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Cultivar‐specific avirulence and virulence functions assigned to avrPphF in Pseudomonas syringae pv. phaseolicola, the cause of bean halo‐blight disease

Cultivar‐specific avirulence and virulence functions assigned to avrPphF in Pseudomonas syringae... The EMBO Journal Vol. 19 No. 13 pp. 3204-3214, 2000 Cultivar-specific avirulence and virulence functions assigned to avrPphF in Pseudomonas syringae pv. phaseolicola, the cause of bean halo-blight disease ated with activation of the hypersensitive reaction (HR), a George Tsiamis, John W.Mansfield , form of rapid programmed cell death, which restricts Ruth Hockenhull, Robert W.Jackson , colonization to the site of inoculation in resistant cultivars Ane Sesma , Evangelos Athanassopoulos, of the host plant (De Wit, 1995; Mansfield et al., Mark A.Bennett, Conrad Stevens, 2 3 1997a). This article concerns the genetics of avirulence Alan Vivian , John D.Taylor and and virulence in the bean halo-blight bacterium Jesus Murillo Pseudomonas syringae pathovar (pv.) pftaseolicola (here­ Department of Biological Sciences, Wye College, University of after Pph). Nine races of Pph have been differentiated London, Ashford, Kent TN25 5AH, Department of Biological and based on their virulence to a range of bean cultivars, as Biomedical Sciences, University of the West of England, Coldharbour 3 summarized in Table I (Taylor et al., 1996). Lane, Bristol BS16 l Y, Horticulture Research International, The gene-for-gene theory explaining cultivar-specific Wellesbourne, Warwick CV35 9EF, UK and 4Escuela Tecnica Superior de Ingenieros Agr6nomos, Universidad Publica de Navarra, resistance to plant disease was developed by Flor (1942, 31006 Pam plona, Spain 1971) based on his analysis of the differential reactions of flax to the rust fungus Melampsora lini. Person et al. Corresponding author e-mail: [email protected] (1962) summarized the essence ofFlor's hypothesis in the statement that ' ... for every resistance gene in flax there is a The avrPphF gene was cloned from Pseudomonas specific and related gene for virulence in those rusts to syringae pathovar phaseolicola (Pph) races 5 and 7, which it is susceptible'. The emphasis on susceptibility based on its ability to confer avirulence towards bean suggested that disease was the result of the positive cultivars carrying the Rl gene for halo-blight resist­ function of cultivar-specific virulence determinants. The ance, such as Red Mexican. avrPphF comprised two advances of molecular genetics have proved the gene-for­ open reading frames, which were both required for gene theory by the cloning not of virulence (vir) genes, but function, and was located on a 154 kb plasmid of avirulence (avr) genes from bacteria and fungi, and the (pA V511) in Pph. Strain RW60 of Pph, lacking matching resistance (R) genes from plant hosts (Dangl, pA V511, displayed a loss in virulence to a range of 1994; De Wit, 1995; Jones and Jones, 1996; Vivian et al., previously susceptible cultivars such as Tendergreen 1997). The proteins encoded by avr and R genes are and Canadian Wonder. In Tendergreen virulence was envisaged to interact either directly or indirectly to restored to RW60 by avrPphF alone, whereas sub­ activate signalling cascades leading to resistance, which cloned avrPphF in the absence of pA V511 greatly is expressed by the HR (Hammond-Kosack and Jones, accelerated the hypersensitive resistance reaction 1996; Van den Ackerveken et al., 1996). Bacterial avr caused by RW60 in Canadian Wonder. A second gene genes have been cloned by function through the transfer of from pA V511, avrPphC, which controls avirulence to genomic libraries from avirulent to virulent races of the soybean, was found to block the activity of avrPphF in pathogen (Staskawicz et al., 1987). Genomic clones Canadian Wonder, but not in Red Mexican. avrPphF causing changes in the virulence phenotype on certain also conferred virulence in soybean. The multiple cultivars have been isolated and genes for avirulence functions of avrPphF illustrate how effector proteins subsequently characterized, e.g. avrPphB and avrPphE from plant pathogens have evolved to be recognized from Pph, which correspond to A3 and A2, respectively, in by R gene products and, therefore, be classified as Table I (Mansfield et al., 1994; Vivian et al., 1997). encoded by avirulence genes. Similarly, genes for avirulence but not virulence have also Keywords: avirulence/disease resistance/gene-for-gene been cloned, which operate at the level of host specificity interactions/hypersensitive reaction/virulence (Kobayashi et al., 1989; Fillingham et al., 1992; Wood et al., 1994). Clearly, the continued presence of genes that restrict host range in certain strains or races of plant pathogens is Introduction somewhat of a paradox, but some avr genes have been The bacteria and fungi that colonize living plants and implicated in basic parasitism, e.g. avrRpml from P.s. pv. cause disease have evolved the ability to overcome the maculicola and avrE from P.s. pv. tomato. Mutations in innate resistance of their hosts to infection. Superimposed avrRpml or avrE lead to greatly reduced ability to on this establishment of basic parasitism ( or patho­ colonize host plants (Lorang et al., 1994; Ritter and genicity) is the phenomenon of cultivar (cv.)-specific Dangl, 1995). The emerging pattern is that plant pathogens resistance. A plant pathogen may, therefore, be fully may produce numerous proteinaceous virulence factors, virulent causing disease on certain cultivars of its host, but which act synergistically to promote disease (Alfano and avirulent on others (De Wit, 1995; Alfano and Collmer, Collmer, 1997; Collmer, 1998). Some of these factors may 1997; Grant and Mansfield, 1999). Avirulence is associ- become recognized by the products of R genes and, 3204 © European Molecular Biology Organization Avirulence-virulence gene from Pseudomonas syringae Table L Gene-for-gene relationship (based on five matching gene pairs) proposed from analysis of the reactions• of bean cultivars to races of P.s. pv. phaseolicola (from Taylor et al., 1996) Races/avirulence (A) genes 1 2 3 4 5 7 9 6 8 Al Al Al Al A2 A2 A2 A2 A3 A3 A4 Differential cultivars Resistance genes A5 A5 A5 Canadian Wonder + + + + + + + + + A52 (ZAA54) R4 + + + + + + + + Tendergreen R3 + + + + + + + Red Mexican Rl R4 + + + + + A53 (ZAA55) R3 R4 + + + + + + A43 (ZAA12) R2 R3 R4 R5 + + '+, susceptible; -, resistant. therefore, be identified as determinants of avirulence. Here we describe molecular characterization of the Because of functional redundancy, however, the loss of avrPphF locus in Pph. The gene was found to comprise an operon with two open reading frames (ORFs), both of what has become an avr gene rather than a vir gene does which were required for function. Although we identified not always compromise the basic pathogenicity on which avrPphF as an a, avr gene that determined race structure cultivar specificity is superimposed. in the halo-blight bacterium, it was also found to The vir gene concept has been strengthened by the confer cultivar-specific virulence to cv. Tendergreen. identification of a gene, virPphA from Pph, based on its Furthermore, in the absence of the plasmid pAV511, ability to restore virulence to strains of the bean pathogen containing the P Al, avrPphF also acted as a � avr gene in cured of a 154 kb plasmid designated pAV511 (Jackson cv. Canadian Wonder, which is susceptible to all known et al., 1999). Cured strains (such as that named RW60) lost races of the halo-blight bacterium. The gene within virulence towards bean, causing hypersensitive reactions pAV511 that masked the � type of avr activity of in all previously susceptible cultivars tested. A cluster of avrPphF to Canadian Wonder was found to be avrPphC, potential vir genes including virPphA was located to a which was previously identified as an avr gene acting on region described as a pathogenicity island (P Al), on soybean cultivars. In short, an intriguing web of interact­ pA V511. The ability of RW60 to elicit the HR suggested ing avirulence and virulence functions has been revealed that it contained avr genes, located on the chromosome or for the avr genes from Pph. In the bean-P.syringae other plasmids, whose function was usually masked by interaction, the gene-for-gene concept, although continu­ virulence factors encoded by the PAL Other avr genes, ing to provide a useful framework for analysis of cultivar­ such as avrPphB or avrPphE, continued to function specific resistance, is far more complex than is apparent despite the operation of the plasmid-borne P Al in wild­ from simple analysis of race structures and a, avr-R gene type strains and were, therefore, classified as a, avr genes. interactions as outlined in Table I. It is proposed that The genes detected only in the absence of the PAI were similar complexity will emerge in other host-pathogen classified as� avr genes (Jackson et al., 1999). systems once the problem of the redundancy of multiple The resistance responses activated by different avr-R virulence factors has been overcome by the removal of gene combinations may all be classified as the HR, but certain P Als. they may be phenotypically different because of, for example, differential timing of plant cell collapse. Differences are clear in bean as the avrPphB-R3 inter­ Results action provokes a more rapid HR than avrPphE-R2 (Mansfield et al., 1994, 1997a,b). In many race--cultivar Cloning of avrPphF from race 5 interactions, several different a, avr genes and their Based on the race structure proposed in Table I (Taylor matching R genes may be present in the pathogen and et al., 1996), we sought to clone the putative Al and A4 plant, respectively (Mansfield et al., 1997a). The potential genes from race 5. A genomic library of race 5 strain for epistatic interaction between various avr-R gene 1375A, predicted to contain Al and A4 and known to combinations is apparent from analysis of Arabidopsis harbour A2 (cloned as avrPphE by Stevens et al., 1998), harbouring the RPMJ and RPS2 genes. Although the HR was screened for avr genes by mating into race 6 strain controlled by avrRpt2-RPS2 is slower than that activated 1448A, which is virulent on all cultivars of bean used by the avrRpml-RPMJ interaction, the phenotypically to define race structure (Mansfield et al., 1994). slower response is observed when both avr genes are Transconjugants were initially tested for virulence on present (Reuber and Ausubel, 1996; Ritter and Dangl, pods of cvs Red Mexican UI3 (proposed Rl, R4) and A53 1996). It has been proposed that such epistasis involves (R3, R4). Two genomic clones, pPPY501 and pPPY502, direct interaction between avr and R gene products (Ritter containing overlapping regions of insert DNA conferred and Dangl, 1996). avirulence on Red Mexican but none from the 1500 tested 3205 G.Tsiamis et al. EMBL/GenBank accession Nos AF231452 and AF231453). The sequence revealed two ORFs with a single upstream promoter region containing the hrp box motif, indicating probable regulation by HrpL (Innes et al., 1993; Pirhonen et al., 1996). Both ORFs were preceded by purine-rich regions, which would be expected to act as ribosome