Molecular Evolution of Pathogenicity-Island Genes in Pseudomonas Viridiflava
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Copyright Ó 2007 by the Genetics Society of America DOI: 10.1534/genetics.107.077925 Molecular Evolution of Pathogenicity-Island Genes in Pseudomonas viridiflava Hitoshi Araki,*,†,1 Hideki Innan,‡ Martin Kreitman* and Joy Bergelson* *Department of Ecology and Evolution, University of Chicago, Chicago, Illinois 60637, †Department of Biology, Nanjing University, Nanjing 210093, China and ‡Graduate University of Advanced Studies, Hayama 240-0193, Japan Manuscript received June 20, 2007 Accepted for publication August 10, 2007 ABSTRACT The bacterial pathogen Pseudomonas viridiflava possesses two pathogenicity islands (PAIs) that share many gene homologs, but are structurally and phenotypically differentiated (T-PAI and S-PAI). These PAIs are paralogous, but only one is present in each isolate. While this dual presence/absence polymorphism has been shown to be maintained by balancing selection, little is known about the molecular evolution of individual genes on the PAIs. Here we investigate genetic variation of 12 PAI gene loci (7 on T-PAI and 5 on S- PAI) in 96 worldwide isolates of P. viridiflava. These genes include avirulence genes (hopPsyA and avrE ), their putative chaperones (shcA and avrF ), and genes encoding the type III outer proteins (hrpA, hrpZ, and hrpW ). Average nucleotide diversities in these genes (p ¼ 0.004–0.020) were close to those in the genetic background. Large numbers of recombination events were found within PAIs and a sign of positive selection was detected in avrE. These results suggest that the PAI genes are evolving relatively freely from each other on the PAIs, rather than as a single unit under balancing selection. Evolutionarily stable PAIs may be preferable in this species because preexisting genetic variation enables P. viridiflava to respond rapidly to natural selection. ENE-for-geneinteractionsbetween plants and plant R genes, however, the Phyophthora infestans scr74 gene G pathogens involve specific resistance (R) gene pro- family of effectors may be subject to diversifying se- ducts in plants that are responsible for the recognition of lection (Liu et al. 2005). Limited evidence of balancing elicitors encoded by avirulence (Avr) genes in pathogens selection acting upon virulence-related genes is found (Flor 1971). Plant genomes possess many R gene loci in Fusarium graminearum (Ward et al. 2002) and in (Arabidopsis Genome Initiative 2000); annotation of Borrelia burgdorferi (Qiu et al. 2004). A comprehensive the Arabidopsis thaliana genomic sequence, in particular, survey of Pseudomonas syringae effectors identified an identifies 150, most of which occur in clusters of 2–9 additional seven gene families with domains subject to loci (Meyers et al. 2003). It is clear that selection has diversifying selection, although the majority of gene acted to diversify paralogous R gene family members in families were shaped by purifying selection (Rohmer the Arabidopsis genome (Meyers et al. 2003). However, et al. 2004). Rohmer et al. (2004) also showed that 9 of 24 many R gene polymorphisms in A. thaliana have been effector genes were acquired via horizontal gene trans- shown to be subject to balancing or transient balancing fer (HGT), indicating that HGT is one of the important selection, rather than directional selection (Stahl et al. factors in bacterial effector gene evolution. 1999; Bergelson et al. 2001; Tianet al. 2002; Bakker et al. In a previous study, we identified two paralogous PAIs 2006; Shen et al. 2006), in contrast to expectations under in P. viridiflava (Araki et al. 2006), a prevalent natural the simplest arms race model (Dawkins and Krebs pathogen of A. thaliana (Jakob et al. 2002). Although 1979). In fact, there is little evidence for direct R–Avr these PAIs (T- and S-PAI, respectively) share 25 gene protein interaction (e.g., Dodds et al. 2006). These facts homologs, they are structurally distinct. The T-PAI is may be explained by the ‘‘guard hypothesis’’, in which R composed of three parts: a gene cluster encoding the gene products are assumed to recognize Avr gene pro- type III protein-secretion apparatus (hrp/hrc gene clus- ducts only indirectly via modifications of host proteins ter), the 59-effector locus (exchangeable effector locus, that are targets of the Avr gene products (Dangl and EEL), and the 39-effector locus (conserved effector Jones 2001). locus, CEL). The S-PAI, on the other hand, has a single Much less is known about the molecular evolution component hrp/hrc cluster with a 10 kb-long insertion. of genes encoding bacterial effectors. Like Arabidopsis The two PAIs are integrated at different locations in the P. viridiflava genome, and both islands are segregating for alleles in which the entire island is deleted (Figure Sequence