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Proc Natl Acad Sci U S A. Apr 11, 2006; 103(15): 5887–5892.
Published online Mar 31, 2006. doi:  10.1073/pnas.0601431103
PMCID: PMC1458668
Genetics

Presence/absence polymorphism for alternative pathogenicity islands in Pseudomonas viridiflava, a pathogen of Arabidopsis

Abstract

The contribution of arms race dynamics to plant–pathogen coevolution has been called into question by the presence of balanced polymorphisms in resistance genes of Arabidopsis thaliana, but less is known about the pathogen side of the interaction. Here we investigate structural polymorphism in pathogenicity islands (PAIs) in Pseudomonas viridiflava, a prevalent bacterial pathogen of A. thaliana. PAIs encode the type III secretion system along with its effectors and are essential for pathogen recognition in plants. P. viridiflava harbors two structurally distinct and highly diverged PAI paralogs (T- and S-PAI) that are integrated in different chromosome locations in the P. viridiflava genome. Both PAIs are segregating as presence/absence polymorphisms such that only one PAI ([T-PAI, [nabla]S-PAI] and [[nabla]T-PAI, S-PAI]) is present in any individual cell. A worldwide population survey identified no isolate with neither or both PAI. T-PAI and S-PAI genotypes exhibit virulence differences and a host-specificity tradeoff. Orthologs of each PAI can be found in conserved syntenic locations in other Pseudomonas species, indicating vertical phylogenetic transmission in this genus. Molecular evolutionary analysis of PAI sequences also argues against “recent” horizontal transfer. Spikes in nucleotide divergence in flanking regions of PAI and [nabla]-PAI alleles suggest that the dual PAI polymorphism has been maintained in this species under some form of balancing selection. Virulence differences and host specificities are hypothesized to be responsible for the maintenance of the dual PAI system in this bacterial pathogen.

Keywords: bacteria, balancing selection, plant–pathogen interaction, arms race, horizontal gene transfer

Arms race dynamics were once thought to dominate plant–pathogen coevolution through the process of rapid substitutions of adaptive mutation in both sides of the interaction (1). However, the presence of balanced polymorphisms in some resistance (R) genes in the plant host Arabidopsis thaliana suggests a different type of coevolutionary dynamic (25). For example, diversifying selection, rather than directional selection, is observed in the extremely polymorphic A. thaliana R-gene RPP13 and the downey mildew avirulence gene, ATR13, which triggers RPP13-mediated resistance (6). In bacteria, effector proteins such as ATR13 are transported into host cells via the Type III secretion system (TTSS), which is found in both animal and plant pathogens (7). Effectors delivered by the TTSS are essential for causing disease in susceptible hosts and for eliciting defense responses in resistant hosts. In a variety of Gram-negative bacterial pathogens, the genes encoding the TTSS and its effectors comprise a physical gene cluster called a pathogenicity island (PAI) (8).

Pathogenicity-related genes, including entire PAIs, are often introduced into bacterial species by horizontal (or lateral) gene transfer (HGT) (810), which is believed to be important because it allows the recipient pathogen to immediately use already-evolved pathogenicity strategies and, thereby, accelerate the pace of pathogen evolution (11). In fact, several human pathogens are defined and differentiated from close relatives by horizontally acquired virulence factors (12). However, a survey of effectors in Pseudomonassyringae finds effectors that have been acquired recently and others that have been transmitted predominantly by descent, indicating that pathogenicity may evolve in both genomic contexts (13).

In this study, we investigated PAIs in P. viridiflava, which is a prevalent bacterial pathogen of wild A. thaliana populations (14). P. viridiflava is in the P. syringae group (15). Although P. syringae is intensively studied as a bacterial plant pathogen (13, 16–18), little is known about the genetic basis of pathogenicity in P. viridiflava. Here we report a previously undescribed arrangement of PAIs and a long-term presence of an unusual dual PAI polymorphism in this bacterial pathogen. This polymorphism is not caused by recent HGT but rather is analogous to polymorphism in host defense genes: evolutionarily long-lived polymorphism for two paralogous PAIs in a single pathogen species.