binding sites (Singer and Berg, 1991) and they were separated by a 57 bp sequence. A rho-dependent pPPY507 s transcription termination loop was located after the pPPY512 translation stop signal in ORF2. The avrPphF locus pPPY514----- therefore had the structural features of an operon. The Fig. 1. Location of avrPphF within pPPY503 and the 1.8 kb of DNA G+C content of avrPphF was 47%, with ORFl 40% and sequenced. The direction of transcription is indicated by the horizontal ORF2 52.5%, both significantly lower than the overall arrowheads. Sites of insertion of Tn3gus in pPPY503 are indicated by figures of 59--61 % reported for pvs of P.syringae (De Ley, vertical arrowheads; only those marked black in the expanded fragment 1968). abolished the avirulence activity of avrPphF. All transposon insertions Transposon insertions that compromised avirulence were mapped from restriction digests, except A33, which was located by sequencing. The region found to have similarity to ISJOO is were located in both ORFl and ORF2 of avrPphF, and indicated. Restriction sites for BamHI (B), Bgill (Bg), EcoRI (E) and further subcloning revealed that both ORFs were required Pstl (P) are marked; an asterisk indicates site in the vector pLAFR3. for avr function (Figure 1). To examine further the role of The reactions caused in bean cv. Red Mexican by transconjugants of the two ORFs, non-polar mutations were created for ORFl race 6 harbouring subclones are noted. HR, hypersensitive reaction; S, susceptible response. and ORF2, resulting in race 7 AORFl and race 7 AORF2, respectively; both mutants failed to elicit the HR in cv. Red Mexican. Protein production from ORFs 1 and 2 was examined in affected reactions on A53. Avirulence activity was Escherichia coli using constructs of avrPphF cloned for retained by a 5.3 kb fragment, designated pPPY503. expression in pBluescript. Proteins of 17 and 24 kDa were Transposon mutagenesis with Tn3 gus located avrPphF to detected on SDS-P AGE gels after Coomassie Blue a 1.3 kb region flanked by BamHI and EcoRI restriction staining, corresponding to the peptides predicted to be sites (Figure 1). The 1.8 kb BamHI fragment containing encoded by ORFs 1 and 2, i.e. 15.6 and 21.9 kDa, this region was cloned as pPPY511, and found to activate respectively (data not shown). Searches of the databases the HR in a cultivar-specific manner; avrPphF therefore using the BLAST or Propsearch approaches (Hobohm and acted as a typical ex avr gene. Sander, 1995; Altschul et al., 1997) failed to reveal Segregation of the Rl resistance gene matching significant homology with known protein sequences. Both avrPphF was examined in 88 F progeny derived from a peptides are predicted to be hydrophilic; they lack leader cross between Red Mexican and Tendergreen (R3). sequences or potentially membrane-spanning domains. Progeny undergoing the HR to the transconjugant race 6(pPPY503) were also resistant to race 1 (Al). The pattern of incompatibility observed fitted that expected if avrPphF is plasmid borne and present only in avrPphF matched the Rl gene in Red Mexican, the races expressing the A 1 phenotype numbers of resistant and susceptible plants (66:22) exactly The presence of homologues of avrPphF was examined in fitting the 3: 1 ratio expected if the Rl gene was dominant. strains of Pph representing all nine races and also in other The F progeny from the cross between Red Mexican and pathovars of P.syringae. Using specific probes for ORFl Tendergreen were also tested with a transconjugant of and ORF2 in PCR or Southern analysis, signals identical to race 6 containing the clone pMS2330, which contains those found in race 5, the origin of the avrPphF clone, avrPphA. isolated by Shintaku et al. (1989). Although were present only in races 1, 7 and 9, but not in races 2, 3, race 6(pMS2330) did cause a weak HR on Red Mexican, 4, 6 or 8, which are virulent on Red Mexican (Table I; reactions on pods from the F progeny of Red Mexican and Figure 2A and B). However, when the entire 1.8 kb BamHI Tendergreen showed that there was no co-segregation with fragment containing avrPphF was used as a probe the race 1 (data not shown). pattern changed. Multiple signals were detected in all Multiplication of race 1 and race 6 was compared with races of Pph (Figure 2C). Further probing revealed that race 6(pPPY503) in leaves of cv. Red Mexican. The HR multiple signals were due to the 0.5 kb EcoRI-BamHI determined by the presence of the cloned avrPphF gene fragment downstream of avrPphF (Figure 1 ), which was was associated with the failure of bacteria to multiply. 6 found to contain an incomplete ORF with predicted Following inoculation to introduce ~0.2 X 10 c.f.u. per similarity to the /Sl 00 transposase homologue from the excised leaf disc, after 2 days race 6 had reached 6 PAI in Pph (Jackson et al., 1999). Sequences hybridizing 3.7 X 10 , whereas race 1 and race 6(pPPY503) were 2 2 to avrPphF were found in strains of P.s. pvs pisi and present at only 9.3 X 10 and 16 X 10 c.f.u. per disc, glycinea, but not in pvs coronafaciens, maculicola, tabaci, respectively. tomato or syringae, or in Pseudomonas cichorii. Interestingly, only P.s. pv. syringae and P.s. pv. pisi Sequence analysis and expression of avrPphF contained sequences hybridizing to the putative transpo­ The nucleotide sequences of the functional 1.8 kb frag­ sase, but, in contrast to Pph, only one band was observed ments from race 5, and as also recovered from race 7 on Southern blots (data not shown), indicating that it is containing avrPphF, were found to be identical (DDBJ/ highly specific to pv. phaseolicola. 3206 Avirulence-virulence gene from Pseudomonas syringae E EH B BE EB EHH BE EBE EH pAV520 1 2 3 4 5a5b 6 7 8 9 pi "' pAV521------------- uvrl'phC wrf'pM. ORF4 {Nf/) uvrPphJ, l . 8kb --- ..,_ Fig. 3. Location of avrPphF in relation to avrD, virPphA, avrPphC and ORF4 in the 154 kb plasmid identified in race 7 strain 1449B (Jackson et al., 1999). The insert cloned in pAV520 (vector pLAFR3) is shown; the region present in pAV521 is underlined. Restriction sites for Bamffi (B), EcoRI (E) and Hindlll (H) are marked. I 2 3 4 Sa5b 6 7 8 9 pi ... a.._ ..,_L8kb symptoms produced were still classified as susceptible . The two non-polar mutants, race 7 AORFl and race 7AORF2, respectively, were also virulent in cv. Tendergreen. The continued presence of plasmid-borne virPphA, and possibly other vir genes in race 7 strains, l 2 3 4 Sa Sb 6 7 8 9 may allow the avrPphF mutants to grow but not to reach wild-type levels . avrPphF behaves as a masked avirulence gene in cv. Canadian Wonder 1::.:!;;:1 The plasmid-cured strain RW60 was found to cause a slow HR (subsequently designated hr) in cv. Canadian Wonder, l. 81cb -- • • ._ whereas wild-type race 7 causes water soaking character­ istic of a susceptible reaction. Unexpectedly, transconju­ Fig. 2. Southern hybridization (high stringency) of Bamffi-digested gant RW60(pPPY511), expressing avrPphF, caused a very total DNA from different strains of P.syringae using (A) ORF! and rapid HR on leaves and pods of cv. Canadian Wonder quite (B) ORF2 of avrPphF and (C) the 0.5 kb EcoRI-Bamffi fragment with distinct from the hr caused by RW60 alone (Figure 4B). similarity to 1S100 as probes. Digests from races 1, 2, 3, 4, 5 (a, strain 52A; b, strain 1375A), 6, 7, 8 and 9 of P.s. pv. phaseolicola, and P.s. Transconjugants of RW60 containing pA V520 (which pv. pisi (pi) are shown. harbours avrPphF; Figure 3), like wild-type race 7, caused a susceptible reaction on cv. Canadian Wonder, indicating Hybridization experiments also located avrPphF to the that a gene in pA V520 must suppress the avirulence 154 kb indigenous plasmid pA V51 l. avrPphF was pos­ function of avrPphF. Analysis of subcloned regions of itioned at the left of the PAI recently identified in Pph pA V520 revealed that the gene suppressing the avr (Figure 3), close to avirulence genes avrPphC and a activity of avrPphF was the non-host avirulence gene homologue of avrD, both of which were cloned as soybean avrPphC (Yucel et al., 1994). Transconjugants of RW60 interactors (Kobayashi et al., 1990; Yucel et al., 1994). containing both avrPphF and avrPphC (in pPPY511 and pDAHR15, respectively) gave a phenotype identical to that caused by RW60 (Table II; Figure 4B). Multiplication avrPphF acts as a gene for virulence in of race 7, RW60, RW60(pPPY511) and RW60(pPPY511, cv. Tendergreen pDAHR15) in leaves was compared. The rapid HR Although race 7 of Pph is virulent on cv. Tendergreen, the determined by the presence of avrPphF in plasmid-cured strain RW60 causes an HR. Virulence RW60(pPPY511) was associated with the recovery of towards Tendergreen was restored by the genomic clones very low numbers of bacteria 96 h after inoculation, pA V520 or pA V521 harbouring avrPphF recovered from whereas RW60(pPPY511, pDAHR15) achieved bacterial race 7 strain 1449B (Jackson et al., 1999). A transposon numbers similar to RW60 alone, confirming the pheno­ insertion in pAV521, which reduced ability to restore virulence only to cv. Tendergreen, was located to avrPphF types observed (Figure 6). and we subsequently found that avrPphF alone (pPPY511) effectively restored water-soaking ability to RW60 in pods Analysis of segregating plant populations and leaves of Tendergreen, as well as the distinct The phenotypes caused by RW60, RW60(avrPphF) and avrPphF-Rl interaction phenotype of the HR in cv. Red RW60(avrPphF, avrPphC) were very distinct, as shown in Mexican (Table II; Figure 4A). The complementation Figure 4A and B. We were, therefore, able to analyse the achieved showed, therefore, that avrPphF acts as a segregation of reactions in crosses between cvs Canadian cultivar-specific virulence gene on cv. Tendergreen. Wonder and Tendergreen. Analyses of F populations Transconjugants, RW60(pPPY514) and RW60 (pPPY512), summarized in Tables ill and IV are based on the separately harbouring ORFl and ORF2 of avrPphF, following proposals: (i) Canadian Wonder has two R respectively, were unable to restore virulence (Table II). genes, RF matching avrPphF and R/12 matching a � avr The increased virulence of transconjugants of RW60 gene unmasked in RW60; (ii) Tendergreen has R/31 harbouring avrPphF was also demonstrated by increases matching a second � avr gene in RW60, but does not in bacterial populations in Tendergreen leaves (Figure 5). harbour RF; (iii) the plasmid-cured strain RW60 causes a Interestingly, bacterial growth of the marker exchange rapid HR on cv. Tendergreen and hr on cv. Canadian avrPphF mutant, race 7::avrPphF Tn3gus A33 (Figure 1), Wonder due to the avr/31-R/31 and avr/J2-R/J2 inter­ was restricted compared with the wild type, but the actions, respectively; (iv) avrPphF acts as a virulence 3207 G.Tsiamis et al. Table II. Reactions• caused by strains of P.s. pv. phaseolicola on bean and soybean cultivars Strain Bean cultivar Soybean cultivar Red Mexican Tendergreen Canadian Wonder Osumi Choska Race 7 HR* s s HR s- RW60 HR HR hr N N RW60(pA V520) HR* s s HR s- RW60(pPPY511) HR* s- HR s- s- RW60(pPPY512) HR HR hr N N RW60(pPPY514) HR HR hr N N RW60(pPPY511, pDAHR15) HR* s- hr nt nt Race 7::avrPphFfn3gus A33 s s s nt nt P.s. pv. glycinea HR HR HR s s •s, fully susceptible water-soaked lesion; s-, slower development of lesions than S; HR, hypersensitive reaction; HR*, characteristic of the avrPphF-Rl interaction; hr, slow development of the HR; N, null reaction; nt, not tested. hpAV520 is a genomic clone; plasmids encoding single ORFs or avr genes are pPPY51 l (avrPphF), pPPY512 (avrPphF, ORF2), pPPY514 (avrPphF, ORFl) and pDAHR15 (avrPphC). 