (Appligene). For dot-blot analysis of DNAs, each bacterial strain to be tested was treated as described by Arnold et al. (1996) . The DNA samples were dot-blotted on a nylon membrane [Biotrans (+) nylon membrane (ICN)] using a commercial apparatus (Bio-Rad). Plasmid inserts and PCR bands, extracted from agarose gels, were labelled with deoxy[
-32P]cytidine 5'-triphosphate using the random primer oligo-labelling method (Feinberg & Vogelstein, 1983
) with a High Prime kit (Roche Diagnostics). Blots were hybridized (65 °C, 16 h) using the labelled probes in the hybridization solution (Sambrook et al., 1989
). Membranes were washed to high stringency as described by Arnold et al. (1996)
.
Production of ORF-specific probes.
Specific DNA fragments were produced for all three ORFs as follows: pAV624 (ORF1) was digested with EcoRI and SmaI to give an 800 bp fragment. PCR was performed using primers DA90/91 and DA94/95 (Table 2) on pAV625 to amplify internal fragments of ORF2 (285 bp) and ORF3 (268 bp), respectively. All probes were excised from gels and purified using QIAEX II (Qiagen).
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RESULTS |
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Predictive analysis indicated that the insert in pAV624 contained a single ORF (1086 bp) encoding a potential protein of 362 aa (ORF1) and that pAV625 contained two ORFs (807 and 654 bp) encoding potential proteins of 269 and 218 aa (ORF2 and ORF3, respectively) with a gap of 658 bp between them (Fig. 1b). All three ORFs appeared to possess a ShineDalgarno ribosome-binding site just upstream of the predicted translation start and an upstream hrp box consensus sequence (Innes et al., 1993
; Table 3
).
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Marker-exchange mutagenesis and phenotypes on plant hosts
A derivative (PG8) of the P. syringae pv. pisi strain 974B a Tn3gus insertion in avrPpiC1, and a derivative (PT10.1) of the race 4B strain PT10 with a Tn3gus insertion in ORF3, were inoculated in the pea differential series (Bevan et al., 1995 ). No difference in symptoms between the original strains and their marker-exchanged mutants was detected. A similar result was observed when the marker-exchanged mutants and parent strains were inoculated in the leaves of four bean cultivars: no difference was detected in the symptoms observed. Repeated attempts to marker-exchange the avrPpiG gene in P. syringae pv. pisi group II races 2, 3A, 4A and 6 were unsuccessful.
Distribution and genomic location of avrPpiG, avrPpiC and ORF3 in P. syringae pv. pisi
ORF-specific DNA fragments for all three genes were used to probe dot-blots of a range of P. syringae isolates. The results (Table 6) indicated that avrPpiG was present only in races 2, 3A, 4A and 6 of P. syringae pv. pisi, whereas avrPpiC and ORF3 were widely distributed among P. syringae and always present together in the same strain.
Digests of total genomic and plasmid DNAs from races of P. syringae pv. pisi were blotted and probed with radiolabelled ORF-specific probes. The results showed that avrPpiG homologues were plasmid-borne in races 2, 3A, 4A and 6 (data not shown). Both avrPpiC and ORF3 were located on the chromosome and present in all races (data not shown). Based on hybridization analysis using undigested plasmids (data not shown), avrPpiG homologues are located on pAV222 (85 kb) in race 2 strain 203, pAV232 (110 kb) in race 3A strain 870A, pAV241 (45 kb) in race 4A strain 895A and pAV398 (110 kb) in race 6 strain 1704B.
Search for novel genes flanked by the conserved sequences
Primers DA72 and DA73 (Table 2) were used in PCR amplifications with a further group of P. syringae pathovars (Table 7
). A 2493 bp band was found in all the strains tested that produced a positive hybridization signal with avrPpiC2- and ORF3-specific probes. A band corresponding to avrPpiA was found in P. syringae pv. maculicola strains 1819A and 65 and in P. syringae pv. apii strain 0988-2. A band corresponding to avrPpiB was found in P. syringae pv. tomato strain DC3000 and P. syringae pv. lachrymans strain ICMP 3988. Two novel bands were amplified: a 973 bp band in P. syringae pv. tabaci strain 11528 and a 1461 bp band in P. syringae. pv. coronafaciens strain ICMP 3113. These two bands were cloned and sequenced. The P. syringae pv. tabaci strain 11528 band did not appear to contain an ORF. The 1461 bp band from P. syringae. pv. coronafaciens strain ICMP 3113 contained a single ORF with very high DNA identity (94·5%) to avrPpiG1. The difference in size between the PCR fragment from P. syringae pv. coronafaciens strain ICMP 3113 and P. syringae pv. pisi strain 895A represents a gap of 144 bp in the P. syringae. pv. coronafaciens sequence between the hrp box and the ATG start codon.