Results

We first examined the structure and DNA sequence of PAIs in five P. viridiflava strains (LP23.1a, PNA3.3a, ME3.1b, RMX23.1a, and RMX3.1b) that were collected from naturally occurring A. thaliana plants in populations in the Midwest United States, and their entire PAIs and flanking sequences were isolated. LP23.1a and PNA3.3a possess a PAI similar in structure to that found in the congener P. syringae (16). It has a tripartite mosaic structure composed of a gene cluster encoding the Type III protein-secretion apparatus (hrp/hrc gene cluster), the 5′ effector loci (exchangeable effector loci or EEL), and the 3′ effector loci (conserved effector loci or CEL). We designate this structural form as the T- (tripartite) PAI (Fig. 1A; see also Table 1, which is published as supporting information on the PNAS web site). The other three isolates (ME3.1b, RMX23.1a, and RMX3.1b) possess a PAI with a single component hrp/hrc cluster (Fig. 1B; see also Table 2, which is published as supporting information on the PNAS web site), a structure previously not reported in any pathogenic bacteria. We designate this type as the S- (single) PAI.

Fig. 1.
Two PAIs in P. viridiflava. Gene compositions of Region 1 (A) and Region 2 (B), locations of the T- and S-PAIs, respectively, in P. viridiflava. Boxes represent ORFs, and numbers above or below boxes are ORF numbers corresponding to Tables 1 and 2. The ...

The T-PAIs of LP23.1a and PNA3.3a are 47 kb and 43 kb, respectively, differing primarily by an insertion/deletion (indel) difference in the EEL region (Fig. 1A). Gene compositions in the hrp/hrc gene cluster and in the CEL region are otherwise identical to one another and are nearly identical to the T-PAI of P. syringae pv. tomato (Pto) DC3000 (16). The gene compositions in the EEL region are nearly identical in the two P. viridiflava isolates but different from that of Pto DC3000. This region is known to be hypervariable among P. syringae pathovars (1617). The T-PAI contains three known avirulence (avr) gene homologs, hopPsyA, avrE, and avrF (18, 20, 21) and two effector gene candidates (hopPtoA1 and hopPtoM).

The S-PAIs in ME3.1b, RMX23.1a, and RMX3.1b are ≈30 kb in length and contain a 10-kb-long insertion in the middle of the hrp/hrc cluster relative to the T-PAI (Fig. 1B). The S-PAI contains only two avr gene homologs (avrE and avrF), and these homologs are located in the 10-kb insertion. The T- and S-PAIs share 25 gene homologs and many operon structures (Tables 1 and 2), and yet are distinct in gene composition and order, especially for effector gene loci. The sequences of these gene homologs are also highly diverged between the two PAIs. Nucleotide divergence (22, 23) between the 25 shared genes averages 0.701 across all sites and 1.44 for synonymous sites.

In a previous study (23) we investigated nucleotide polymorphism at five genomic regions in a worldwide collection of P. viridiflava isolates. Both T-PAI and S-PAI bearing isolates are present in this sample. Total and synonymous site divergence between T- and S-PAI genotypes at these five loci (0.042 and 0.182, respectively) is 16.9 and 7.7 times lower, respectively, than the corresponding divergence between genes located in the S- and T-PAI. This observation suggests a paralogous (rather than an allelic) relationship of the two PAIs. Analysis of genomic sequences flanking their boundaries confirms that they are indeed located in different genomic regions (T-PAI in Region 1 and S-PAI in Region 2;Fig. 1). Given this lack of allelism, we were surprised to discover that isolates with one type of PAI did not contain the other: the presence of one PAI was perfectly associated with the absence of the other. In a larger sample of 286 P. viridiflava isolates collected from worldwide populations of A. thaliana, four of seven populations are polymorphic for the PAI type (Table 3, which is published as supporting information on the PNAS web site). In total, 10% contain a T-PAI and 90% contain an S-PAI. Each isolate harbors one, and only one, PAI. Isolates containing a T-PAI in Region 1 have a 200-bp sequence in Region 2, instead of an S-PAI ([T-PAI, [nabla]S-PAI]). Similarly, isolates containing an S-PAI in Region 2 have a 3-kb-long sequence in Region 1 instead of a T-PAI ([[nabla]T-PAI, S-PAI]). This sequence is similar to part of the EEL sequence in the T-PAI but lacks any known effector gene homologs. Note that we defined the 5′ end of the T-PAI as being immediately downstream of tgt, queA, and tRNALeu following a definition of PAIs in P. syringae (16). This genomic region does not necessarily represent a unit that shares the same evolutionary history in P. viridiflava.