100-.------------------ -, -,­ ..L V) '-'0.1 Time after inoculation (h) Fig. 5. Bacterial multiplication in leaves of bean cv. Tendergreen inoculated with suspensions of 2 X 10 cells/ml of race 7 (open), RW60 (striped), RW60 (pPPY5ll) (light grey), RW60 (pAV521) (dark grey) and the avrPphF marker exchange mutant race 7::avrPphF Fig. 4. (A) Reaction phenotypes in a pod of cv. Tendergreen 2 days Tn3gusA33 (black); bars, ± SEM. after inoculation with (from left to right) race 7, RW60, RW60 (pAV520) containing the PAI and RW60(pPPY511) expressing avrPphF alone. (B) Reaction phenotypes in a pod of cv. Canadian slower hr, was epistatic to avr/31. Phenotypes segregated Wonder 2 days after inoculation with (from left to right) race 7, RW60, hr:HR 31:7, closely approximating the predicted 13:3 ratio RW60(pPPY511) expressing avrPphF, and RW60(pPPY511, (X = 0.0025). Reaction to RW60(avrPphF) clearly indi­ pDAHR15) expressing both avrPphF and avrPphC. Note that the cated the presence of the matching dominant RF gene in lesions caused by RW60 and RW60(pPPY511, pDAHR15) are sunken at this stage compared with the water-soaked susceptible response to Canadian Wonder with phenotypes observed, HR:hr or S, race 7, whereas RW60(pPPY511) has already caused a brown lesion 27:11 (Table IV). Amongst the 38 F progeny examined, characteristic of the rapid HR induced. (C) Reactions at infiltration two plants developed a rapid HR when challenged by sites in soybean leaves (cv. Osumi) 3 days after inoculation with (left RW60 (indicating avr/31-R/31 interaction), but were to right) RW60, which causes a null reaction, and the transconjugant RW60(pPPY5ll) expressing avrPphF, which causes a susceptible susceptible to RW60(avrPphF), indicating the absence response recognized by yellowing as shown and later some water­ of RF. The virulence function of avrPphF therefore soaking. appeared to be directly related to blocking the proposed avr/31-R/31 interaction. determinant on cv. Tendergreen. The phenotypes observed Nineteen of the 38 progeny were also tested with the RW60(avrPphF, avrPphC) transconjugant. Amongst the corresponded to those expected from these proposals, the parental genotypes predicted being R/31, R/31; r/12, r/J2; 64 possible genotypes predicted from the Canadian rF, rF for Tendergreen, and r/31, r/31; R/12, R/12; RF, RF Wonder X Tendergreen cross only three, r/31, r/31; r/12, for Canadian Wonder. r/J2; RF, RF and r/31, r/Jl; r/J2, r/J2; RF, rF (or rF, RF) The reaction of F progeny to RW60 (Table III) would be expected to produce a rapid HR when challenged indicated that the avr/J2 gene, although conferring a by RW60(avrPphF) but be susceptible to RW60(avrPphF, 3208 Avirulence-virulence gene from Pseudomonas syringae avrPphC). One plant was indeed detected with this 1000 ....--------------------, phenotype, clearly supporting the gene-specific virulence function of avrPphC masking the avrPphF-RF inter­ action, and also the probable absence of other R genes, ... which would have prevented the establishment of a � 10 0 ..L susceptible response . avrPphF acts as a virulence gene in soybean Race 7 of Pph typically causes a rapid HR in soybean .... leaves, but in certain cvs such as Choska it was found to cause a weak susceptible response recognized by yellow­ ing and water soaking at inoculation sites in leaves. The plasmid-cured strain RW60 caused a null response on all cvs of soybean tested. avrPphF was able to restore virulence to RW60 in cvs Choska and Osumi (Figure 4C; Table II). Restoration of virulence by avrPphF was also demonstrated by increases in bacterial populations in leaves of cv. Osumi (Figure 7). Time after inocul ation {h) Fig. 6. Bacterial multiplication in leaves of bean cv. Canadian Wonder Discussion inoculated with bacterial suspensions of 2 X 10 cells/ml of race 7 The pattern of avirulence conferred by avrPphF in wild­ (open), RW60 (stippled), RW60 (pPPY511) (grey) and RW60(pPPY511, pDAHR15) (black); bars, :<:: SEM. type races of Pph fits that predicted for an ex avr gene Table III. Segregation of resistance to RW60(avr PI , avr/J2,) amongst the F progeny of a cross between Tendergreen (RPI , RPI ; r/J2,, rfJ2,)• and Canadian Wonder (rPI , rPI ; R/J2,, RfJ2,)• Predicted genotype Phenotype of response if R�2 epistatic Number of plants Observed Expected (13:3) RPI .RPI ; R/J2,,R/J2, RPI ,RPI ; r/J2,,R/J2, rPI ,RPI ; R/J2,,R/J2, rPI ,RPI ; r/J2,,R/J2, Slow hr 31 RPI .RPI ; R/J2,,r/J2, rPI RPI ; R/J2,,r/J2, or 31 30.9 rPI ,RPI ; R/J2,,R/J2, rPI ,RPI ; r/J2,, R/J2, susceptible 0 rPI ,rPI; R/J2,,R/J2, rPI ,r PI ; r/J2,,R/J2, rpI ,RPI ; R/J2,,r/J2, rpI ,r pI ; R/J2,, r/J2, rPI ,rPI; r/J2,,rf32,b RPI .RPI ; r/J2,,r/J2, RPI ,rpI; r/J2,,r/J2, Rapid HR 7 7.1 rPI ,RPI ; r/J2,,r/J2, •RPI confers a rapid HR and R/J2, a slow hr. lYfhe predicted phenotype would be a susceptible reaction, unless there are other � avr genes in RW60. Table IV. Segregation of resistance to RW60 (avrPI, avr/J2,, avrPphF") amongst the F progeny of a cross between Tendergreen (RPI , RPI ; r/J2,, r/J2,; rF, rF) and Canadian Wonder (rPI , rPI ; R/J2,, R/J2,; RF, RF) Predicted genotypes Frequency Phenotype Number of plants Observed Expected (3: 1) Dominant RPI , Dominant R/J2,, Dominant RF 27 Dominant RPI , recessive r/J2,, Dominant RF 9 recessive rPI , Dominant R/J2,, Dominant RF 9 Rapid HR 27 28.5 recessive rPI , recessive r/J2,, Dominant RF 3 Total 48 Dominant RPI , Dominant R/J2,, recessive rF 9 Dominant RPI , recessive r/J2,, recessive rF" 3 Slow hr recessive rPI , Dominant R/J2,, recessive rF 3 or recessive rPI , recessive r/J2,, recessive rF" 1 Susceptible Total •avrPphF present on pPPY511. The parental phenotypes were rapid HR and susceptibility on cvs Canadian Wonder and Tendergreen, respectively. Dominant means homozygous (RR) or heterozygous (Rr). Predicted to develop a susceptible reaction due to suppression of Avr�l by AvrPphF or with all R genes recessive, if no other avr-R gene interactions occurring. 9.5 G.Tsiamis et al. matching the Rl gene for resistance in Phaseolus (Taylor ORFs, but only the second is required for avirulence activity (Ronald and Staskawicz, 1988). Here, we show et al., 1996). The cloning strategy adopted failed to isolate the fourth avr gene predicted to match R4 (Table I), that avrPphF is organized into an operon with two ORFs, possibly because the gene may have lethal effects in both of which are needed for function. E. coli. The first avr gene cloned from Pph (Shintaku et al., The mechanism by which avrPphF causes effects in 1989), and designated avrPphA according to the nomen­ plant cells remains unknown. According to published reports, all of the bacterial avr genes that have been tested clature proposed by Vivian and Mansfield (1993), did not elicit the HR if they are expressed transiently in plant cells; appear to match any of the R genes recognized in examples are avrPphB and avrPphE from Pph (Stevens Phaseolus. The proposed gene-for-gene pattern of race structure (Taylor et al., 1996) has, therefore, not et al., 1998). The implication from in planta expression is been entirely confirmed by molecular genetics. that the A vr proteins act as elicitors following their The avrPphF locus is organized into an operon with a delivery into plant cells by the hrp-dependent type Ill secretion apparatus (Alfano and Collmer, 1997; Bonas and characteristic 'h box' promoter indicating regulation by rp Van den Ackerveken, 1997; Galan and Collmer, 1999). Jn HrpL. Both ORFs within the operon were required for planta expression of the ORFs from avrPphF should either avirulence or virulence functions. The requirement reveal whether one functions as the elicitor per se and one for more than one transcriptional unit for the function of an avr gene was described in detail for avrE from P.s. pv. perhaps acts as a chaperone for protein delivery. The two ORFs were always found together, arranged into the tomato by Lorang and Keen (1995). The avrE locus is avrPphF operon, but only in strains expressing the Al linked to the right end of the hrp region and comprises two phenotype (Table I; Figure 2). The adjacent ISJ 00 convergently transcribed units, which are both required for homologue was distributed throughout strains irrespective the avirulence phenotype. avrBsl from Xan thornonas of the race designation. ISJO 0 is present in species of campestris pv. vesicatoria comprises an operon of two Yers inia and several copies have been located in plasmids encoding type Ill secretion systems or other virulence factors (Buchrieser et al., 1998; Hu et al., 1998). Sequences hybridizing to the insertion sequence were, 10 00....----------------- however, not widely distributed amongst other pathovars of P. syringae. Given the pathogenicity functions attributed to other avr .I. genes from P.syringae, e.g. avrE and avrRpml (Lorang ,...._ 100 et al., 1994; Ritter and Dangl, 1995), the finding that ,­ avrPphF was able to restore virulence to compromised .L plasmid-cured strains such as RW60 was not unexpected. However, avrPphF is, to our knowledge, the first gene to be shown to have cultivar- and gene-specific virulence activity. By contrast, the discovery that avrPphF also acts as a � avr gene, i.e. has functions revealed only in the absence of the PAI, operating in a defined gene-for-gene manner with a matching R gene in Canadian Wonder, was most unexpected. The interaction between avrPphC and avrPphF blocking the HR in cv. Canadian Wonder would appear to occur in the plant, possibly with a receptor for 48 72 the A vrPphF protein, because avrPphC has no effect on Time after inoculation (h) the expression of the HR caused by avrPphF in cv. Red Mexican. It is important to emphasize that the virulence Fig. 7. Bacterial growth in leaves of soybean cv. Osumi inoculated and � avirulence functions of avrPphF are only observed with bacterial suspensions of 10cells/ml of P.s. pv. glycinea (open), RW60 (stippled) and RW60 (pPPY511) (grey); bars, ± SEM. in the absence of other genes from the PAI. The A vrPphF Table V. Genes with dual avirulence and virulence functions, either eliciting or blocking the HR, respectively, identified using strains of P.s. pv. phaseolicola lacking the 154 kb plasmid which contains the putative pathogenicity island Gene designation Plant in which phenotype observed• Avirulence Virulence avrPphF Red Mexican , Canadian Wonder' Tendergreen, Soybean avrPphC Soybean Canadian Wonder virPphA Soybean Canadian Wonder, Red Mexican, Tendergreen •Bean cultivars are named. b A virulence due to interaction with different R genes. Yucel et al. (1994). dJackson et al. (1999). 