Fasolotoksyna u Pseudomonas syringae, Publikacje naukowe
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//-->Microbiology(2004),150,473–482DOI10.1099/mic.0.26635-0Pseudomonas syringaepv. phaseolicola can beseparated into two genetic lineages distinguishedby the possession of the phaseolotoxinbiosynthetic cluster´Jose A. Oguiza,1Arantza Rico,1Luis A. Rivas,1Laurent Sutra,23´Alan Vivian3and Jesus Murillo1CorrespondenceJesus Murillo´jesus@unavarra.es1Instituto de Agrobiotecnologıa y Recursos Naturales, CSIC-UPNA, and Laboratorio de´´Patologıa Vegetal, Departamento de Produccion Agraria, Universidad Publica de Navarra,´´31006 Pamplona, Spain´ ´´´UMR de Pathologie Vegetale INRA-INH-Universite, Beaucouze, 49071 FranceCentre for Research in Plant Science, University of the West of England, Coldharbour Lane,Bristol BS16 1QY, UK23Received10 July 2003Revised30 October 2003Accepted31 October 2003The bean (Phaseolus spp.) plant pathogenPseudomonas syringaepv. phaseolicola ischaracterized by the ability to produce phaseolotoxin (Tox+). We recently reported that themajority of the SpanishP. syringaepv. phaseolicola population is unable to synthesize thistoxin (Tox”). These Tox”isolates appear to lack the entire DNA region for the biosynthesis ofphaseolotoxin (argK-tox gene cluster), as shown by PCR amplification and DNA hybridizationusing DNA sequences specific for separated genes of this cluster. Tox+and Tox”isolates alsoshowed genomic divergence that included differences in ERIC-PCR and arbitrarily primed-PCRprofiles. Tox+isolates showed distinct patterns of IS801 genomic insertions and contained achromosomal IS801 insertion that was absent from Tox”isolates. Using a heteroduplex mobilityassay, sequence differences were observed only among the intergenic transcribed spacer ofthe five rDNA operons of the Tox”isolates. The techniques used allowed the unequivocaldifferentiation of isolates ofP. syringaepv. phaseolicola from the closely related soybean (Glycinemax)pathogen,P. syringaepv. glycinea. Finally, a pathogenicity island that is essential for thepathogenicity ofP. syringaepv. phaseolicola on beans appears to be conserved among Tox+, butnot among Tox”isolates, which also lacked the characteristic large plasmid that carries thispathogenicity island. It is proposed that the results presented here justify the separation of theTox+and Tox”P. syringaepv. phaseolicola isolates into two distinct genetic lineages, designatedPph1 and Pph2, respectively, that show relevant genomic differences that include thepathogenicity gene complement.INTRODUCTIONPseudomonas syringaepv. phaseolicola is a seed-bornepathogen of bean (Phaseolusvulgaris)worldwide that causes3Deceased(d. 16 December 2002); this paper is dedicated to hismemory.Abbreviations:AP-PCR, arbitrarily primed PCR; ERIC-PCR, extragenicrepetitive consensus PCR; REP-PCR, repetitive extragenic palindromicPCR; EEL, exchangeable effector locus; HMA, heteroduplex mobilityassay; ITS, internal transcribed spacer; PAI, pathogenicity island.The EMBL accession numbers for the sequences reported in this paperare AJ568000 (IS50, 734 bp), AJ568001 (IS50, 295 bp), AJ568002(ERIC, 1289 bp), AJ550186 (EEL-Pph1), AJ550187 (EEL-Pph2) andAJ550188 (EEL-Pseudomonassyringaepv. glycinea).the halo blight disease. Disease symptoms are typicallywatersoaked lesions that eventually develop a surroundingyellow halo produced by the release of the non-specifictoxin, phaseolotoxin (Mitchell, 1978). Based on theirvirulence to a range of bean cultivars, nine races ofP. syringaepv. phaseolicola have been distinguished (Tayloret al.,1996). Recently, the ability of this pathogen to pro-duce disease in bean has been shown to be based on thepossession of a pathogenicity island (PAI), localized to a150 kb plasmid, that includes genes that are either essentialfor pathogenicity on bean and soybean or that contributeto aggressiveness in an additive fashion (Jacksonet al.,1999;Tsiamiset al.,2000). In addition to the PAI,P. syringaepv. phaseolicola strains are defined by possession oftheargK-toxgene cluster, which directs phaseolotoxin4730002-6635G2004 SGMPrinted in Great