We have reported previously the presence of two diverged (and nonrecombining) clades in P. viridiflava, which likely represent two distinct subspecies (19). However, these clades do not correspond to the PAI haplotypes: in a survey of 96 isolates, the two PAI haplotypes coexist within clade A (10 AT and 57 AS), whereas clade B is fixed for [[nabla]T-PAI, S-PAI] (29 BS). Recombination between AT and AS isolates is clearly evident at other loci spread around the genome (19), so divergent clades cannot explain this disassociation between the S- and T-PAIs.

Other possible explanations for the absence of recombinant PAI genotypes include tight physical linkage and/or natural selection. According to the similarities of the flanking regions (Tables 1 and 2), Region 1 and Region 2 are 2.1–2.4 Mb apart in three divergent genomes of P. syringae (6.1–6.3 Mb circular genomes), suggesting that these regions may not be physically tightly linked in P. viridiflava (2426). Strong selection on this association implies that genotypes with both or no PAI are at a selective disadvantage relative to the genotypes with only one PAI. The biological importance of PAIs in pathogenic bacteria is well understood; the disadvantage of carrying two PAIs, on the other hand, awaits experimental analysis.

The fact that the two PAI haplotypes reside in a single species means that both of the PAI indels are true polymorphisms. Two issues related to the possible adaptive significance of these polymorphisms can be addressed: (i) the age of origination of each PAI in the lineage leading to (or represented by) P. viridiflava and (ii) the age of each PAI indel polymorphism. For clarity, we analyze data for the T-PAI first and in greater detail, and then we show that the same data patterns and arguments hold for the S-PAI.

Phylogenetic analysis of the PAIs revealed that the time of the most recent common ancestor of T-PAI and S-PAI predates the split of P. viridiflava from other Pseudomonas species (Fig. 2A). This genealogical relationship means that one of the PAIs cannot have originated as a recent duplication event of the other. As discussed above, HGT is thought to be a common mechanism for bacterial pathogens to acquire distinct PAIs. Is the PAI indel polymorphism a consequence of a recent HGT in P. viridiflava? Four lines of evidence argue against such a HGT event being recent. First, the structures of the T-PAIs in P. viridiflava and P. syringae Pto DC3000 are similar, and the genes flanking the PAIs are orthologous, indicating synteny. Second, there are no obvious indicators of recent HGT, like heterogeneity of G+C contents, integration-related fragments, and presence of tRNAs around this PAI (911). The EEL region of the T-PAI does have a tRNA in its 5′ flanking region (which are sometimes associated with HGT integration) and has slightly lower G+C content (52.8% on average) compared with other regions (58–61%). This tRNA is conserved between Pseudomonas species and may mark the original insertion point of a more ancient HGT of this PAI in an ancestor of the Pseudomonas plant pathogens (16). Alternatively, these features may reflect the exchangeability of the EEL region (1617). Third, a phylogeny of T-PAI sequences is consistent in both depth (i.e., divergence) and branching order with a corresponding phylogeny based on other resident genes in these species (Fig. 2A and B). Finally, the two T-PAI alleles in P. viridiflava (of LP23.1a and PNA3.3a) differ at ≈200 segregating sites within the T-PAI, which argues against a very recent HGT event. Indeed, the level of genetic variation of several genes on the T-PAI is indistinguishable from that on the background of T-PAI containing isolates from natural populations (H.A., M.K., and J.B., unpublished data). All of these results suggest that a recent HGT cannot explain the presence/absence polymorphism of the T-PAI.

Fig. 2.
Genealogical relationships of PAI and non-PAI genes. Neighbor joining trees of 25 PAI genes (A), non-PAI genes (16S and gyrB; B) and the PAI gene avrE (C) for P. viridiflava, Pto DC3000,and P. cichorii 83-1 are shown. The third positions of codons in ...