32 10 Avirulence-virulence gene from Pseudomonas syringae Table VI. Bacterial strains and pla smids used in this study" Strain/plasmid Relevant properties Source or reference Bacteria. P.syringae pv. phaseolicola Principal isolates used 13 75A race 5, wild-type isolate D.Teverson 1375AN race 5, Nal from 13 75A D.Teverson 1375AR race 5, Rif1l- from 1375A D.Teverson race 1, wild-type isolate 12 81A D.Teverson 12 81A R race 1, Rif1l- from 12 81A D.Teverson 1448A race 6, wild-type isolate Fillingham et al. (1 992) 1448AR race 6, Rif1l-from 1448A Fillingham et al. (1 992) 1449B race 7, wild-type isolate D.Teverson 1449BR race 7, Rif1l- from 1449B D.Teverson RW60 Vir, pAV 51 1-, Ap , Rif1l- Jackson et al. (1 999) Additional isolates 882 race 2 D.Teverson 13 01A race 3 Hitchin et al. (19 89) 1302A race 4 Jenner et al. (19 91) 52A race 5 Hitchin et al. (19 89) 2656A race 8 D.Teverson 2709A race 9 D.Teverson P.cichorii 2379 lettuce pathogen NCPPB P.s. pv. corna faciens 1354 oat pathogen Harper et al. (19 87) P.s. pv. pisi 299A pea pathogen J.Taylor' P.s. pv. glycinea 11 39 soybean pathogen this study P.s. pv. maculicola 18 20 brassica pathogen NCPPB P.s. pv. tabaci 11 528 tobacco pathogen J.Turnerd P.s. pv. tomato DC3000 tomato pathogen Whalen et al. (1 99 1) P.s. pv. syringae 28 1 lilac pathogen NCPPB E.coli C211 0 Nal,polAJ Leong et al. (19 82) DH5a Nal , recA, laczt:.M15 Bethesda Research Laboratories HB101 Sm , recA Boyer and Roulland-Dussoix (19 69) Plasmids + R pBluescript SK Ap , multiple cloning sites and priming sites Stratagene R R pHoKmGus Ap , Km , tnpA , promoterless �-glucuronidase gene Bonas et al. (19 89) in Tn3, pWB15A replicon R + pLAFR3 Tc , IncPl replicon, Tra-, Mob, cosmid Sta skawicz et al. (19 87) Sp, Inc Q pDSK600 replicon, 3X lac UV5promoter Murillo et al. (1 994) R + + pRK20 13 Km , Co!El replicon, Tra, Mob, helper plasmid Figurski and Helinski (19 79) R + pSShe Cm , tnpA , pACYC 184 replicon Stachel et al. (19 85) Clones containing avrPphF pPPY501 pLAFR3-based genomic clone from race 5 strain 13 75 this study pPPY502 pLAFR3-based genomic clone from race 5 strain 13 75 this study pPPY503 5.3 kb BamHI-H indIII subclone of pPPY502 harbouring avrPphF this study pPPY505 1. 8 kb BamHI fragment from pPPY503 in pBluescript II SK this study pPPY507 1.1 kb EcoRI fragment in pLAFR3 thi s study pPPY5 11 insert as in pPPY505 but in pDSK600 this study pPPY5 12 avrPphF ORF2 in pDSK600 this study pPPY5 14 avrPphF ORFl in pDSK600 this study Additional plasmids pMS2330 pLAFRl based genomic clone harbouring avrPphA from HB33 Shintaku et al. (19 89) pAV 51 1 native plasmid from 1449B, ~15 4 kb Jackson et al. (1 999) pAV520 genomic clone harbouring pA V5 11 sequences in pLAFR3 Jackson et al. (1 999) pAV521 genomic clone harbouring pAV 511 sequences in pLAFR3. Jackson et al. (1 999) pDAHR 15 1. 4 kb Clal-BamHI fragment containing avrPphC in pDSK600 Yucel et al. (1 994) R R R R R R •Nal , Rif1l-, Sm , Ap , Km , Tc and Cm indicate re sistance to nalidixic acid, rifampicin, streptomycin, ampicillin, kanamycin, tetracycline and chloramphenicol, respectively. Horticulture Research International, Wellesboume, UK. National Collection of Plant Pathogenic Bacteria, York, UK. dUniversity of East of Anglia, Norwich, UK. proteins appear to block the HR caused by � avr genes The ability of certain A vr proteins to express virulence such as avrfi l, which is predicted to be present in the functions, blocking the phenotype conferred by other avr chromosome of Pph. The � avr function of avrPphF is in genes, may involve three different types of interaction: turn blocked by avrPphC. In contrast to avrPphF, the other (i) between the A vr proteins themselves; (ii) between A vr genes recognized to act as virulence determinants, and masked R proteins; (iii) downstream of hypothetical virPphA and to a lesser extent ORF4 from the PAI, do Avr-R protein interactions to interfere with gene-specific not have differential effects in the cultivars tested (Jackson signalling cascades leading to the HR. A possible target for et al. , 1999). (iii) would be one of the MAP kinases which have been 3211 G.Tsiamis et al. Pathogenicity tests and in planta bacterial population implicated in plant defence (Grant and Mansfield, 1999; counts Romeis et al., 1999). The virulence determinant YopJ Pods and leaves of French bean were inoculated as described previously from Yers inia has recently been found to bind and (Harper et al., 1987; Hitchin et al., 1989). The inoculum concentration inactivate MAP kinase kinases in mammalian cells, routinely used in bean leaves was 2 X 10cells/ml. Soybean plants were inoculated using a l ml syringe (without needle) to infiltrate bacterial leading to suppression of defence responses (Orth et al., suspensions of 10cells/ml into the underside of fully expanded primary 1999). There are no reports of direct interactions between leaves. Bacterial multiplication in leaves was examined by cutting tissue Avr proteins, but Avr-R protein binding has been from the inoculation sites with a 0.6-cm-diameter borer, homogenization demonstrated between A vrPto and Pto (Tang et al., in 10 mM MgCh, and serial dilution of the homogenate, which was then spread onto LB agar with appropriate antibiotics to allow colony 1996). However, the action of intermediates linking Avr development at 25 C. and R proteins has also been proposed (Grant and Mansfield, 1999). General molecular techniques The multiple function of avr genes found in Phaseolus Basic procedures were carried out as described in Sambrook et al. (1989). The methods of Jenner et al. (1991) and Mansfield et al. (1994) were was also extended to the Pph-soybean interaction. followed for the construction of the genomic library from race 5 strain Whether or not soybean should continue to be considered 1375A, preparation of minipreps, Southern blotting and hybridization. as a non-host to Pph (Yucel et al., 1994) is questionable as The library was screened for determinants of avirulence by conjugation of certain strains clearly show weak pathogenicity. individual clones into race 6 strain l 448AR with the helper plasmid pRK2013. Transconjugants were tested for pathogenicity on pods of cvs Interestingly, however, genes recognized for virulence or Red Mexican and A53 as described by Harper et al. (1987). Transposon avirulence function in Phaseolus have so far all displayed mutagenesis of pA V521 and pPPY503, and marker exchange of the opposite activity in soybean leaves, as summarized in avrPphF::Tn3gusA33 into race 7 were carried out as described by Table V. Pathogenicity towards the legume hosts has Mansfield et al. (1994); the position of the transposon A33 in the target DNA was confirmed by sequencing. probably involved interaction with common virulence targets. As targets mutated to activate resistance (i.e. PCR and DNA sequencing acting as R gene products), perhaps by hyperactivation of Standard PCRs were performed with Taq polymerase and buffers from signal transduction cascades blocked by Vir factors, the Bioline using a Perkin Elmer Gene Amp 2400. Sequencing was performed using the dideoxy chain termination method with the pathogen would need to acquire further vir genes whose Sequenase enzyme (Pharmacia) as described in the manufacturer 's products would override the activation of resistance. instructions. Primers used for the amplification of avrPphF ORF l were: Restoration of virulence could be achieved by suppression ORFlF, 5'-ATGAAGAATTCGTTCGACCG-3'; ORFlR, 5'-TCAGAC­ of signal transduction, or, as outlined above, simply by CGAACTCTCAGACA-3'. For the amp lification of avrPphF ORF2, the primers used were: ORF2F, 5'-ATGGGTAATATCTGCAATTCG-3'; blocking any direct avr-R gene interactions. ORF2R, 5'-GGCCAGTTATAGGAGCTAAT-3'. An intriguing question is whether or not the avr or vir genes now identified from a highly evolved pathogen such Construction of non-polar mutations as Pph represent virulence determinants that have had a For the generation of the non-polar mutants, race 7.1.ORFl and race 7.1.ORF2, the following steps were followed. ORFl was deleted fundamental role in the evolution to parasitism from a from position 332-439 in pPPY505 by a double digest with Aatll-Agel. saprophytic ancestry. The layers of interactions and Digested pPPY505 was filled in using Kienow polymerase and re-ligated. possibly interconnected targets that exist amongst the Deletions were selected by PCR and the deleted form cloned into pOK numerous avr and vir genes in the present populations of (Huguet et al., 1998). ORF2 was deleted from position 767-1157 in pPPY505 by a double digest with Cs p451-Bst9 8I. The plasmid with the plant pathogens may make it difficult to distinguish the digested insert was filled in and re-ligated. Cloned deletions were again evolutionary significance of individual effector proteins. It selected by PCR and the deleted form was then introduced to pOK. is possible that gene-specific virulence, as shown by Mutated genes were introduced to race 7 strain 1449BR by homologous avrPphF, represents a second wave of activity, whereas recombination in two steps as described by Kaniga et al. (1991) and Huguet et al. (1998). In both mutants, the presence of the correct deletion virPphA, apparently with less cultivar specificity, may in race 7 was verified by PCR. have more direct effects on signal transduction pathways and represent the first class of virulence factor. The co­ evolution of hosts and pathogens would mean that clear Ackn owledgements distinction between primary and secondary waves of Many thanks are due to Elisabeth Huguet and Ulla Bonas for advice on activity may have become very blurred, and it is certainly the creation of non-polar mutants, to Noel Keen for clones containing impossible to predict the function of avirulence and avrPphC and to Suresh Patil for avrPphA. We also wish to acknowledge virulence factors we have identified. In order to unravel the support from the BBSRC, EC grants BIO-CT97-2244 and TMR, CICYT grant BI097-0598 and the British Council-MEC Acciones Integradas complex web of interacting effectors, it is now a priority to programme. 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(1989) Cloning and partial characterization of an avirulence determinant from race 1 of Pseudomonas syringae pv. phaseolicola. Physiol. Mol. Plant Pathol., 35, 313-322. Singer,M. and Berg,P. (1991) Genes and Genomes: A Changing Perspective. University Science Books, Mill Valley, CA. Stachel, S.E., An,G., Flores,C.W. and Nester,E.W. (1985) A Tn3lacZ transposon for the random generation of 13-galactosidase gene fusions: Application to the analysis of gene expression in Agrobacterium. EMBO J., 4, 891-898. Staskawi cz,B., Dahlbeck ,D., Keen,N. and Napoli,C. (1987) Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea. J. Bacterial., 169, 5789-5794. Stevens,C., Bennet t,M.A., Athanassopoulo s,E., Tsiam is,G., Taylor ,J.D. and Mansfield,J.W. (1998) Sequence variations in alleles of the avirulence gene avrPphE.R2 from Pseudomonas syringae pv. phaseolicola lead to loss of recognition of the A vrPphE protein within bean cells and a gain in cultivar-specific virulence. Mol. Microbial., 29, 165-177. Tang,X .Y., Frederick ,R.D., Zhou,J., Halterman ,D.A., Jia,Y. and Martin, G.B. (1996) Initiation of plant-disease resistance by physical interaction of AvrPto and Pto kinase. Science, 274, 2060-2063. Taylor ,J.D., Teverso n,D.M., Allen,D.J. and 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. Van den Ackerveken ,G.F.J.M., Marois,E. and Bonas,U. (1996) Recognition of the bacterial avirulence protein A vrBs3 occurs inside the host plant cell. Cell, 87, 1307-1316. Vivian,A. and Mansfield,J. (1993) A proposal for a uniform genetic nomenclature for avirulence genes in phytopathogenic pseudomonads. Mol. Plant Microbe Inte ract., 6, 9-10. Vivian, A., Gibbon, M.J. and Murillo,J. (1997) The molecular genetics of specificity determinants in plant pathogenic bacteria. In Crute ,I.R., Holub,E.B. and Burdon,J.J. (eds), The Gene- for-Gene Relationship in Host-Parasite Inte ractions. CAB International, Wallingford, UK, pp. 293-328. Whalen ,M.C., Innes ,R.W., Bent, A.F. and Staskawi cz,B.J. (1991) Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining avirulence on both Arab idopsis and soybean. Plant Cell, 3, 49-59. Wood,J.R., Vivian,A., Jenner,C., Mansfield,J.W. and Taylor,J.D. (1994) Detection of a gene in pea controlling nonhost resistance to Pseudomonas syringae pv. phaseolicola. Mol. Plant Microbe Inte ract., 1, 534-537. Yucel,I., Slaymaker,D., Boyd,C., Murillo,J., Buzzell, R.I. and 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., 1, 677---679. Received March 6, 2000; revised May 3, 2000; accepted May JO, 2000 32 14 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Cultivar‐specific avirulence and virulence functions assigned to avrPphF in Pseudomonas syringae pv. phaseolicola, the cause of bean halo‐blight disease