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DISCUSSION |
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A novel gene, avrPpiG, was present only in races of P. syringae pv. pisi belonging to the genomic group II of Arnold et al. (1996) and was also isolated from P. syringae. pv. coronafaciens strain ICMP 3113, but on a smaller PCR fragment. This gene, when present in P. syringae pv. phaseolicola, resulted in a non-host HR in all bean cultivars tested. Database searches revealed that AvrPpiG is similar to two proteins, AvrRxv and AvrBsT, both specified by avr genes from X. campestris pv. vesicatoria, and matching resistance in tomato (Lycopersicon esculentum) (Whalen et al., 1993
; Ciesiolka et al., 1999
). Another similar gene was recently found by Alfano et al. (2000)
in an area they termed an Exchangeable Effector Locus (EEL) from P. syringae pv. syringae (B728a) flanking the hrp/hrc gene cluster. These genes provide direct evidence of a link to virulence determinants in animal pathogens, seen in their similarity to YopP, which is presumed to act as an effector in the induction of apoptosis in macrophages by Yersinia enterocolitica (Mills et al., 1997
). A marker-exchange mutant of avrPpiG could not be obtained because the group II strains of P. syringae pv. pisi, which are the only ones containing this gene, do not readily accept broad-host-range plasmids (Moulton et al., 1993
).
The gene avrPpiC1 was previously identified by screening a genomic library of P. syringae pv. pisi race 5 strain 974B for avr genes functioning in P. syringae pv. phaseolicola race 5 strain 52 (Fillingham, 1994 ; Goss, 1995
). The gene conferred the ability to cause a strong HR on all bean cultivars normally compatible with race 5. Insertional mutagenesis with Tn3gus enabled a marker-exchange mutant of strain 974B to be created, but no change in symptoms was detected on either pea or bean. A similar lack of change of phenotype was seen with a marker-exchange mutant of strain PT10 with an insertion in ORF3. The present study has shown that avrPpiC is linked to a second, putatively hrp-regulated gene, ORF3, which invariably accompanies it in the strains tested. Both genes are flanked by the conserved sequences and although avrPpiC clearly can function as an avr gene alone, both genes may be required for an (as yet undetected) virulence function. The failure to observe any changes in virulence toward pea or avirulence towards bean suggests that both virulence and non-host avirulence are redundantly encoded by these pathogens. The distribution of these genes among pathovars of P. syringae may reflect aspects of their precise function in planta and whether they involve specific roles towards a limited number of host plants or a more general and perhaps fundamental role. While avrPpiG appears to be restricted to a single genomic group (II) within P. syringae pv. pisi, avrPpiC and ORF3 are widely distributed in strains representing pathovars in the genomospecies 1 (pvs aptata and pisi), 2 (pvs glycinea and lachrymans) and 3 (pvs maculicola and tomato) as proposed by Gardan et al. (1999)
.
The flanking region primers in some cases amplified up to three bands per strain, implying duplication of the region bounded by the rulB gene. The finding that avrPpiA, avrPpiB, avrPpiC and avrPpiG are all located between the flanking primers suggests that this region may serve as a hotspot for the integration (and possibly the excision) of avr genes. The relatively wide conservation of the flanking sequence regions across members of genomospecies 1, 2, 3 and 4 imply a role in pathogen evolution over a relatively long period of time or, alternatively, efficient horizontal transfer of these regions. An important question remaining unanswered is the extent to which this genomic region offers a vehicle for rapid evolution of novel pathogen specificities. In summary, the primers described here provide a novel method for detecting potential avr and vir genes and through these may identify novel PAIs in bacterial genomes.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Arnold, D. L., Athey-Pollard, A., Gibbon, M. J., Taylor, J. D. & Vivian, A. (1996). Specific oligonucleotide primers for the identification of Pseudomonas syringae pv. pisi yield one of two possible DNA fragments by PCR amplification: evidence for phylogenetic divergence. Physiol Mol Plant Pathol 49, 233-245.
Arnold, D. L., Jackson, R. W. & Vivian, A. (2000). Evidence for the mobility of an avirulence gene, avrPpiA1, between the chromosome and plasmids of races of Pseudomonas syringae pv. pisi. Mol Plant Pathol 1, 195-199.