The age of the T-PAI indel polymorphism can provide clues about the type of selection acting on this polymorphism. Following established methods (34), we investigated the genetic divergence in the regions immediately flanking the site of the T-PAI indel polymorphism between [T-PAI, [nabla]S-PAI] and [[nabla]T-PAI, S-PAI] isolates. Window plot analysis reveals a clear spike of genetic variation in the junction region (nucleotide diversity approaching 30%), almost all of which can be accounted for by the divergence between [T-PAI, [nabla]S-PAI] and [[nabla]T-PAI, S-PAI] isolates (Fig. 3A). We do not believe this divergence is the result of mutational processes associated with insertion or excision of the PAI but rather represents the accumulation of substitutions between the two alleles over time. First, nucleotide divergence is nearly equally divided between groups when compared with PtoDC3000. Specifically, nucleotide divergence for synonymous sites in the ORFs that neighbor the T-PAI indel junction (R1-ORF41 and R1-ORF79) is 1.24 for [T-PAI, [nabla]S-PAI] isolates and 1.26 for [[nabla]T-PAI, S-PAI] isolates. Second, the level of polymorphism in the flanking region within each allele class is similar to that of the background loci in this species. E[θ] (expected genetic diversity conditioned on allele frequency; ref. 28) of the 1-kb-long flanking regions centered on the T-PAI junction is 0.032 [T-PAI, [nabla]S-PAI] and 0.020 [[nabla]T-PAI, S-PAI] for isolates within clade A and 0.014 [[nabla]T-PAI, S-PAI] for isolates within clade B; these values are comparable to 0.022 (clade A) and 0.009 (clade B) for the background loci (19). Therefore, both with respect to the mutational differences that have accumulated between the T-PAI and [nabla]T-PAI alleles and the mutational polymorphism that has accumulated within each allelic class, we find no evidence for unequal mutation rates.

Fig. 3.
Genetic divergence in flanking regions of T- and S-PAIs. Average genetic diversity of all samples (π) and between isolates containing T- and S-PAIs (Dxy) by using the Jukes and Cantor correction (22, 23) were plotted for the flanking regions of ...

Window plot analysis of AT and AS sequences revealed significant departures from selective neutrality (D* and F*) (27) around the indel-junction (Fig. 3A), a reflection of the large relative divergence between the two alleles. Such a departure from neutrality is consistent with balancing selection acting on the T-PAI polymorphism. The genealogical relationship of the sequence flanking the T-PAI (Region 1) indicates that the deletion of the T-PAI predated the most recent common ancestor of the A and B clades. This finding is significant because, based on the divergence of the two clades (Ks = 0.334; ref. 19), we estimate that these clades split off from each other 43–49 million years ago (29). There is virtually no chance that a polymorphism can remain segregating for this length of time by genetic drift alone but rather requires some form of balancing selection.

The same logic allows us to conclude that the S-PAI has also been maintained as an ancient balanced polymorphism in P. viridiflava. We did not detect the presence of a homolog of the S-PAI in P. syringae, but we were able to identify a homolog in P. cichorii 83-1, another relative of P. viridiflava (30). The S-PAIs in P. viridiflava and P. cichorii share similarity in their flanking sequence (Table 2) and show no indication of HGT. In addition, the genealogy of the S-PAI is consistent with the phylogenic relationship of the species (Fig. 2A and B), including the divergence between species and divergence between the A and B clades of P. viridiflava (19). The same branching pattern was further confirmed by other isolates collected from wild A. thaliana populations, based on the third codon position of avrE in the PAIs (Fig. 2C). Finally, window plot analysis for the flanking regions of the S-PAI reveals a clear spike of genetic variation when [T-PAI, [nabla]S-PAI] and [[nabla]T-PAI, S-PAI] isolates are compared (Fig. 3B). Again, these polymorphisms are not associated with the insertion or excision of the PAIs: nucleotide divergence (compared with Pto DC3000) for synonymous sites in the ORFs that neighbor the S-PAI indel-junction (R2-ORF6 and R2-ORF40) are 0.78 and 0.75 for [T-PAI, [nabla]S-PAI] and [[nabla]T-PAI, S-PAI] isolates, respectively. In the flanking regions of the S-PAI (1 kb centered on the junction) among the 96 isolates, E[θ] = 0.037 ([T-PAI, [nabla]S-PAI]) (27) and 0.024 ([[nabla]T-PAI, S-PAI]) for isolates in clade A and 0.008 for isolates within clade B, which again are close to those in the background loci (E[θ] = 0.022 for clade A and 0.009 for clade B). Window plot analysis between [T-PAI, [nabla]S-PAI] and [[nabla]T-PAI, S-PAI] isolates in the clade A revealed significant departures from selective neutrality (D* and F*) (27) around the S-PAI junction (Fig. 3B), consistent with balancing selection on the S-PAI presence/absence polymorphism. The genealogical relationship of these flanking sequences was similar to that of flanking sequences of the T-PAI (data not shown), suggesting that the indel of the S-PAI also had occurred before the split of the clades.