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The EMBO Journal Vol. 19 No. 13 pp. 3204-3214, 2000 Cultivar-specific avirulence and virulence functions assigned to avrPphF in Pseudomonas syringae pv. phaseolicola, the cause of bean halo-blight disease ated with activation of the hypersensitive reaction (HR), a George Tsiamis, John W.Mansfield , form of rapid programmed cell death, which restricts Ruth Hockenhull, Robert W.Jackson , colonization to the site of inoculation in resistant cultivars Ane Sesma , Evangelos Athanassopoulos, of the host plant (De Wit, 1995; Mansfield et al., Mark A.Bennett, Conrad Stevens, 2 3 1997a). This article concerns the genetics of avirulence Alan Vivian , John D.Taylor and and virulence in the bean halo-blight bacterium Jesus Murillo Pseudomonas syringae pathovar (pv.) pftaseolicola (here­ Department of Biological Sciences, Wye College, University of after Pph). Nine races of Pph have been differentiated London, Ashford, Kent TN25 5AH, Department of Biological and based on their virulence to a range of bean cultivars, as Biomedical Sciences, University of the West of England, Coldharbour 3 summarized in Table I (Taylor et al., 1996). Lane, Bristol BS16 l Y, Horticulture Research International, The gene-for-gene theory explaining cultivar-specific Wellesbourne, Warwick CV35 9EF, UK and 4Escuela Tecnica Superior de Ingenieros Agr6nomos, Universidad Publica de Navarra, resistance to plant disease was developed by Flor (1942, 31006 Pam plona, Spain 1971) based on his analysis of the differential reactions of flax to the rust fungus Melampsora lini. Person et al. Corresponding author e-mail: [email protected] (1962) summarized the essence ofFlor's hypothesis in the statement that ' ... for every resistance gene in flax there is a The avrPphF gene was cloned from Pseudomonas specific and related gene for virulence in those rusts to syringae pathovar phaseolicola (Pph) races 5 and 7, which it is susceptible'. The emphasis on susceptibility based on its ability to confer avirulence towards bean suggested that disease was the result of the positive cultivars carrying the Rl gene for halo-blight resist­ function of cultivar-specific virulence determinants. The ance, such as Red Mexican. avrPphF comprised two advances of molecular genetics have proved the gene-for­ open reading frames, which were both required for gene theory by the cloning not of virulence (vir) genes, but function, and was located on a 154 kb plasmid of avirulence (avr) genes from bacteria and fungi, and the (pA V511) in Pph. Strain RW60 of Pph, lacking matching resistance (R) genes from plant hosts (Dangl, pA V511, displayed a loss in virulence to a range of 1994; De Wit, 1995; Jones and Jones, 1996; Vivian et al., previously susceptible cultivars such as Tendergreen 1997). The proteins encoded by avr and R genes are and Canadian Wonder. In Tendergreen virulence was envisaged to interact either directly or indirectly to restored to RW60 by avrPphF alone, whereas sub­ activate signalling cascades leading to resistance, which cloned avrPphF in the absence of pA V511 greatly is expressed by the HR (Hammond-Kosack and Jones, accelerated the hypersensitive resistance reaction 1996; Van den Ackerveken et al., 1996). Bacterial avr caused by RW60 in Canadian Wonder. A second gene genes have been cloned by function through the transfer of from pA V511, avrPphC, which controls avirulence to genomic libraries from avirulent to virulent races of the soybean, was found to block the activity of avrPphF in pathogen (Staskawicz et al., 1987). Genomic clones Canadian Wonder, but not in Red Mexican. avrPphF causing changes in the virulence phenotype on certain also conferred virulence in soybean. The multiple cultivars have been isolated and genes for avirulence functions of avrPphF illustrate how effector proteins subsequently characterized, e.g. avrPphB and avrPphE from plant pathogens have evolved to be recognized from Pph, which correspond to A3 and A2, respectively, in by R gene products and, therefore, be classified as Table I (Mansfield et al., 1994; Vivian et al., 1997). encoded by avirulence genes. Similarly, genes for avirulence but not virulence have also Keywords: avirulence/disease resistance/gene-for-gene been cloned, which operate at the level of host specificity interactions/hypersensitive reaction/virulence (Kobayashi et al., 1989; Fillingham et al., 1992; Wood et al., 1994). Clearly, the continued presence of genes that restrict host range in certain strains or races of plant pathogens is Introduction somewhat of a paradox, but some avr genes have been The bacteria and fungi that colonize living plants and implicated in basic parasitism, e.g. avrRpml from P.s. pv. cause disease have evolved the ability to overcome the maculicola and avrE from P.s. pv. tomato. Mutations in innate resistance of their hosts to infection. Superimposed avrRpml or avrE lead to greatly reduced ability to on this establishment of basic parasitism ( or patho­ colonize host plants (Lorang et al., 1994; Ritter and genicity) is the phenomenon of cultivar (cv.)-specific Dangl, 1995). The emerging pattern is that plant pathogens resistance. A plant pathogen may, therefore, be fully may produce numerous proteinaceous virulence factors, virulent causing disease on certain cultivars of its host, but which act synergistically to promote disease (Alfano and avirulent on others (De Wit, 1995; Alfano and Collmer, Collmer, 1997; Collmer, 1998). Some of these factors may 1997; Grant and Mansfield, 1999). Avirulence is associ- become recognized by the products of R genes and, 3204 © European Molecular Biology Organization Avirulence-virulence gene from Pseudomonas syringae Table L Gene-for-gene relationship (based on five matching gene pairs) proposed from analysis of the reactions• of bean cultivars to races of P.s. pv. phaseolicola (from Taylor et al., 1996) Races/avirulence (A) genes 1 2 3 4 5 7 9 6 8 Al Al Al Al A2 A2 A2 A2 A3 A3 A4 Differential cultivars Resistance genes A5 A5 A5 Canadian Wonder + + + + + + + + + A52 (ZAA54) R4 + + + + + + + + Tendergreen R3 + + + + + + + Red Mexican Rl R4 + + + + + A53 (ZAA55) R3 R4 + + + + + + A43 (ZAA12) R2 R3 R4 R5 + + '+, susceptible; -, resistant. therefore, be identified as determinants of avirulence. Here we describe molecular characterization of the Because of functional redundancy, however, the loss of avrPphF locus in Pph. The gene was found to comprise an operon with two open reading frames (ORFs), both of what has become an avr gene rather than a vir gene does which were required for function. Although we identified not always compromise the basic pathogenicity on which avrPphF as an a, avr gene that determined race structure cultivar specificity is superimposed. in the halo-blight bacterium, it was also found to The vir gene concept has been strengthened by the confer cultivar-specific virulence to cv. Tendergreen. identification of a gene, virPphA from Pph, based on its Furthermore, in the absence of the plasmid pAV511, ability to restore virulence to strains of the bean pathogen containing the P Al, avrPphF also acted as a � avr gene in cured of a 154 kb plasmid designated pAV511 (Jackson cv. Canadian Wonder, which is susceptible to all known et al., 1999). Cured strains (such as that named RW60) lost races of the halo-blight bacterium. The gene within virulence towards bean, causing hypersensitive reactions pAV511 that masked the � type of avr activity of in all previously susceptible cultivars tested. A cluster of avrPphF to Canadian Wonder was found to be avrPphC, potential vir genes including virPphA was located to a which was previously identified as an avr gene acting on region described as a pathogenicity island (P Al), on soybean cultivars. In short, an intriguing web of interact­ pA V511. The ability of RW60 to elicit the HR suggested ing avirulence and virulence functions has been revealed that it contained avr genes, located on the chromosome or for the avr genes from Pph. In the bean-P.syringae other plasmids, whose function was usually masked by interaction, the gene-for-gene concept, although continu­ virulence factors encoded by the PAL Other avr genes, ing to provide a useful framework for analysis of cultivar­ such as avrPphB or avrPphE, continued to function specific resistance, is far more complex than is apparent despite the operation of the plasmid-borne P Al in wild­ from simple analysis of race structures and a, avr-R gene type strains and were, therefore, classified as a, avr genes. interactions as outlined in Table I. It is proposed that The genes detected only in the absence of the PAI were similar complexity will emerge in other host-pathogen classified as� avr genes (Jackson et al., 1999). systems once the problem of the redundancy of multiple The resistance responses activated by different avr-R virulence factors has been overcome by the removal of gene combinations may all be classified as the HR, but certain P Als. they may be phenotypically different because of, for example, differential timing of plant cell collapse. Differences are clear in bean as the avrPphB-R3 inter­ Results action provokes a more rapid HR than avrPphE-R2 (Mansfield et al., 1994, 1997a,b). In many race--cultivar Cloning of avrPphF from race 5 interactions, several different a, avr genes and their Based on the race structure proposed in Table I (Taylor matching R genes may be present in the pathogen and et al., 1996), we sought to clone the putative Al and A4 plant, respectively (Mansfield et al., 1997a). The potential genes from race 5. A genomic library of race 5 strain for epistatic interaction between various avr-R gene 1375A, predicted to contain Al and A4 and known to combinations is apparent from analysis of Arabidopsis harbour A2 (cloned as avrPphE by Stevens et al., 1998), harbouring the RPMJ and RPS2 genes. Although the HR was screened for avr genes by mating into race 6 