Bender, C., Liyanage, H., Palmer, D., Ullrich, M., Young, S. & Mitchell, R. (1993). Characterization of the genes controlling the biosynthesis of the polyketide phytotoxin coronatine including conjugation between coronafacic acid and coronamic acid. Gene 133, 31-38.[Medline]
Bevan, J. R., Taylor, J. D., Crute, I. R., Hunter, P. J. & Vivian, A. (1995). Genetic analysis of resistance in Pisum sativum cultivars to specific races of Pseudomonas syringae pathovar pisi. Plant Pathol 44, 98-108.
Bonas, U., Stall, R. E. & Staskawicz, B. J. (1989). Genetic and structural characterization of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria. Mol Gen Genet 218, 127-136.[Medline]
Ciesiolka, L. D., Hwin, T., Gearlds, J. D. & 11 other authors (1999). Regulation of expression of avirulence gene avrRxv and identification of a family of host interaction factors by sequence analysis of avrBsT. Mol Plant-Microbe Interact 12, 3544.[Medline]
Cournoyer, B., Sharp, J. D., Astuto, A., Gibbon, M. J., Taylor, J. D. & Vivian, A. (1995). Molecular characterization of the Pseudomonas syringae pv. pisi plasmid-borne avirulence gene avrPpiB which matches the R3 resistance locus in pea. Mol Plant-Microbe Interact 8, 700-708.[Medline]
Cuppels, D. A. (1986). Generation and characterization of Tn5 insertion mutations in Pseudomonas syringae pv. tomato. Appl Environ Microbiol 51, 323-327.
Dangl, J. L., Ritter, C., Gibbon, M. J., Mur, L. A. J., Wood, J. R., Goss, S., Mansfield, J., Taylor, J. D. & Vivian, A. (1992). Functional homologs of the Arabidopsis RPM1 disease resistance gene in bean and pea. Plant Cell 4, 1359-1369.
DeLey, J. (1968). DNA base composition and hybridization in the taxonomy of phytopathogenic bacteria. Annu Rev Phytopathol 6, 63-90.
Errington, J. & Vivian, A. (1981). An indigenous system of gene transfer in the plant pathogen Pseudomonas morsprunorum. J Gen Microbiol 124, 439-442.
Feinberg, A. P. & Vogelstein, B. (1983). A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132, 6-13.[Medline]
Figurski, D. H. & Helinski, D. R. (1979). Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function derived in trans. Proc Natl Acad Sci USA 76, 1648-1652.[Abstract]
Fillingham, A. J. (1994). Avirulence genes from Pseudomonas syringae pv. pisi controlling species specificity towards Phaseolus vulgaris L. PhD thesis, Wye College, University of London.
Fillingham, A. J., Wood, J., Bevan, J. R., Crute, I. R., Mansfield, J. W., Taylor, J. D. & Vivian, A. (1992). Avirulence genes from Pseudomonas syringae pathovars phaseolicola and pisi confer specificity towards both host and non-host species. Physiol Mol Plant Pathol 40, 1-15.
Gardan, L., Shafik, H., Belouin, S., Broch, R., Grimont, F. & Grimont, P. A. D. (1999). DNA relatedness among the pathovars of Pseudomonas syringae and description of Pseudomonas tremae sp. nov. and Pseudomonas cannabina sp. nov. (ex Sutic and Dowson 1959). Int J Syst Bacteriol 49, 469-478.[Abstract]
Gibbon, M. J., Jenner, C., Mur, L. A. J., Puri, N., Mansfield, J. W., Taylor, J. D. & Vivian, A. (1997). Avirulence gene avrPpiA from Pseudomonas syringae pv. pisi is not required for full virulence on pea. Physiol Mol Plant Pathol 50, 219-236.
Gilmartin, C. R. (1997).The detection and characterisation of avirulence genes in Pseudomonas syringae pathovars. PhD thesis, University of the West of England, Bristol.
Goss, S. C. (1995). The role of avirulence genes in the interactions between Pseudomonas syringae pathovars and non-host plant species. PhD thesis, Wye College, University of London.
Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557-580.[Medline]
Harper, S., Zewdie, N., Brown, I. R. & Mansfield, J. W. (1987). Histological, physiological and genetical studies of the responses of leaves and pods of Phaseolus vulgaris to three races of Pseudomonas syringae pv. phaseolicola and Pseudomonas syringae pv. coronafaciens. Physiol Mol Plant Pathol 31, 153-172.