data from this article have been deposited with the GenBank 1). Extensive surveys of P. viridiflava isolates from Data Library under accession nos. AY859094–AY859371. worldwide collections have identified only two com- 1Corresponding author: Department of Zoology, Oregon State University, 3029 Cordley Hall, Corvallis, OR 97331. binations, each containing one of the two islands (i.e., E-mail: [email protected] ½T-PAI, =S-PAI and ½=T-PAI, S-PAI). Several lines of Genetics 177: 1031–1041 (October 2007) 1032 H. Araki et al. evidence indicate that this dual presence/absence poly- differentiated clades, A and B (Goss et al. 2005). Both T- morphism has been maintained by balancing selection. and S-PAI isolates are present in clade A, but only S-PAI Furthermore, the ½T-PAI, =S-PAI and ½=T-PAI, S-PAI isolates have been identified in clade B. We therefore isolates exhibit virulence differences (Araki et al. 2006). analyzed genetic variation and molecular evolution of ½T-PAI, =S-PAI isolates elicit a rapid defense response the 12 loci for each of the three groupings: 7 loci on ½AT known as the hypersensitive response (HR) in A. (indicating isolates with the T-PAI in clade A) and 5 on thaliana significantly more slowly than ½=T-PAI, S-PAI ½AS (indicating isolates with the S-PAI in clade A) and ½BS isolates, whereas they elicit an HR in tobacco signifi- (indicating isolates with the S-PAI in clade B) (Figure 1). cantly more quickly than the ½=T-PAI, S-PAI isolates. Our results suggest that these loci have evolved relatively This functional differentiation of PAIs is required for freely from other genes on the same PAI, despite the fact selection to maintain this unusual dual-haplotype con- that they are located on genetic islands and that entire figuration as a polymorphism. Why the entire PAI, PAIs are under balancing selection. rather than a particular effector gene on it, has been selected as a unit has been unclear and how individual genes on the PAI evolve remains unanswered. MATERIALS AND METHODS To address these questions, we investigate molecular Samples: Ninety-three isolates out of the 96 investigated evolution in 12 PAI gene loci (7 on T-PAI and 5 on S-PAI) here are described in Goss et al. (2005). Eighty-three of them in P. viridiflava. These loci include five genes shared by were isolated from A. thaliana plants and the remaining 10 the two PAIs (avrE, avrF, hrpA, hrpZ, and hrpW designated were isolated from other weedy plant species alongside A. as avrE , avrF , hrpA , hrpZ , and hrpW on S-PAI thaliana. Three additional isolates included in this study—- [S] [S] [S] [S] [S] LU1.1a and LU18.1a (from Lund, Sweden) and KY12.1d and avrE[T], avrF[T], hrpA[T], hrpZ[T], and hrpW[T] on T- (from Kyoto, Japan)—were isolated from A. thaliana plants. PAI) as well as two T-PAI-specific genes (hopPsyA[T] and The PAI presence/absence genotype was identified by PCR raki shcA[T]) (Figure 1). hopPsyA and shcA encode an Avr pro- (see A et al. 2006 for details). In this manner, 10 isolates tein that elicits a HR on tobacco and its putative chaper- were identified as ½AT, 57 were identified as ½AS and 29 were one, respectively (Alfano and Collmer 1997; Van Dijk identified as ½BS. Sequence data analyses and the tests of selective neutrality: et al. 2002). avrE encodes a known Avr protein that elicits Sequences of the seven PAI genes were obtained by direct PCR HR on tobacco and soybean plants (Lorang and Keen and sequencing using the same conditions as those described 1995). avrF is putatively an AvrE-specific chaperone in Araki et al. (2006). Primers for the PCR are listed in sup- (Bogdanove et al. 1998) and might be required for plemental Table 1 at http://www.genetics.org/supplemental/. ° ° efficient delivery of AvrE (Ham et al. 2006). A potential Primer annealing temperatures were between 55 and 60 . Sequences of hrpW from three isolates (LU5.1a, LU9.1e, role of AvrE as a suppressor of basal immunity and pro- [T] and PT220.1a) and hrpW[S] from four isolates (KNOX230.1a, motion of host cell death is reported in Debroy et al. ME751a, ME753a, and ME756a) were eliminated from the fol- (2004). hrpA, hrpZ, and hrpW encode outer proteins lowing analyses because we could obtain only partial sequences. involved in the type-III secretion system (TTSS). hrpA Sequences were edited by Sequencher v.4.1.2 (Gene Codes, encodes the pilus, which plays a key role in the secretion Ann Arbor, MI). After editing, sequences were aligned by CLUSTAL X (Thompsonet al. 1997) with minor manual adjust- of Avr and other type-III effector proteins and is subject ments. Reference sequences for alignments were obtained from to natural selection in P. syringae (Jin and He 2001; the GenBank online database as follows: P. viridiflava LP23.1a, Guttman et al. 2006). hrpZ and hrpW encode harpin PNA29.1a, and P. syringae pv. tomato (Pto) DC3000 for T-PAI uang reston and P.