strain controlled by avrRpt2-RPS2 is slower than that activated 1448A, which is virulent on all cultivars of bean used by the avrRpml-RPMJ interaction, the phenotypically to define race structure (Mansfield et al., 1994). slower response is observed when both avr genes are Transconjugants were initially tested for virulence on present (Reuber and Ausubel, 1996; Ritter and Dangl, pods of cvs Red Mexican UI3 (proposed Rl, R4) and A53 1996). It has been proposed that such epistasis involves (R3, R4). Two genomic clones, pPPY501 and pPPY502, direct interaction between avr and R gene products (Ritter containing overlapping regions of insert DNA conferred and Dangl, 1996). avirulence on Red Mexican but none from the 1500 tested 3205 G.Tsiamis et al. EMBL/GenBank accession Nos AF231452 and AF231453). The sequence revealed two ORFs with a single upstream promoter region containing the hrp box motif, indicating probable regulation by HrpL (Innes et al., 1993; Pirhonen et al., 1996). Both ORFs were preceded by purine-rich regions, which would be expected to act as ribosome binding sites (Singer and Berg, 1991) and they were separated by a 57 bp sequence. A rho-dependent pPPY507 s transcription termination loop was located after the pPPY512 translation stop signal in ORF2. The avrPphF locus pPPY514----- therefore had the structural features of an operon. The Fig. 1. Location of avrPphF within pPPY503 and the 1.8 kb of DNA G+C content of avrPphF was 47%, with ORFl 40% and sequenced. The direction of transcription is indicated by the horizontal ORF2 52.5%, both significantly lower than the overall arrowheads. Sites of insertion of Tn3gus in pPPY503 are indicated by figures of 59--61 % reported for pvs of P.syringae (De Ley, vertical arrowheads; only those marked black in the expanded fragment 1968). abolished the avirulence activity of avrPphF. All transposon insertions Transposon insertions that compromised avirulence were mapped from restriction digests, except A33, which was located by sequencing. The region found to have similarity to ISJOO is were located in both ORFl and ORF2 of avrPphF, and indicated. Restriction sites for BamHI (B), Bgill (Bg), EcoRI (E) and further subcloning revealed that both ORFs were required Pstl (P) are marked; an asterisk indicates site in the vector pLAFR3. for avr function (Figure 1). To examine further the role of The reactions caused in bean cv. Red Mexican by transconjugants of the two ORFs, non-polar mutations were created for ORFl race 6 harbouring subclones are noted. HR, hypersensitive reaction; S, susceptible response. and ORF2, resulting in race 7 AORFl and race 7 AORF2, respectively; both mutants failed to elicit the HR in cv. Red Mexican. Protein production from ORFs 1 and 2 was examined in affected reactions on A53. Avirulence activity was Escherichia coli using constructs of avrPphF cloned for retained by a 5.3 kb fragment, designated pPPY503. expression in pBluescript. Proteins of 17 and 24 kDa were Transposon mutagenesis with Tn3 gus located avrPphF to detected on SDS-P AGE gels after Coomassie Blue a 1.3 kb region flanked by BamHI and EcoRI restriction staining, corresponding to the peptides predicted to be sites (Figure 1). The 1.8 kb BamHI fragment containing encoded by ORFs 1 and 2, i.e. 15.6 and 21.9 kDa, this region was cloned as pPPY511, and found to activate respectively (data not shown). Searches of the databases the HR in a cultivar-specific manner; avrPphF therefore using the BLAST or Propsearch approaches (Hobohm and acted as a typical ex avr gene. Sander, 1995; Altschul et al., 1997) failed to reveal Segregation of the Rl resistance gene matching significant homology with known protein sequences. Both avrPphF was examined in 88 F progeny derived from a peptides are predicted to be hydrophilic; they lack leader cross between Red Mexican and Tendergreen (R3). sequences or potentially membrane-spanning domains. Progeny undergoing the HR to the transconjugant race 6(pPPY503) were also resistant to race 1 (Al). The pattern of incompatibility observed fitted that expected if avrPphF is plasmid borne and present only in avrPphF matched the Rl gene in Red Mexican, the races expressing the A 1 phenotype numbers of resistant and susceptible plants (66:22) exactly The presence of homologues of avrPphF was examined in fitting the 3: 1 ratio expected if the Rl gene was dominant. strains of Pph representing all nine races and also in other The F progeny from the cross between Red Mexican and pathovars of P.syringae. Using specific probes for ORFl Tendergreen were also tested with a transconjugant of and ORF2 in PCR or Southern analysis, signals identical to race 6 containing the clone pMS2330, which contains those found in race 5, the origin of the avrPphF clone, avrPphA. isolated by Shintaku et al. (1989). Although were present only in races 1, 7 and 9, but not in races 2, 3, race 6(pMS2330) did cause a weak HR on Red Mexican, 4, 6 or 8, which are virulent on Red Mexican (Table I; reactions on pods from the F progeny of Red Mexican and Figure 2A and B). However, when the entire 1.8 kb BamHI Tendergreen showed that there was no co-segregation with fragment containing avrPphF was used as a probe the race 1 (data not shown). pattern changed. Multiple signals were detected in all Multiplication of race 1 and race 6 was compared with races of Pph (Figure 2C). Further probing revealed that race 6(pPPY503) in leaves of cv. Red Mexican. The HR multiple signals were due to the 0.5 kb EcoRI-BamHI determined by the presence of the cloned avrPphF gene fragment downstream of avrPphF (Figure 1 ), which was was associated with the failure of bacteria to multiply. 6 found to contain an incomplete ORF with predicted Following inoculation to introduce ~0.2 X 10 c.f.u. per similarity to the /Sl 00 transposase homologue from the excised leaf disc, after 2 days race 6 had reached 6 PAI in Pph (Jackson et al., 1999). Sequences hybridizing 3.7 X 10 , whereas race 1 and race 6(pPPY503) were 2 2 to avrPphF were found in strains of P.s. pvs pisi and present at only 9.3 X 10 and 16 X 10 c.f.u. per disc, glycinea, but not in pvs coronafaciens, maculicola, tabaci, respectively. tomato or syringae, or in Pseudomonas cichorii. Interestingly, only P.s. pv. syringae and P.s. pv. pisi Sequence analysis and expression of avrPphF contained sequences hybridizing to the putative transpo­ The nucleotide sequences of the functional 1.8 kb frag­ sase, but, in contrast to Pph, only one band was observed ments from race 5, and as also recovered from race 7 on Southern blots (data not shown), indicating that it is containing avrPphF, were found to be identical (DDBJ/ highly specific to pv. phaseolicola. 3206 Avirulence-virulence gene from Pseudomonas syringae E EH B BE EB EHH BE EBE EH pAV520 1 2 3 4 5a5b 6 7 8 9 pi "' pAV521------------- uvrl'phC wrf'pM. ORF4 {Nf/) uvrPphJ, l . 8kb --- ..,_ Fig. 3. Location of avrPphF in relation to avrD, virPphA, avrPphC and ORF4 in the 154 kb plasmid identified in race 7 strain 1449B (Jackson et al., 1999). The insert cloned in pAV520 (vector pLAFR3) is shown; the region present in pAV521 is underlined. Restriction sites for Bamffi (B), EcoRI (E) and Hindlll (H) are marked. I 2 3 4 Sa5b 6 7 8 9 pi ... a.._ ..,_L8kb symptoms produced were still classified as susceptible . The two non-polar mutants, race 7 AORFl and race 7AORF2, respectively, were also virulent in cv. Tendergreen. The continued presence of plasmid-borne virPphA, and possibly other vir genes in race 7 strains, l 2 3 4 Sa Sb 6 7 8 9 may allow the avrPphF mutants to grow but not to reach wild-type levels . avrPphF behaves as a masked avirulence gene in cv. Canadian Wonder 1::.:!;;:1 The plasmid-cured strain RW60 was found to cause a slow HR (subsequently designated hr) in cv. Canadian Wonder, l. 81cb -- • • ._ whereas wild-type race 7 causes water soaking character­ istic of a susceptible reaction. Unexpectedly, transconju­ Fig. 2. Southern hybridization (high stringency) of Bamffi-digested gant RW60(pPPY511), expressing avrPphF, caused a very total DNA from different strains of P.syringae using (A) ORF! and rapid HR on leaves and pods of cv. Canadian Wonder quite (B) ORF2 of avrPphF and (C) the 0.5 kb EcoRI-Bamffi fragment with distinct from the hr caused by RW60 alone (Figure 4B). similarity to 1S100 as probes. Digests from races 1, 2, 3, 4, 5 (a, strain 52A; b, strain 1375A), 6, 7, 8 and 9 of P.s. pv. phaseolicola, and P.s. Transconjugants of RW60 containing pA V520 (which pv. pisi (pi) are shown. harbours avrPphF; Figure 3), like wild-type race 7, caused a susceptible reaction on cv. Canadian Wonder, indicating Hybridization experiments also located avrPphF to the that a gene in pA V520 must suppress the avirulence 154 kb indigenous plasmid pA V51 l. avrPphF was pos­ function of avrPphF. Analysis of subcloned regions of itioned at the left of the PAI recently identified in Pph pA V520 revealed that the gene suppressing the avr (Figure 3), close to avirulence genes avrPphC and a activity of avrPphF was the non-host avirulence gene homologue of avrD, both of which were cloned as soybean avrPphC (Yucel et al., 1994). Transconjugants of RW60 interactors (Kobayashi et al., 1990; Yucel et al., 1994). containing both avrPphF and avrPphC (in pPPY511 and pDAHR15, respectively) gave a phenotype identical to that caused by RW60 (Table II; Figure 4B). Multiplication avrPphF acts as a gene for virulence in of race 7, RW60, RW60(pPPY511) and RW60(pPPY511, cv. Tendergreen pDAHR15) in leaves was compared. The rapid HR Although race 7 of Pph is virulent on cv. Tendergreen, the determined by the presence of avrPphF in plasmid-cured strain RW60 causes an HR. Virulence RW60(pPPY511) was associated with the recovery of towards Tendergreen was restored by the genomic clones very low numbers of bacteria 96 h after inoculation, pA V520 or pA V521 harbouring avrPphF recovered from whereas RW60(pPPY511, pDAHR15) achieved bacterial race 7 strain 1449B (Jackson et al., 1999). A transposon numbers similar to RW60 alone, confirming the pheno­ insertion in pAV521, which reduced ability to restore virulence only to cv. Tendergreen, was located to avrPphF types observed (Figure 6). and we subsequently found that avrPphF alone (pPPY511) effectively restored water-soaking ability to RW60 in pods Analysis of segregating plant populations and leaves of Tendergreen, as well as the distinct The phenotypes caused by RW60, RW60(avrPphF) and avrPphF-Rl interaction phenotype of the HR in cv. Red RW60(avrPphF, avrPphC) were very distinct, as shown in Mexican (Table II; Figure 4A). The complementation Figure 4A and B. We were, therefore, able to analyse the achieved showed, therefore, that avrPphF acts as a segregation of reactions in crosses between cvs Canadian cultivar-specific virulence gene on cv. Tendergreen. Wonder and Tendergreen. Analyses of F populations Transconjugants, RW60(pPPY514) and RW60 (pPPY512), summarized in Tables ill and IV are based on the separately harbouring ORFl and ORF2 of avrPphF, following proposals: (i) Canadian Wonder has two R respectively, were unable to restore virulence (Table II). genes, RF matching avrPphF and R/12 matching a � avr The increased virulence of transconjugants of RW60 gene unmasked in RW60; (ii) Tendergreen has R/31 harbouring avrPphF was also demonstrated by increases matching a second � avr gene in RW60, but does not in bacterial populations in Tendergreen leaves (Figure 5). harbour RF; (iii) the plasmid-cured strain RW60 causes a Interestingly, bacterial growth of the marker exchange rapid HR on cv. Tendergreen and hr on cv. Canadian avrPphF mutant, race 7::avrPphF Tn3gus A33 (Figure 1), Wonder due to the avr/31-R/31 and avr/J2-R/J2 inter­ was restricted compared with the wild type, but the actions, respectively; (iv) avrPphF acts as a virulence 3207 G.Tsiamis et al. Table II. Reactions• caused by strains of P.s. pv. phaseolicola on bean and soybean cultivars Strain Bean cultivar Soybean cultivar Red Mexican Tendergreen Canadian Wonder Osumi Choska Race 7 HR* s s HR s- RW60 HR HR hr N N RW60(pA V520) HR* s s HR s- RW60(pPPY511) HR* s- HR s- s- RW60(pPPY512) HR HR hr N N RW60(pPPY514) HR HR hr N N RW60(pPPY511, pDAHR15) HR* s- hr nt nt Race 7::avrPphFfn3gus A33 s s s nt nt P.s. pv. glycinea HR HR HR s s •s, fully susceptible water-soaked lesion; s-, slower development of lesions than S; HR, hypersensitive reaction; HR*, characteristic of the avrPphF-Rl interaction; hr, slow development of the HR; N, null reaction; nt, not tested. hpAV520 is a genomic clone; plasmids encoding single ORFs or avr genes are pPPY51 l (avrPphF), pPPY512 (avrPphF, ORF2), pPPY514 (avrPphF, ORFl) and pDAHR15 (avrPphC). 