BritainJ. A. Oguiza and othersbiosynthesis and appears to increase virulence (Patilet al.,´1974; Mitchell, 1978; de la Fuente-Martınezet al.,1992).Additionally, phaseolotoxin has been considered a usefuldeterminative character unique toP. syringaepv. phaseoli-cola among the bacterial bean pathogens. It is generallybelieved that only isolates able to synthesize phaseolotoxin(Tox+isolates) are of epidemiological significance and,hence, this DNA region is commonly used as a target forPCR detection and identification ofP. syringaepv. phaseoli-cola (Schaadet al.,1995).P. syringaepv. phaseolicola can readily be distinguishedfrom other pathovars ofP. syringaepathogenic to beans,such as pathovars syringae and glycinea, by nutritionalcharacteristics and because onlyP. syringaepv. phaseolicolaisolates produce water-soaked lesions on bean pods¨(Palleroni, 1984; Volksch & Weingart, 1997; Marqueset al.,2000). In general,P. syringaepv. phaseolicola appearsto be a more or less homogeneous pathovar, although itdisplays a degree of genetic and phenotypic variation thatoverlaps with isolates fromP. syringaepv. glycinea (Marqueset al.,2000). On the basis of phenotypic characteristicsand ERIC-PCR-generated profiles, strains ofP. syringaepv. glycinea,P. syringaepv. phaseolicola isolated frombean andP. syringaepv. phaseolicola isolated from kudzu(Puerarialobata),can be divided into three distinct groups¨(Volksch & Weingart, 1997). Additionally, intrapathovarvariation inP. syringaepv. phaseolicola can be linked, insome cases, to the host plant species of isolation (Marqueset al.,2000). Isolates that produce natural infections onkudzu vine are distinguished, among other characters, forcarrying a plasmid-borneefegene (Nagahamaet al.,1994)and, similar to isolates fromVigna radiata,by their REP-¨PCR profile with ERIC primers (Volksch & Weingart, 1997;Marqueset al.,2000).Most isolates ofP. syringaepv. phaseolicola are reported tobe Tox+and naturally occurring isolates unable to syn-thesize phaseolotoxin (Tox2isolates), which usually possessthe correspondingargK-toxgene cluster region, are rare(Rudolph, 1995; Schaadet al.,1995). We reported recently,however, that over 60 % of the Spanish field isolates ofP. syringaepv. phaseolicola were Tox2and did not producethe expected PCR amplification using a primer pair specificfor ORF6 (Ricoet al.,2003), which is essential for phaseo-lotoxin biosynthesis and is routinely used as a target for thedetection of this pathogen (Schaadet al.,1995). Addition-ally, Tox2isolates did not show hybridization to an ORF6-specific DNA probe (Ricoet al.,2003), suggesting theabsence of part or of the entireargK-toxgene cluster. Thisraised the possibility that the Spanish Tox2isolates weregenetically separable from the more common isolates thatsynthesize phaseolotoxin. In this study, we analyse thegenetic variability within the SpanishP. syringaepv. phaseo-licola population, in comparison withP. syringaepv.phaseolicola andP. syringaepv. glycinea isolates frominternational collections. Collectively, our results allowedthe differentiation of two genetic lineages inP. syringae474pv. phaseolicola and suggest the separate evolution oftheir pathogenicity gene complement.METHODSBacterial strains and growth conditions.Escherichia coliDH5awas used for cloning purposes and was propagated in LB at 37uC(Sambrooket al.,1989). The type races ofP. syringaepv. phaseoli-cola 1281A (race 1), 1301A (race 3), 1302A (race 4), 1449B (race 7),2656A (race 8) and 2709A (race 9) have been described elsewhere(Tayloret al.,1996). Strains Hb-1b and M2/1 ofP. syringaepv.phaseolicola were isolated from beans in an unknown place and¨Germany, respectively, and do not produce phaseolotoxin (Volksch& Weingart, 1997). Another 13 Tox+and 24 Tox2P. syringaepv. phaseolicola isolated in Spain were characterized previously(Ricoet al.,2003).P. syringaepv. phaseolicola CFBP1390 andP.syringaepv. glycinea CFBP2214 are the pathotype strains and wereobtained from C. Manceau (INRA, Angers, France).P. syringaepv. glycinea strains PG4180 and 49a/90 (both race 4) were obtainedfrom M. Ullrich (Bremen University, Bremen, Germany).P. syringaestrains