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ABSTRACT: The 154-kb plasmid was cured from race 7 strain 1449B of the phytopathogen Pseudomonas syringae pv. phaseolicola (Pph). Cured strains lost virulence toward bean, causing the hypersensitive reaction in previously susceptible cultivars. Restoration of virulence was achieved by complementation with cosmid clones spanning a 30-kb region of the plasmid that contained previously identified avirulence (avr) genes avrD, avrPphC, and avrPphF. Single transposon insertions at multiple sites (including one located in avrPphF) abolished restoration of virulence by genomic clones. Sequencing 11 kb of the complementing region identified three potential virulence (vir) genes that were predicted to encode hydrophilic proteins and shared the hrp-box promoter motif indicating regulation by HrpL. One gene achieved partial restoration of virulence when cloned on its own and therefore was designated virPphA as the first (A) gene from Pph to be identified for virulence function. In soybean, virPphA acted as an avr gene controlling expression of a rapid cultivar-specific hypersensitive reaction. Sequencing also revealed the presence of homologs of the insertion sequence IS100 from Yersinia and transposase Tn501 from P. aeruginosa. The proximity of several avr and vir genes together with mobile elements, as well as G+C content significantly lower than that expected for P. syringae, indicates that we have located a plasmid-borne pathogenicity island equivalent to those found in mammalian pathogens.
Proceedings of the National Academy of Sciences 10/1999; 96(19):10875-80. · 9.68 Impact Factor
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ABSTRACT: Many strains of the phytopathogen Pseudomonas syringae contain mutually compatible plasmids that share extensive regions of sequence homology and essential replication determinants. The replication regions of two compatible large plasmids involved in virulence or pathogenicity, pPT23A from P. syringae pv. tomato strain PT23 and pAV505 from P. syringae pv. phaseolicola strain HRI1302A, were isolated. DNA sequencing of the origins of replication revealed homologous ORFs, designated ORF-Pto and ORF-Pph, respectively. Both ORFs are 1311 bp long and encode peptides of 437 amino acids with predicted molecular masses of 48259 (Pto) and 48334 (Pph) Da. Expression of the two ORFs in Escherichia coli produced peptides of 50 kDa (Pto) and 56 kDa (Pph). The predicted peptides showed an overall identity of 897 %, being highly conserved from residues 1 to 373, but showing considerable variation in their C-terminal regions (50% identity over the last 64 aa). The two ORFs had significant similarity with the putative replication protein from plasmid pTiK12 of Thiobacillus intermedius and other CoIE2-related plasmids. However, both peptides were 100 residues longer than any of the known CoIE2-related rep sequences. Subcloning of fragments from the replication region of pPT23A revealed the presence of at least three incompatibility determinants, designated IncA, IncB and IncC. Partial sequencing of the region downstream of ORF-Pto revealed homology to the ru/AB genes, involved in UV resistance, from plasmid pPSR1. It is proposed that the replication origin of pPT23A serves as the prototype of a family of related plasmids.
Microbiology 03/1999; 145 ( Pt 2):325-34. · 3.06 Impact Factor
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ABSTRACT: The open reading frame encoding the avirulence gene acrPpiA1, previously cloned from race 2 of Pseudomonas syringae pv. pisi, has been subcloned and shown to express peptides corresponding to all six possible start codons. The half-life of the peptides was found to be approximately 5 min in Escherichia coli. Transposon insertions which inactivate the gene in P. syringae pv. Pisi were located both within the open reading frame and in the upstream regulatory sequence, the hrp-box. Homologues of the gene were present only in races 5 and 7, which expressed the avirulence phenotype. The gene was present on the chromosome in all race 2 isolates tested, but was plasmid-borne in the type strains of races 5 and 7. In contrast to the homologue avrRpm1 cloned from P. syringae pv. maculicola, marker-exchange mutagenesis of the gene in races 2 and 7 of P. syringae pv. pisi was not found to affect pathogenicity towards the susceptible host as indicated by symptom development or bacterial growth in the plant. (C) 1997 Academic Press Limited.
Physiological and Molecular Plant Pathology. 01/1997; 50(4):219-236.