The long-term presence of this unusual dual polymorphism, together with the absence of recombinant haplotypes, are strong indicators of selection-maintaining alternative PAIs in this pathogen species. The biology of the two PAIs must differ in some way relevant to that selection. PAIs encode effectors that, when delivered to plant cells by the TTSS, may be recognized by the host and elicit a rapid defense response known as the hypersensitive response (HR). To test this hypothesis, we conducted infection experiments of [T-PAI, [nabla]S-PAI] and [[nabla]T-PAI, S-PAI] isolates in A. thaliana and in tobacco (Fig. 4). These isolates were selected from within clade A, so that the effects of genetic background are minimized. [T-PAI, [nabla]S-PAI] isolates elicit an HR in A. thaliana Col-0 significantly slower than [[nabla]T-PAI, S-PAI] isolates ([T-PAI, [nabla]S-PAI] mean = 21.8 h, [[nabla]T-PAI, S-PAI] mean = 14.8 h; P < 0.0001). However, in tobacco, we observed the opposite pattern; the same [T-PAI, [nabla]S-PAI] isolates elicit an HR significantly more rapidly ([T-PAI, [nabla]S-PAI] mean = 10.0 h, [[nabla]T-PAI, S-PAI] mean = 14.9 h; P < 0.0001) and produce significantly larger lesions ([T-PAI, [nabla]S-PAI] mean = 100%, [[nabla]T-PAI, S-PAI] mean = 85.6%; P < 0.0002) than [[nabla]T-PAI, S-PAI] isolates. These virulence and the host-specificity differences raise the possibility that the two distinct PAIs are maintained by selection as alternative means of interacting with different hosts. P. viridiflava is known to have a broad host range and does appear to infect other plant species co-occurring with A. thaliana in Midwest populations (19).

Fig. 4.
Virulence phenotype variations in P. viridiflava. Virulence phenotypes of P. viridiflava isolates measured by HR tests. Time until isolates are recognized (causing HR) by host plants was plotted (Materials and Methods). Two host plants were used, A. thaliana ...

Discussion

Multiple functionally distinct PAIs previously have been identified in bacterial pathogen species and are generally associated with HGT (31). The two-PAI system in P. viridiflava is unusual in several respects. First, individual isolates possess only one PAI rather than both. Second, although we cannot exclude an ancient HGT event for the origination of the two PAIs in the lineages leading to P. viridiflava, recent HGT can be effectively ruled out. Third, the pattern of genetic variation observed in and around the PAIs, and the phylogeny of the two alleles, suggests that balancing selection has been responsible for the maintenance of the two-PAI system. This evidence shows balancing selection on PAIs of bacterial pathogens.

The conventional view of pathogen evolution has been that a single optimal level of virulence should evolve to balance the costs and benefits of virulence (growth and competition vs. host survival and transmission). However, long-term polymorphism in pathogen populations can be obtained in models in which levels of virulence vary, particularly in the face of host heterogeneity, superinfection, and spatial structure (3235). Such polymorphisms have not been reported yet for any pathogen, although evidence of balancing selection in bacteria has been accumulating (6, 36–37). We predict that population genetic analysis of virulence factors in other pathogens will provide additional examples of evolutionarily stable polymorphisms. Why the entire PAI, rather than a particular effector gene, has been selected as a unit remains to be addressed.