were routinely grown on King’s medium B (KMB) (Kinget al.,1954) at 25–28uC.PCR analysis.Genetic variability amongP. syringaestrains wasexamined by PCR fingerprinting of repetitive DNA sequences usingprimers for extragenic repetitive consensus (ERIC), repetitive extra-genic palindromic (REP) and the arbitrarily primed PCR (AP-PCR)techniques. For ERIC and REP analyses, primers and reaction condi-tions were as described by McManus & Jones (1995). AP-PCR wascarried out using the universal M13 reverse primer (59-AGCGGA-TAACAATTTCACAGG-39) or a single 20 bp oligonucleotide primer(59-GGTTCCGTTCAGGACGCTAC-39) complementary to the IS50portion of Tn5, as described by Sundin & Murillo (1999). For theamplification of phaseolotoxin biosynthetic genes, we assayed twodifferent primer pairs which are specific for DNA regions separatedin the genome that are essential for phaseolotoxin biosynthesis.Primers PHA19 and PHA95 amplify a 480 bp internal fragmentfrom the amidinotransferase geneamtA(Marqueset al.,2000;´´Hernandez-Guzman & Alvarez-Morales, 2001) and primers OCTF-03 and OCT-R amplify a 632 bp DNA fragment of the ornithinecarbamoyltransferase geneargK(Sawadaet al.,2002), which confersresistance to phaseolotoxin. Amplification of genes included in thepathogenicity island was performed with primers DL-04523 (59-GT-AATCGAGTCGCCGCTCTG-39) and DR-05216 (59-GAAAGTGAA-GCGAACGCAAG-39) foravrD,and primers CL-19541 (59-GATCG-TAAGAACGGGCGATT-39) and CR-20852 (59-CGTGCATGGTAG-CATGTATGAA-39) foravrPphC.The exchangeable effector locus(EEL) region of thehrppathogenicity island (Alfanoet al.,2000)was amplified using primersavrPphE-FOR(Stevenset al.,1998) andqueA-2(59-AATCAGGGAATCGGGGAGTT-39) within the codingregions of thehrpKandqueAgenes, respectively. A 627 bp fragmentfrom the insertion sequence element IS801 (Romantschuket al.,1991)was amplified fromP. syringaepv. phaseolicola strain 1449B usingprimers IS801F (59-AGTCCTGCCTACACACCTCGA-39) and IS801R1(59-GCCTCTTTGTGGAACGACAG-39). The occurrence of a chromo-somal insertion of IS801 inP. syringaepv. phaseolicola was tested by´amplification with primers RP-1 and RP-2 (Gonzalezet al.,1998).For amplifications, bacterial cell suspensions of isolates grown onKMB were prepared in 500ml sterile distilled water and subjected tofreeze–thaw lysis. Standard PCR reactions were performed in a finalvolume of 25ml containing as template 50 ng total genomic DNA or5ml bacterial lysates, using eitherTaqDNA polymerase (Biotaq;Bioline) or Ready To Go PCR Beads (Amersham Pharmacia Biotech).General molecular techniques.Total DNA was extracted using aPuregene DNA isolation kit (Gentra Systems), according to theMicrobiology150Genetic lineages ofP. syringaepv. phaseolicolamanufacturer’s instructions. Plasmids were isolated by a modifiedalkaline lysis procedure (Zhouet al.,1990) and intact native plasmidswere separated by electrophoresis on 0?6 % agarose gels in 16 TAEas described previously (Murilloet al.,1994). PCR products werepurified using the GFX PCR DNA purification kit (AmershamPharmacia Biotech). DNA sequencing was performed by MWG-Biotech AG. Nucleotide sequences were aligned usingCLUSTALW(Thompsonet al.,1997) and database comparisons were made viatheBLASTN,BLASTPandTBLASTXalgorithms (Altschulet al.,1997).Preliminary sequence data fromP. syringaepv. tomato DC3000and pv. syringae B728a genome projects were obtained from TheFor Southern blots, chromosomal DNA was routinely digested withappropriate restriction enzymes, and DNA fragments separated byelectrophoresis in 1 % agarose gels were transferred to a nylonmembrane (Roche Diagnostics). For the preparation of DNA probes,specific DNA fragments were gel-extracted and cloned into thepGEM-T Easy vector (Promega). After restriction digestion, the insertswere separated by electrophoresis, excised from the gels and used asprobes. Preparation of labelled probes with digoxigenin, Southernhybridization and detection of the hybridized DNA were carried outwith the DIG DNA labelling and detection kit (Roche Diagnostics).Heteroduplex mobility assay (HMA).The sequence polymorph-ism of the internal transcribed spacer (ITS) region between 16S and23S rRNA genes was analysed using a DNA HMA (Delwartet al.,1993). The ITS region was amplified using primers D21 and D22(Manceau & Horvais, 1997) and PCR products were migrated in5 % polyacrylamide gels (Delwartet al.,1993).RESULTSA group ofP. syringaepv. phaseolicola isolateslack the phaseolotoxin biosynthetic geneclusterIn a previous study (Ricoet al.,2003), 94 Spanish Tox2isolates lacked ORF6, which is contained in theargK-toxgene cluster and used for detection purposes (Schaadet al.,1995; Zhang & Patil, 1997). By PCR amplification andDNA hybridization we tested the conservation of theargK-toxgene cluster among a collection of sixP. syringaepv. phaseolicola type races, 13 Tox+Spanish isolates, 24Spanish Tox2isolates and the two Tox2strains Hb-1b andM2/1, isolated elsewhere. We focused on genesargKandamtA,which currently define the ends of the cluster, fortheir importance in the detection of this pathogen (Schaad´´et al.,1995; Hernandez-Guzman & Alvarez-Morales, 2001).PCR amplification using primers internal toamtA(Fig. 1a)andargK(not shown) yielded the expected 480 and 632 bpamplification products, respectively, for all the Tox+iso-lates tested, as well as for the Tox2isolates Hb-1b andM2/1. Conversely, no strong specific amplicons wereobserved for any of the Spanish Tox2isolates or forP.syringaepv. glycinea strains PG4180 and 49a/90 (Fig. 1a).We determined that the published sequence of primerPHA19 (Marqueset al.,2000) showed two mismatches inits 59 end with the sequence of theamtAgene deposited in´´the databases (accession no. AF186235; Hernandez-Guzman& Alvarez-Morales, 2001). Although theargKgene wasFig. 1.Detection of theargK-toxgene cluster. (a) PCR amplifi-cation of a 480 bp fragment from theamtAgene using primers´´PHA19/PHA95 (Marqueset al.,2000; Hernandez-Guzman &Alvarez-Morales, 2001). Lanes: 1,P. syringaepv. phaseolicola(Pph) isolate 1281A; 2, 2709A; 3, 1449B; 4, CYL215;5, CYL281; 6, CYL285; 7, CYL207; 8, CYL283; 9, CYL286;10, CYL233; 11, CYL275; 12, CYL325; 13, CYL352; 14,CYL309; 15, CYL314;P. syringaepv. glycinea (Pgy) isolates16, 49a/90; 17, PG4180. Pph1 and Pph2 correspond to thetwo genetic lineages ofP. syringaepv. phaseolicola. (b)Southern hybridization ofEcoRI-digestedtotal DNA. An internalfragment of theamtAgene was amplified fromP. syringaepv.phaseolicola strain 1449B with primers PHA19/PHA95, labelledwith digoxigenin and used as probe. Lanes are as describedabove. Sizes are indicated to the left in kb.shown to be highly conserved (Sawadaet al.,1999), theseresults suggest that the observed lack of amplificationobserved for some of the Tox2isolates might be due topossible sequence variations in theirargK-toxgene clusterwith respect to the primers used. We therefore examined theconservation of this cluster by DNA hybridization.Internal fragments ofamtAandargKwere amplified asabove from the Tox+strain 1449B, labelled with digoxi-genin and used as probes in Southern analysis of the selected45 isolates detailed above. As expected, all the strains thatproduced specific PCR bands with the two primer pairsalso showed hybridization to theamtAandargKprobes(Fig. 1b and not shown). In all cases, the homologous DNAwas located to a 0?8 kbEcoRIfragment for theamtAprobe(Fig. 1b) and to an 8 kbHindIIIfragment for theargKprobe (not shown). On the other hand, the strains that didnot produce specific PCR amplification products did not475J. A. Oguiza and othershybridize with either of the two probes, suggesting that theymay lack the entireargK-toxgene cluster. We propose todesignate the group of strains containing theargK-toxgene cluster Pph1, and the group of strains lacking thiscluster Pph2.Isolates containing or lacking theargK-toxgene cluster can be differentiated into twogroups by REP-PCRThe phaseolotoxin biosynthetic cluster appears to havebeen acquired by horizontal gene transfer (Sawadaet al.,1997, 1999) and, as a consequence it is possible that theP. syringaepv. phaseolicola isolates containing this DNAand those lacking it might represent distinct geneticlineages. We used PCR fingerprinting of repetitive DNAsequences (REP-PCR) to assess