100-.------------------ -, -,­ ..L V) '-'0.1 Time after inoculation (h) Fig. 5. Bacterial multiplication in leaves of bean cv. Tendergreen inoculated with suspensions of 2 X 10 cells/ml of race 7 (open), RW60 (striped), RW60 (pPPY5ll) (light grey), RW60 (pAV521) (dark grey) and the avrPphF marker exchange mutant race 7::avrPphF Fig. 4. (A) Reaction phenotypes in a pod of cv. Tendergreen 2 days Tn3gusA33 (black); bars, ± SEM. after inoculation with (from left to right) race 7, RW60, RW60 (pAV520) containing the PAI and RW60(pPPY511) expressing avrPphF alone. (B) Reaction phenotypes in a pod of cv. Canadian slower hr, was epistatic to avr/31. Phenotypes segregated Wonder 2 days after inoculation with (from left to right) race 7, RW60, hr:HR 31:7, closely approximating the predicted 13:3 ratio RW60(pPPY511) expressing avrPphF, and RW60(pPPY511, (X = 0.0025). Reaction to RW60(avrPphF) clearly indi­ pDAHR15) expressing both avrPphF and avrPphC. Note that the cated the presence of the matching dominant RF gene in lesions caused by RW60 and RW60(pPPY511, pDAHR15) are sunken at this stage compared with the water-soaked susceptible response to Canadian Wonder with phenotypes observed, HR:hr or S, race 7, whereas RW60(pPPY511) has already caused a brown lesion 27:11 (Table IV). Amongst the 38 F progeny examined, characteristic of the rapid HR induced. (C) Reactions at infiltration two plants developed a rapid HR when challenged by sites in soybean leaves (cv. Osumi) 3 days after inoculation with (left RW60 (indicating avr/31-R/31 interaction), but were to right) RW60, which causes a null reaction, and the transconjugant RW60(pPPY5ll) expressing avrPphF, which causes a susceptible susceptible to RW60(avrPphF), indicating the absence response recognized by yellowing as shown and later some water­ of RF. The virulence function of avrPphF therefore soaking. appeared to be directly related to blocking the proposed avr/31-R/31 interaction. determinant on cv. Tendergreen. The phenotypes observed Nineteen of the 38 progeny were also tested with the RW60(avrPphF, avrPphC) transconjugant. Amongst the corresponded to those expected from these proposals, the parental genotypes predicted being R/31, R/31; r/12, r/J2; 64 possible genotypes predicted from the Canadian rF, rF for Tendergreen, and r/31, r/31; R/12, R/12; RF, RF Wonder X Tendergreen cross only three, r/31, r/31; r/12, for Canadian Wonder. r/J2; RF, RF and r/31, r/Jl; r/J2, r/J2; RF, rF (or rF, RF) The reaction of F progeny to RW60 (Table III) would be expected to produce a rapid HR when challenged indicated that the avr/J2 gene, although conferring a by RW60(avrPphF) but be susceptible to RW60(avrPphF, 3208 Avirulence-virulence gene from Pseudomonas syringae avrPphC). One plant was indeed detected with this 1000 ....--------------------, phenotype, clearly supporting the gene-specific virulence function of avrPphC masking the avrPphF-RF inter­ action, and also the probable absence of other R genes, ... which would have prevented the establishment of a � 10 0 ..L susceptible response . avrPphF acts as a virulence gene in soybean Race 7 of Pph typically causes a rapid HR in soybean .... leaves, but in certain cvs such as Choska it was found to cause a weak susceptible response recognized by yellow­ ing and water soaking at inoculation sites in leaves. The plasmid-cured strain RW60 caused a null response on all cvs of soybean tested. avrPphF was able to restore virulence to RW60 in cvs Choska and Osumi (Figure 4C; Table II). Restoration of virulence by avrPphF was also demonstrated by increases in bacterial populations in leaves of cv. Osumi (Figure 7). Time after inocul ation {h) Fig. 6. Bacterial multiplication in leaves of bean cv. Canadian Wonder Discussion inoculated with bacterial suspensions of 2 X 10 cells/ml of race 7 The pattern of avirulence conferred by avrPphF in wild­ (open), RW60 (stippled), RW60 (pPPY511) (grey) and RW60(pPPY511, pDAHR15) (black); bars, :<:: SEM. type races of Pph fits that predicted for an ex avr gene Table III. Segregation of resistance to RW60(avr PI , avr/J2,) amongst the F progeny of a cross between Tendergreen (RPI , RPI ; r/J2,, rfJ2,)• and Canadian Wonder (rPI , rPI ; R/J2,, RfJ2,)• Predicted genotype Phenotype of response if R�2 epistatic Number of plants Observed Expected (13:3) RPI .RPI ; R/J2,,R/J2, RPI ,RPI ; r/J2,,R/J2, rPI ,RPI ; R/J2,,R/J2, rPI ,RPI ; r/J2,,R/J2, Slow hr 31 RPI .RPI ; R/J2,,r/J2, rPI RPI ; R/J2,,r/J2, or 31 30.9 rPI ,RPI ; R/J2,,R/J2, rPI ,RPI ; r/J2,, R/J2, susceptible 0 rPI ,rPI; R/J2,,R/J2, rPI ,r PI ; r/J2,,R/J2, rpI ,RPI ; R/J2,,r/J2, rpI ,r pI ; R/J2,, r/J2, rPI ,rPI; r/J2,,rf32,b RPI .RPI ; r/J2,,r/J2, RPI ,rpI; r/J2,,r/J2, Rapid HR 7 7.1 rPI ,RPI ; r/J2,,r/J2, •RPI confers a rapid HR and R/J2, a slow hr. lYfhe predicted phenotype would be a susceptible reaction, unless there are other � avr genes in RW60. Table IV. Segregation of resistance to RW60 (avrPI, avr/J2,, avrPphF") amongst the F progeny of a cross between Tendergreen (RPI , RPI ; r/J2,, r/J2,; rF, rF) and Canadian Wonder (rPI , rPI ; R/J2,, R/J2,; RF, RF) Predicted genotypes Frequency Phenotype Number of plants Observed Expected (3: 1) Dominant RPI , Dominant R/J2,, Dominant RF 27 Dominant RPI , recessive r/J2,, Dominant RF 9 recessive rPI , Dominant R/J2,, Dominant RF 9 Rapid HR 27 28.5 recessive rPI , recessive r/J2,, Dominant RF 3 Total 48 Dominant RPI , Dominant R/J2,, recessive rF 9 Dominant RPI , recessive r/J2,, recessive rF" 3 Slow hr recessive rPI , Dominant R/J2,, recessive rF 3 or recessive rPI , recessive r/J2,, recessive rF" 1 Susceptible Total •avrPphF present on pPPY511. The parental phenotypes were rapid HR and susceptibility on cvs Canadian Wonder and Tendergreen, respectively. Dominant means homozygous (RR) or heterozygous (Rr). Predicted to develop a susceptible reaction due to suppression of Avr�l by AvrPphF or with all R genes recessive, if no other avr-R gene interactions occurring. 9.5 G.Tsiamis et al. matching the Rl gene for resistance in Phaseolus (Taylor ORFs, but only the second is required for avirulence activity (Ronald and Staskawicz, 1988). Here, we show et al., 1996). The cloning strategy adopted failed to isolate the fourth avr gene predicted to match R4 (Table I), that avrPphF is organized into an operon with two ORFs, possibly because the gene may have lethal effects in both of which are needed for function. E. coli. The first avr gene cloned from Pph (Shintaku et al., The mechanism by which avrPphF causes effects in 1989), and designated avrPphA according to the nomen­ plant cells remains unknown. According to published reports, all of the bacterial avr genes that have been tested clature proposed by Vivian and Mansfield (1993), did not elicit the HR if they are expressed transiently in plant cells; appear to match any of the R genes recognized in examples are avrPphB and avrPphE from Pph (Stevens Phaseolus. The proposed gene-for-gene pattern of race structure (Taylor et al., 1996) has, therefore, not et al., 1998). The implication from in planta expression is been entirely confirmed by molecular genetics. that the A vr proteins act as elicitors following their The avrPphF locus is organized into an operon with a delivery into plant cells by the hrp-dependent type Ill secretion apparatus (Alfano and Collmer, 1997; Bonas and characteristic 'h box' promoter indicating regulation by rp Van den Ackerveken, 1997; Galan and Collmer, 1999). Jn HrpL. Both ORFs within the operon were required for planta expression of the ORFs from avrPphF should either avirulence or virulence functions. The requirement reveal whether one functions as the elicitor per se and one for more than one transcriptional unit for the function of an avr gene was described in detail for avrE from P.s. pv. perhaps acts as a chaperone for protein delivery. The two ORFs were always found together, arranged into the tomato by Lorang and Keen (1995). The avrE locus is avrPphF operon, but only in strains expressing the Al linked to the right end of the hrp region and comprises two phenotype (Table I; Figure 2). The adjacent ISJ 00 convergently transcribed units, which are both required for homologue was distributed throughout strains irrespective the avirulence phenotype. avrBsl from Xan thornonas of the race designation. ISJO 0 is present in species of campestris pv. vesicatoria comprises an operon of two Yers inia and several copies have been located in plasmids encoding type Ill secretion systems or other virulence factors (Buchrieser et al., 1998; Hu et al., 1998). Sequences hybridizing to the insertion sequence were, 10 00....----------------- however, not widely distributed amongst other pathovars of P. syringae. Given the pathogenicity functions attributed to other avr .I. genes from P.syringae, e.g. avrE and avrRpml (Lorang ,...._ 100 et al., 1994; Ritter and Dangl, 1995), the finding that ,­ avrPphF was able to restore virulence to compromised .L plasmid-cured strains such as RW60 was not unexpected. However, avrPphF is, to our knowledge, the first gene to be shown to have cultivar- and gene-specific virulence activity. By contrast, the discovery that avrPphF also acts as a � avr gene, i.e. has functions revealed only in the absence of the PAI, operating in a defined gene-for-gene manner with a matching R gene in Canadian Wonder, was most unexpected. The interaction between avrPphC and avrPphF blocking the HR in cv. Canadian Wonder would appear to occur in the plant, possibly with a receptor for 48 72 the A vrPphF protein, because avrPphC has no effect on Time after inoculation (h) the expression of the HR caused by avrPphF in cv. Red Mexican. It is important to emphasize that the virulence Fig. 7. Bacterial growth in leaves of soybean cv. Osumi inoculated and � avirulence functions of avrPphF are only observed with bacterial suspensions of 10cells/ml of P.s. pv. glycinea (open), RW60 (stippled) and RW60 (pPPY511) (grey); bars, ± SEM. in the absence of other genes from the PAI. The A vrPphF Table V. Genes with dual avirulence and virulence functions, either eliciting or blocking the HR, respectively, identified using strains of P.s. pv. phaseolicola lacking the 154 kb plasmid which contains the putative pathogenicity island Gene designation Plant in which phenotype observed• Avirulence Virulence avrPphF Red Mexican , Canadian Wonder' Tendergreen, Soybean avrPphC Soybean Canadian Wonder virPphA Soybean Canadian Wonder, Red Mexican, Tendergreen •Bean cultivars are named. b A virulence due to interaction with different R genes. Yucel et al. (1994). dJackson et al. (1999). 