Stable polymorphism for alternative forms of a critical component of virulence in P. viridiflava is strikingly similar to stable polymorphism for resistance and susceptibility alleles in certain defense genes in their plant host, A. thaliana (24). This study indicates the potential for evolutionarily stable polymorphism on both sides of the plant–pathogen interaction. We do not yet know whether plant–pathogen polymorphism is mechanistically coupled in a “trench warfare”; theoretically, such coevolutionary dynamics should be a viable possibility (4).

Materials and Methods

Sample Materials.

Five P. viridiflava samples, from which entire PAIs were sequenced, were collected from naturally occurring A. thaliana plants in populations in the Midwest United States (14). Samples from worldwide populations were collected in the same manner (19). P. cichorii 83-1 was kindly provided by J. Greenberg (University of Chicago).

PAI Isolation and Sequencing.

We constructed genomic libraries of all five strains of P. viridiflava employing a Lambda FIX II vector kit in Escherichia coli XL-1 Blue MRA-P2 (Stratagene) and using standard molecular cloning procedures (38). Probes were designed based on partial hrpS sequences (14) or by amplifying HrcN sequence based on Pto DC3000 (16). PCR products were purified and radioactively labeled with Ready-To-Go DNA Labeling Beads (Amersham Pharmacia Biosciences). Hybridization was performed at 50–65°C overnight in 6× SSC/5× Denhardt’s/0.5% SDS solution. Membranes were washed with 2× SSC and 1% SDS solution twice at 50°C for 5 min and then once for 15 min. Positive phage clones were partially digested by MboI and subcloned into a plasmid by using a Zero Background/Kan Cloning Kit (Invitrogen). PCR products from the inserts were sequenced by using a CEQ8000 capillary sequencer (Beckman Coulter). To cover larger regions of the PAIs in LP23.1a and PNA3.3a, secondary screening was performed by using primer sets LP40222f (5′-GATGATGAAACCACGGGCTGTA-3′)-LP40589r (5′-TCCTCACGCAATGGCACCGTTA-3′) and LP38131f (5′-CGAGCAACTGAAAAACCTCGGGC-3′)-LP37891r (5′-TTGAGCGTTACCAGATCAAGCG-3′). All PCR reactions used Takara Taq HS (Takara Bio, Shiga, Japan) with 25 cycles of 95°C for 30 sec, 55–60°C for 40 sec, and 72°C for 2 min.

Because of the large sizes of the target sequences, we also constructed Fosmid libraries for ME3.1b, RMX23.1a, RMX3.1b, and P. cichorii 83-1, employing CopyControl Fosmid Library Production Kit (Epicentre Technologies, Madison, WI), and screened them by PCR. We used the EZ::TN <KAN-2> Insertion Kit (Epicentre Technologies) to sequence positive clones.

To obtain a sequence where the S-PAI was deleted in LP23.1a, the primers GR10kF (5′-GGGCTGGGCTACAGCATCGTGCCAC-3′) and GR11kR (5′-GTGGCTTCAGCAGCACGAATGATTG-3′) were used, and positive clones were isolated. To obtain this sequence in PNA3.3a, we directly sequenced PCR products by using primer sets based on conserved sites. To obtain a sequence where the T-PAI was deleted in ME3.1b and RMX23.1a, the primers LP80.5F (5′-GTCGTGCCACCGCCGTCACCTTCGC-3′)-LP83.3R (5′-GACACGGATGAAGTGAGTCTTCTCG-3′) were used and positive clones were isolated. To obtain this sequence in RMX3.1b, we again sequenced PCR products generated by using primers based on conserved sites. Partial sequences of the PAIs in the other isolates of P. viridiflava were obtained in the same manner (PCR), based on the conserved sequences among the isolates above. Corresponding GenBank accession nos. to these sequences are AY597274AY597283, AY859111, AY859112, AY859115, AY859128, AY859131, AY859183, AY859184, AY859351, AY859355, AY859358, and DQ158500158855. The entire PAI sequences from P. cichorii 83-1 were cloned from a Fosmid library in the same manner as above. Partial GyrB sequence also was obtained from this species by PCR with primers Gyr-F (5′-CMGGCGGYAAGTTCGATGACAAYTC-3′) and Gyr-R (5′-TRATBKCAGTCARACCTTCRCGSGC-3′). Corresponding GenBank accession nos. are DQ168848 and DQ220702.