the genetic diversity amongthe above 21 Pph1 and 24 Pph2 isolates. We also analysedtwo strains ofP. syringaepv. glycinea, because strains ofthis pathovar also lack theargK-toxgene cluster and areclosely related phylogenetically toP. syringaepv. phaseoli-cola (Gardanet al.,1999; Marqueset al.,2000; Yamamotoet al.,2000).The REP-PCR amplification profiles were similar among allisolates examined (Fig. 2), although strains ofP. syringaepv. phaseolicola showed several strong differential bandsthat allowed their distinction from theP. syringaepv.glycinea isolates. One of these was a 1700 bp band presentin the ERIC profile (Fig. 2). Additionally, strains belongingto Pph1 and Pph2 could be distinguished on the basis ofsignificant differences in their REP-PCR banding profiles(Fig. 2). Besides several minor differential bands, a strong734 bp band was present in the IS50 profile of all the Pph1strains (Fig. 2), independently of their place of isolation.Hybridization experiments showed that the 45P. syringaepv. phaseolicola isolates examined contained several frag-ments with homology to the sequences included in the734 bp fragment (not shown). However, the pattern ofhybridization to the probe showed significant differencesbetween Pph1 and Pph2 isolates (not shown), indicatingthe existence of more dissimilarities than those revealed byREP-PCR. The analysis of the nucleotide sequence of the734 bp band, obtained in this work, indicated that it is amosaic (Table 1) that probably resulted from a reorganiza-tion event. Comparison with the databases showed thatparts of this sequence are also repeated and scattered indifferent positions of theP. syringaepv. tomato DC3000genome and plasmid pDC3000A (Table 1).All the Pph2 isolates showed a characteristic REP-PCRprofile that included two strong differential bands: a1289 bp band present in the ERIC profile and a 295 bpband amplified by the IS50 primer (Fig. 2). The nucleotidesequences of the 1289 and 295 bp bands were also deter-mined and analysed. The 1289 bp band appears to be wellconserved, since its nucleotide sequence was highly con-served in the genomes ofP. syringaepv. tomato DC3000 andpv. syringae B728a (Table 1) and because theP. syringae476Fig. 2.Repetitive PCR fingerprinting (ERIC, IS50, Reverse andREP) patterns ofP. syringaepv. phaseolicola (Pph) andP.syringaepv. glycinea (Pgy) isolates. M, 1 kb DNA ladder(Promega). Lanes 1–17 are as described in the legend toFig. 1. The size (in bp) of the differential bands observed in theERIC and IS50 profiles are indicated.pv. glycinea strains contained a co-migrating band (Fig. 2).All Pph1 and Pph2 isolates showed a unique 10 kbEcoRIhybridization band in Southern experiments using the1289 bp fragment as a probe (not shown). In contrast, the295 bp band showed strong hybridization only to genomicDNA from Pph2 isolates, and the homologous DNA waslocalized to a native plasmid of 40–50 kb (not shown). Thecomparison of the nucleotide sequence of the 295 bp bandwith the databases suggests that it is a chimera of sequencesthat are separated in otherP. syringaestrains (Table 1).Conservation of the exchangeable effector lociThehrpcluster encodes a type III secretion system thatinjects specialized proteins, or effectors, into the plant hostcell; these effectors appear to be the main host range deter-minants, promoting pathogenicity or defence reactions ofthe plant. InP. syringae,thehrpcluster is bordered by twoDNA regions containing diverse effector genes (Alfanoet al.,Microbiology150Genetic lineages ofP. syringaepv. phaseolicolaTable 1.Features of the ERIC and IS50 profile bands that differentiated strains of Pph1 and Pph2Band specificity/size (bp)*Pph1/734PrimerIS50Position321–466365–466466–73419–276124–249251–277Pph2 andPgy/1289ERIC1–1289Relevant nucleotide homologies (nucleotide position/accession no.)P. syringaepv. tomato DC3000 genome (5 346 878–5 347 023)P. syringaepv. syringae B728a genome Psyr_6 (NZ_AABP020 00006)P. syringaepv. tomato DC3000 plasmid pDC3000A (30 047–30 148)P. syringaepv. tomato DC3000 genome (908 368–908 100)Plasmid pIAA1, DNA region downstream IAA lysine synthetase geneP. syringaepv. savastanoi (M35373)DNA region upstream type III effector HopPmaD gene;P. syringaepv. maculicola (AF458043)P. syringaepv. tomato DC3000 genome (16 683–16 709)DNA IS801 insertion sequence element;P. syringae(X57269)P. syringaepv. tomato DC3000 genome (3 101 476–3 100 187)P. syringaepv. syringae B728a genome Psyr_7 (NZ_AABP020 00007)Identity(%)9292969597961001008284Pph2/295IS50*Pgy,P. syringaepv. glycinea.2000). One of them, the exchangeable effector locus (EEL),begins 3 nt downstream of the stop codon of thehrpgenehrpKand ends near tRNALeu,queAandtgtsequences, whichare highly conserved among differentPseudomonasspecies.The size and gene sequence of the EEL are highly diverseamong different isolates ofP. syringae(Charityet al.,2003;Denget al.,2003).The EEL region from different isolates belonging to bothgroups ofP. syringaepv. phaseolicola and fromP. syringaepv. glycinea strains PG4180 and 49a/90 was amplified byPCR using primers located within the coding regions ofgeneshrpKandqueA.Identical 2?4 kb PCR amplificationproducts were observed for all the isolates examined (notshown), suggesting that the EEL region is conserved amongPph1, Pph2 andP. syringaepv. glycinea. The EEL sequence(1083 bp) between genequeAand the effector geneavrPphE,located immediately downstream ofhrpK,was determinedfor one representative isolate each of Pph1 (strain 1449B),Pph2 (strain CYL325) andP. syringaepv. glycinea (strain49a/90). Pairwise comparison showed from one to a maxi-mum of three nucleotide differences, indicating a highdegree of conservation. The analysis of the 1083 bp EELsequence showed the presence of an ORF homologous(85 % identity) to ORF3 (eelF1) located in the EEL regionofP. syringaepv. tomato DC3000 (Alfanoet al.,2000;Charityet al.,2003).The 150 kb virulence plasmid of Pph1 is notpresent in Pph2Strains ofP. syringaepv. phaseolicola usually contain a largenative plasmid of around 150 kb that, in the race 7 strain1449B, was shown to carry the PAI (Jacksonet al.,1999). Wetherefore decided to evaluate the conservation and physicallocation of the PAI between groups Pph1 and Pph2 byexamination of the plasmid profiles and by Southern hybri-dization with probes specific for effector genesavrDandavrPphC,which are located in the leftmost border and inthe centre of the PAI, respectively (Yucelet al.,1994; Jacksonet al.,1999).avrDis widely distributed inP. syringaeandrestricts infection on certain soybean cultivars by triggeringa defence response, as doesavrPphC.Additionally,avrPphCalso behaves as a virulence gene on bean cultivar CanadianWonder (Tsiamiset al.,2000).The profiles of Pph1 isolates showed diverse native plas-mids and all of them contained a large plasmid similar to the150 kb virulence plasmid present in strain 1449B (Fig. 3a).In contrast, the Pph2 isolates contained one or two nativeplasmids of 30–50 kb, with absence of the typical 150 kbplasmid present in Pph1 (Fig. 3a). DNA probes specific forgenesavrDandavrPphCshowed hybridization with thelarge plasmid present in strain 1449B and in all the otherPph1 isolates (Fig. 3b), indicating that the physical locationof the PAI is conserved in Pph1. Conversely,avrDdid notshow hybridization with any of the plasmids of the Pph2isolates (Fig. 3b), although it hybridized to a 5?6 kbHindIIIfragment when digested total genomic DNA was usedinstead of intact native plasmids (not shown). TheavrPphCprobe, however, hybridized to a single plasmid of 40–50 kbin each Pph2 isolate (Fig. 3b). These results suggest a dif-ferent organization of the pathogenicity genes included inthe PAI among Pph1 and Pph2 isolates.IS801 insertion patterns are different for Pph1and Pph2The 1512 nt insertion sequence element IS801 has a limiteddistribution amongP. syringae(Romantschuket al.,1990,1991) and is thought to produce permanent insertionsdue to its putative replicative transposition mechanism(Mendiolaet al.,1994; Richteret al.,1998). Therefore, weexamined the profile of IS801 insertions as a potentialmethod of fingerprinting strains ofP. syringaepv. phaseoli-cola. Genomic and plasmid DNA of selectedP. syringae477
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