32 10 Avirulence-virulence gene from Pseudomonas syringae Table VI. Bacterial strains and pla smids used in this study" Strain/plasmid Relevant properties Source or reference Bacteria. P.syringae pv. phaseolicola Principal isolates used 13 75A race 5, wild-type isolate D.Teverson 1375AN race 5, Nal from 13 75A D.Teverson 1375AR race 5, Rif1l- from 1375A D.Teverson race 1, wild-type isolate 12 81A D.Teverson 12 81A R race 1, Rif1l- from 12 81A D.Teverson 1448A race 6, wild-type isolate Fillingham et al. (1 992) 1448AR race 6, Rif1l-from 1448A Fillingham et al. (1 992) 1449B race 7, wild-type isolate D.Teverson 1449BR race 7, Rif1l- from 1449B D.Teverson RW60 Vir, pAV 51 1-, Ap , Rif1l- Jackson et al. (1 999) Additional isolates 882 race 2 D.Teverson 13 01A race 3 Hitchin et al. (19 89) 1302A race 4 Jenner et al. (19 91) 52A race 5 Hitchin et al. (19 89) 2656A race 8 D.Teverson 2709A race 9 D.Teverson P.cichorii 2379 lettuce pathogen NCPPB P.s. pv. corna faciens 1354 oat pathogen Harper et al. (19 87) P.s. pv. pisi 299A pea pathogen J.Taylor' P.s. pv. glycinea 11 39 soybean pathogen this study P.s. pv. maculicola 18 20 brassica pathogen NCPPB P.s. pv. tabaci 11 528 tobacco pathogen J.Turnerd P.s. pv. tomato DC3000 tomato pathogen Whalen et al. (1 99 1) P.s. pv. syringae 28 1 lilac pathogen NCPPB E.coli C211 0 Nal,polAJ Leong et al. (19 82) DH5a Nal , recA, laczt:.M15 Bethesda Research Laboratories HB101 Sm , recA Boyer and Roulland-Dussoix (19 69) Plasmids + R pBluescript SK Ap , multiple cloning sites and priming sites Stratagene R R pHoKmGus Ap , Km , tnpA , promoterless �-glucuronidase gene Bonas et al. (19 89) in Tn3, pWB15A replicon R + pLAFR3 Tc , IncPl replicon, Tra-, Mob, cosmid Sta skawicz et al. (19 87) Sp, Inc Q pDSK600 replicon, 3X lac UV5promoter Murillo et al. (1 994) R + + pRK20 13 Km , Co!El replicon, Tra, Mob, helper plasmid Figurski and Helinski (19 79) R + pSShe Cm , tnpA , pACYC 184 replicon Stachel et al. (19 85) Clones containing avrPphF pPPY501 pLAFR3-based genomic clone from race 5 strain 13 75 this study pPPY502 pLAFR3-based genomic clone from race 5 strain 13 75 this study pPPY503 5.3 kb BamHI-H indIII subclone of pPPY502 harbouring avrPphF this study pPPY505 1. 8 kb BamHI fragment from pPPY503 in pBluescript II SK this study pPPY507 1.1 kb EcoRI fragment in pLAFR3 thi s study pPPY5 11 insert as in pPPY505 but in pDSK600 this study pPPY5 12 avrPphF ORF2 in pDSK600 this study pPPY5 14 avrPphF ORFl in pDSK600 this study Additional plasmids pMS2330 pLAFRl based genomic clone harbouring avrPphA from HB33 Shintaku et al. (19 89) pAV 51 1 native plasmid from 1449B, ~15 4 kb Jackson et al. (1 999) pAV520 genomic clone harbouring pA V5 11 sequences in pLAFR3 Jackson et al. (1 999) pAV521 genomic clone harbouring pAV 511 sequences in pLAFR3. Jackson et al. (1 999) pDAHR 15 1. 4 kb Clal-BamHI fragment containing avrPphC in pDSK600 Yucel et al. (1 994) R R R R R R •Nal , Rif1l-, Sm , Ap , Km , Tc and Cm indicate re sistance to nalidixic acid, rifampicin, streptomycin, ampicillin, kanamycin, tetracycline and chloramphenicol, respectively. Horticulture Research International, Wellesboume, UK. National Collection of Plant Pathogenic Bacteria, York, UK. dUniversity of East of Anglia, Norwich, UK. proteins appear to block the HR caused by � avr genes The ability of certain A vr proteins to express virulence such as avrfi l, which is predicted to be present in the functions, blocking the phenotype conferred by other avr chromosome of Pph. The � avr function of avrPphF is in genes, may involve three different types of interaction: turn blocked by avrPphC. In contrast to avrPphF, the other (i) between the A vr proteins themselves; (ii) between A vr genes recognized to act as virulence determinants, and masked R proteins; (iii) downstream of hypothetical virPphA and to a lesser extent ORF4 from the PAI, do Avr-R protein interactions to interfere with gene-specific not have differential effects in the cultivars tested (Jackson signalling cascades leading to the HR. A possible target for et al. , 1999). (iii) would be one of the MAP kinases which have been 3211 G.Tsiamis et al. Pathogenicity tests and in planta bacterial population implicated in plant defence (Grant and Mansfield, 1999; counts Romeis et al., 1999). The virulence determinant YopJ Pods and leaves of French bean were inoculated as described previously from Yers inia has recently been found to bind and (Harper et al., 1987; Hitchin et al., 1989). The inoculum concentration inactivate MAP kinase kinases in mammalian cells, routinely used in bean leaves was 2 X 10cells/ml. Soybean plants were inoculated using a l ml syringe (without needle) to infiltrate bacterial leading to suppression of defence responses (Orth et al., suspensions of 10cells/ml into the underside of fully expanded primary 1999). There are no reports of direct interactions between leaves. Bacterial multiplication in leaves was examined by cutting tissue Avr proteins, but Avr-R protein binding has been from the inoculation sites with a 0.6-cm-diameter borer, homogenization demonstrated between A vrPto and Pto (Tang et al., in 10 mM MgCh, and serial dilution of the homogenate, which was then spread onto LB agar with appropriate antibiotics to allow colony 1996). However, the action of intermediates linking Avr development at 25 C. and R proteins has also been proposed (Grant and Mansfield, 1999). General molecular techniques The multiple function of avr genes found in Phaseolus Basic procedures were carried out as described in Sambrook et al. (1989). The methods of Jenner et al. (1991) and Mansfield et al. (1994) were was also extended to the Pph-soybean interaction. followed for the construction of the genomic library from race 5 strain Whether or not soybean should continue to be considered 1375A, preparation of minipreps, Southern blotting and hybridization. as a non-host to Pph (Yucel et al., 1994) is questionable as The library was screened for determinants of avirulence by conjugation of certain strains clearly show weak pathogenicity. individual clones into race 6 strain l 448AR with the helper plasmid pRK2013. Transconjugants were tested for pathogenicity on pods of cvs Interestingly, however, genes recognized for virulence or Red Mexican and A53 as described by Harper et al. (1987). Transposon avirulence function in Phaseolus have so far all displayed mutagenesis of pA V521 and pPPY503, and marker exchange of the opposite activity in soybean leaves, as summarized in avrPphF::Tn3gusA33 into race 7 were carried out as described by Table V. Pathogenicity towards the legume hosts has Mansfield et al. (1994); the position of the transposon A33 in the target DNA was confirmed by sequencing. probably involved interaction with common virulence targets. As targets mutated to activate resistance (i.e. PCR and DNA sequencing acting as R gene products), perhaps by hyperactivation of Standard PCRs were performed with Taq polymerase and buffers from signal transduction cascades blocked by Vir factors, the Bioline using a Perkin Elmer Gene Amp 2400. Sequencing was performed using the dideoxy chain termination method with the pathogen would need to acquire further vir genes whose Sequenase enzyme (Pharmacia) as described in the manufacturer 's products would override the activation of resistance. instructions. Primers used for the amplification of avrPphF ORF l were: Restoration of virulence could be achieved by suppression ORFlF, 5'-ATGAAGAATTCGTTCGACCG-3'; ORFlR, 5'-TCAGAC­ of signal transduction, or, as outlined above, simply by CGAACTCTCAGACA-3'. For the amp lification of avrPphF ORF2, the primers used were: ORF2F, 5'-ATGGGTAATATCTGCAATTCG-3'; blocking any direct avr-R gene interactions. ORF2R, 5'-GGCCAGTTATAGGAGCTAAT-3'. An intriguing question is whether or not the avr or vir genes now identified from a highly evolved pathogen such Construction of non-polar mutations as Pph represent virulence determinants that have had a For the generation of the non-polar mutants, race 7.1.ORFl and race 7.1.ORF2, the following steps were followed. ORFl was deleted fundamental role in the evolution to parasitism from a from position 332-439 in pPPY505 by a double digest with Aatll-Agel. saprophytic ancestry. The layers of interactions and Digested pPPY505 was filled in using Kienow polymerase and re-ligated. possibly interconnected targets that exist amongst the Deletions were selected by PCR and the deleted form cloned into pOK numerous avr and vir genes in the present populations of (Huguet et al., 1998). ORF2 was deleted from position 767-1157 in pPPY505 by a double digest with Cs p451-Bst9 8I. The plasmid with the plant pathogens may make it difficult to distinguish the digested insert was filled in and re-ligated. Cloned deletions were again evolutionary significance of individual effector proteins. It selected by PCR and the deleted form was then introduced to pOK. is possible that gene-specific virulence, as shown by Mutated genes were introduced to race 7 strain 1449BR by homologous avrPphF, represents a second wave of activity, whereas recombination in two steps as described by Kaniga et al. (1991) and Huguet et al. (1998). In both mutants, the presence of the correct deletion virPphA, apparently with less cultivar specificity, may in race 7 was verified by PCR. have more direct effects on signal transduction pathways and represent the first class of virulence factor. The co­ evolution of hosts and pathogens would mean that clear Ackn owledgements distinction between primary and secondary waves of Many thanks are due to Elisabeth Huguet and Ulla Bonas for advice on activity may have become very blurred, and it is certainly the creation of non-polar mutants, to Noel Keen for clones containing impossible to predict the function of avirulence and avrPphC and to Suresh Patil for avrPphA. We also wish to acknowledge virulence factors we have identified. In order to unravel the support from the BBSRC, EC grants BIO-CT97-2244 and TMR, CICYT grant BI097-0598 and the British Council-MEC Acciones Integradas complex web of interacting effectors, it is now a priority to programme. 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Plant Microbe Interact., 1, 677---679. Received March 6, 2000; revised May 3, 2000; accepted May JO, 2000 32 14

Journal

The EMBO Journal – Springer Journals

Published: Jul 3, 2000

Keywords: avirulence; disease resistance; gene‐for‐gene interactions; hypersensitive reaction; virulence

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