Genotyping PAIs from Worldwide A. thaliana Populations.

We genotyped the PAIs in 298 P. viridiflava isolates from the Midwest and other worldwide populations. For 93 of these isolates, the clade was known from Goss et al. (19). We determined the clade of three additional samples, two from Lund, Sweden (LU1.1a and LU18.1a), and one from Kyoto (KY12.1d). The remaining 202 isolates were genotyped for PAIs only. PAI genotyping was done by PCR. The primer set to identify the presence of the T-PAI included shcAf1 (5′-GGCGCACTTAACCCTCTGKTCAATGA-3′) and hoppsyAr1 (5′-CYGGCGTATGATTGATAAACGCATCG-3′). The primer set to identify the presence of the S-PAI included RMXPAIf6 (5′-TGGTCGAGCTGTTCACTCACCTGT-3′) and RMXPAIr7 (5′-TTGAACTGGTTGATCGGGTTCAGG-3′). There were three primer sets used to identify the absence of the T-PAI: (i) RMXnPAIf5 (5′-CCGTGCTGTGGTCATTGTCCTGAT-3′) and MEnPAIr2u (5′-GTAACAAGCCRTGACACAAACCTAC-3′), (ii) MEnPAIf5 (5′-TGCCATCGTTCATATTGAAGCTCAG-3′) and MEnPAIr4 (5′-CGCACGGCATCGCCAACCTTGAATG-3′), and (iii) MEnPAIf7 (5′-ACCTCAATCAATACTCTGGAGATCA-3′) and MEnPAIr6 (5′-CGAYTCACTGACCATCAACTGCCTG-3′). Finally, to identify the absence of the S-PAI, we used the primers LPnPAIf2 (5′-GTCCGGTCTGCTACCAGAACCTGGC-3′) and LPnPAIr2 (5′-TGCGCACCAGCGGCAGGTATTGCGG-3′).

Statistical Tests of Polymorphism Levels.

Sequences were edited by sequencher 4.1.2 (Gene Codes, Ann Arbor, MI). Homologs were searched by blastx (using the E value threshold >0.001) for the bacterial databases for the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) and TIGR (www.tigr.org) and aligned by clustal x (39) with minor manual corrections. Polymorphism and divergence surveys were performed by using dnasp 3.53 and dnasp 4.00 (40) and proseq 2.91 (41). Fu and Li’s test was performed by using dnasp 4.00 (40) with the window size 100 bp and the step size 25 bp (Fig. 3). Neighbor-joining trees were constructed by mega2.1 (42).

HR Tests.

P. viridiflava strains were incubated on KB plates at 28°C for 48 h. Single colonies were grown in liquid KB overnight, diluted in the morning, and grown to an optical density of 0.7–1.0 colony-forming units (cfu)/ml at 600 nm. For infection, bacteria were diluted to 2 × 108 cfu/ml in 10 mM MgSO4 buffer. Two leaves on each of two A. thaliana Col-0 plants and one leaf on each of three tobacco cv. Burley plants were infected with each strain of P. viridiflava by using a blunt-end syringe. For both host species, 10 isolates with each PAI were assayed. Appearance of HR was scored hourly after infection. For tobacco, percent available leaf area (area within the leaf veins) showing necrosis was estimated visually after 24 h.

Supplementary Material

Supporting Tables:

Acknowledgments

We thank J. Dangl, R. F. Doolittle, G. Dwyer, M. Nordborg, and H. Ochman for helpful discussions; J. T. Greenberg for a sample of P. cichorii 83-1; and J. Gladstone, M. Ludwig, and E. Bakker for technical help. This work was supported by grants from the National Institutes of Health and National Science Foundation and fellowships from the Japan Society for the Promotion of Science and the Dropkin Foundation.

Abbreviations

HGT
horizontal gene transfer
HR
hypersensitive response
PAI
pathogenicity island
TTSS
Type III secretion system.

Footnotes

Conflict of interest statement: No conflicts declared.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession nos. AY597274AY597283, AY859111, AY859112, AY859115, AY859128, AY859131, AY859183, AY859184, AY859351, AY859355, AY859358, DQ158500158855, DQ168848, and DQ220702).

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