Hendson, H., Hildebrand, D. C. & Schroth, M. N. (1992). Relatedness of Pseudomonas syringae pv. tomato, Pseudomonas syringae pv. maculicola and Pseudomonas syringae pv. antirrhini. J Appl Bacteriol 73, 455-464.
Innes, R. W., Bent, A. F., Kunkel, B. N., Bisgrove, S. R. & Staskawicz, B. J. (1993). Molecular analysis of avirulence gene avrRpt2 and identification of a putative regulatory sequence common to all known Pseudomonas syringae avirulence genes. J Bacteriol 175, 4859-4869.[Abstract]
Jackson, R. W., Athanassopoulos, E., Tsiamis, G. & 7 other authors (1999). Identification of a pathogenicity island, which contains genes for virulence and avirulence, on a large native plasmid in the bean pathogen Pseudomonas syringae pathovar phaseolicola. Proc Natl Acad Sci USA 96, 1087510880.
Keen, N. T. (1990). Gene-for-gene complementarity in plant-pathogen interactions. Annu Rev Genet 24, 421-440.
Kim, J. F., Charkowski, A. O., Alfano, J. A., Collmer, A. & Beer, S. V. (1998). Sequences related to transposable elements and bacteriophages flank avirulence genes of Pseudomonas syringae. Mol Plant-Microbe Interact 11, 1247-1252.
Kim, J. J. & Sundin, G. W. (2000). Regulation of the rulAB mutagenic DNA repair operon of Pseudomonas syringae by UV-B (290320 nanometers) radiation and analysis of rulAB-mediated mutability in vitro and in planta. J Bacteriol 182, 6137-6144.
King, E. O., Ward, M. K. & Raney, D. E. (1954). Two simple media for the demonstration of pyocyanin and fluorescein. J Lab Clin Med 44, 301-307.
Kjemtrup, S., Nimchuk, Z. & Dangl, J. L. (2000). Effector proteins of phytopathogenic bacteria: bifunctional signals in virulence and host recognition. Curr Opin Microbiol 3, 73-78.[Medline]
Kobayashi, D. Y., Tamaki, S. J. & Keen, N. T. (1989). Cloned avirulence genes from the tomato pathogen Pseudomonas syringae pv. tomato confer cultivar specificity on soybean. Proc Natl Acad Sci USA 86, 157-161.[Abstract]
Kobayashi, D. Y., Tamaki, S. J. & Keen, N. T. (1990). Molecular characterization of avirulence gene D from Pseudomonas syringae pv. tomato. Mol Plant-Microbe Interact 3, 94-102.[Medline]
Kovach, M. E., Phillips, R. W., Elzer, P. H., Roop, R. M., II & Peterson K. M. (1994). pBBR1MCS: broad-host-range cloning vector. Biotechniques 16, 800802.[Medline]
Kovach, M. E., Elzer, P. H., Hills, D. S., Robertson, G. T., Farris, M. A., Roop, R. M.II & Peterson, K. M. (1995). Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166, 175-176.[Medline]
Mansfield, J., Jenner, C., Hockenhull, R., Bennett, M. A. & Stewart, R. (1994). Characterization of avrPphE, a gene for cultivar-specific avirulence from Pseudomonas syringae pv. phaseolicola which is physically linked to hrpY, a new hrp gene identified in the halo blight bacterium. Mol Plant-Microbe Interact 7, 726-739.[Medline]
Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Mills, S. D., Boland, A., Sory, M., van der Smissen, P., Kerbourch, C., Finlay, B. B. & Cornelis, G. R. (1997). Yersinia enterocolitica induces apoptosis in macrophages by a process requiring functional type III secretion and translocation mechanisms and involving YopP, presumably acting as an effector protein. Proc Natl Acad Sci USA 94, 12638-12643.
Moulton, P. J., Vivian, A., Hunter, P. J. & Taylor, J. D. (1993). Changes in cultivar-specificity toward pea can result from transfer of plasmid RP4 and other incompatibility group P1 replicons to Pseudomonas syringae pv. pisi. J Gen Microbiol 139, 3149-3155.[Medline]
Mudgett, M. B. & Staskawicz, B. J. (1998). Protein signalling via type III secretion pathways in phytopathogenic bacteria. Curr Opin Microbiol 1, 109-114.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sesma, A., Sundin, G. W. & Murillo, J. (1998). Closely related plasmid replicons coexisting in the phytopathogen Pseudomonas syringae show a mosaic organization of the replication region and altered incompatibility behavior. Appl Environ Microbiol 64, 3948-3953.