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ABSTRACT: We showed that a bacterial avirulence (avr) gene function, avrPpiA1, from the pea pathogen Pseudomonas syringae pv pisi, is recognized by some, but not all, genotypes of Arabidopsis. Thus, an avr gene functionally defined on a crop species is also an avr gene on Arabidopsis. The activity of avrPpiA1 on a series of Arabidopsis genotypes is identical to that of the avrRpm1 gene from P.s. pv maculicola previously defined using Arabidopsis. The two avr genes are homologous and encode nearly identical predicted products. Moreover, this conserved avr function is also recognized by some bean and pea cultivars in what has been shown to be a gene-for-gene manner. We further demonstrated that the Arabidopsis disease resistance locus, RPM1, conditioning resistance to avrRpm1, also conditions resistance to bacterial strains carrying avrPpiA1. Therefore, bean, pea, and conceivably other crop species contain functional and potentially molecular homologs of RPM1.
The Plant Cell 12/1992; 4(11):1359-69. · 8.99 Impact Factor
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ABSTRACT: An avirulence gene (designated avrPpiB) from race 3 of Pseudomonas syringae pv. pisi was cloned and sequenced. The gene corresponded to a single open reading frame of 831 nt identified by transposon mutagenesis and subcloning. This ORF encodes a predicted hydrophilic protein of 276 amino acids (MW 31,300). It effects the expression of a resistance mechanism governed by a single genetic locus in pea. Cosegregation of resistance at the R3 locus of pea was observed towards race 3 and a transconjugant carrying the cloned avrPpiB gene according to the predicted 3:1 ratio of resistant:susceptible F2 progeny from a cross between Jade (R3 R3) and Kelvedon Wonder (rr) cultivars. DNA hybridization studies showed avrPpiB to be plasmid-borne in race 3 and suggested the presence of other alleles on one of the endogenous plasmids of races 1 and 7. Disruption of the avrPpiB allele of race 1 and its complementation confirmed its behavior towards pea cultivars expressing the R3 locus. Homologs of avrPpiB were detected in P. syringae pv. phaseolicola, P. syringae pv. maculicola, and P. syringae pv. tomato. The presence of avrPpiB homologs in P. syringae pv. phaseolicola does not match any gene-for-gene pattern of interaction with bean cultivars.
Molecular Plant-Microbe Interactions 8(5):700-8. · 4.43 Impact Factor
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ABSTRACT: A genomic library clone, pPPY40, from Pseudomonas syringae pv. phaseolicola was previously shown to consist of two regions: region I potentially encoded an avirulence gene involved in non-host recognition in pea and region II contained a replicase gene. In this study, region I was sequenced and shown to contain a single open reading frame (ORF) of 2130 nt with a 55% G + C ratio encoding a deduced peptide product of 710 amino acids with a predicted molecular mass of 75.4 kDa. Both the entire ORF and an ORF truncated by five residues were isolated by PCR amplification. The full length ORF was successfully cloned in the broad host range vector pBBR1MCS-2, but not in the RK2-based broad host range vector pLAFR3. Truncated ORFs were successfully cloned in both vectors. The constructs obtained were transferred to Pseudomonas syringae pv. pisi strain PT10 and the transconjugants tested for pathogenicity on pea plants. Only the full length ORF conferred avirulence and the gene was designated avrPphD. Inactivation ofavrPphD by marker-exchange did not affect the ability of P. syringae pv. phaseolicola to cause a non-host hypersensitive reaction. PCR amplification using avrPphD -specific primers and hybridization analysis indicated that homologues of avrPphD occurred in a wide range of P. syringae pathovars.
Physiological and Molecular Plant Pathology 58(2):55-62. · 1.38 Impact Factor
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ABSTRACT: Two unique DNA fragments, generated by RAPD-PCR, were used as probes against dot-blots of representative isolates of the seven races ofPseudomonas syringaepv.pisi. DNA from each isolate hybridized only to one of the two probes. Fragments identified from isolates 1691 (race 7) and 203 (race 2), were cloned into pUC18 and sequenced. The resulting sequences were used to design two pairs of oligonucleotide primers which when used in PCR reactions withP. syringaepv.pisicells gave specific amplification products (either a 272 bp or a 132 bp fragment) corresponding to the original cloned fragments. When all four primers were used in combination with specific DNA amplification reactions in 51 isolates ofP. syringaepv.pisithey produced one of the two PCR bands. No bands were detected in a range of closely relatedP. syringaepathovars following PCR amplification. These results suggest thatP. syringaepv.pisican be unambiguously identified using specific oligonucleotide primers and that isolates can be classified into two phylogenetic groups, I and II.
Physiological and Molecular Plant Pathology 49(4):233-245. · 1.38 Impact Factor