Simonich, M. T. & Innes, R. W. (1995). A disease resistance gene in Arabidopsis with specificity for the avrPph3 gene of Pseudomonas syringae pv. phaseolicola. Mol Plant-Microbe Interact 8, 637-640.[Medline]
Smith, B. T. & Walker, G. C. (1998). Mutagenesis and more: umuDC and the Escherichia coli SOS response. Genetics 148, 1599-1610.
Staskawicz, B., Dahlbeck, D., Keen, N. & Napoli, C. (1987). Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea. J Bacteriol 169, 5789-5794.[Medline]
Sundin, G. W. & Murillo, J. (1999). Functional analysis of the Pseudomonas syringae rulAB determinant in tolerance to ultraviolet B (290320 nm) radiation and distribution of rulAB among P. syringae pathovars. Environ Microbiol 1, 75-87.[Medline]
Taylor, J. D., Bevan, J. R., Crute, I. R. & Reader, S. L. (1989). Genetic relationship between races of Pseudomonas syringae pv. pisi and cultivars of Pisum sativum. Plant Pathol 38, 364-375.
Taylor, J. D., Teverson, D. M., Allen, D. J. & Pastor-Corrales, M. A. (1996). Identification and origin of races of Pseudomonas syringae pv. phaseolicola from Africa and other bean growing areas. Plant Pathol 45, 469-478.
Tsiamis, G., Mansfield, J. W., Hockenhull, R. & 8 other authors (2000). Cultivar-specific avirulence and virulence functions assigned to avrPphF in Pseudomonas syringae pv. phaseolicola, the cause of bean halo-blight disease. EMBO J 19, 32043214.
Ullrich, M., Völksch, B., Fritsche, W. & Geider, K. (1993). Molecular characterization of field isolates of Pseudomonas syringae pv. glycinea differing in coronatine production. J Gen Microbiol 139, 1927-1937.[Medline]
Vivian, A. & Arnold, D. L. (2000). Bacterial effector genes and their role in host-pathogen interactions. J Plant Pathol 82, 163-178.
Vivian, A. & Gibbon, M. J. (1997). Avirulence genes in plant-pathogenic bacteria: signals or weapons? Microbiology 143, 693-704.
Vivian, A. & Mansfield, J. (1993). A proposal for a uniform genetic nomenclature for avirulence genes in phytopathogenic pseudomonads. Mol Plant-Microbe Interact 6, 9-10.
Vivian, A., Atherton, G. T., Bevan, J. R., Crute, I. R., Mur, L. A. J. & Taylor, J. D. (1989). Isolation and charaterization of cloned DNA conferring specific avirulence in Pseudomonas syringae pathovar pisi to pea (Pisum sativum) cultivars, which possess the resistance allele, R2. Physiol Mol Plant Pathol 34, 335-344.
Whalen, M. C., Wang, J. F., Carland, F. M. & 7 other authors (1993). Avirulence gene avrRxv from Xanthomonas campestris pv. vesicatoria specifies resistance on tomato line Hawaii 7998. Mol Plant-Microbe Interact 6, 616627.[Medline]
Wood, J. R., Vivian, A., Jenner, C., Mansfield, J. W. & Taylor, J. D. (1994). Detection of a gene in pea controlling nonhost resistance to Pseudomonas syringae pv. haseolicola. Mol Plant-Microbe Interact 7, 534-537.
Yang, Y., Yuan, Q. & Gabriel, D. W. (1996). Watersoaking function(s) of XcmH1005 are redundantly encoded by members of the Xanthomonas avr/pth gene family. Mol Plant-Microbe Interact 9, 105-113.
Yeo, C. C., Wong, D. T. S. & Poh, C. L. (1998). IS1491 from Pseudomonas alcaligenes NCIB 9867: characterization and distribution among Pseudomonas species. Plasmid 39, 187-195.[Medline]
Yucel, I., Slaymaker, D., Boyd, C., Murillo, J., Buzzell, R. I. & Keen, N. T. (1994). Avirulence gene avrPphC from Pseudomonas syringae pv. phaseolicola 3121: a plasmid-borne homologue of avrC closely linked to an avrD allele. Mol Plant-Microbe Interact 7, 677-679.[Medline]
Received 27 November 2000;
revised 23 January 2001;
accepted 29 January 2001.