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, nablaS-PAI] and [nablaT-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.

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 clade (19) of each isolate and which PAI it contains is indicated.

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 ORFs were used for PAI genes (7.4 kb) and avrE (1.7 kb), and total sequences were used for 16S and gyrB (2.0 kb in total). The 25 PAI genes in A include ORFs R1-ORF45-51, 53–68, 72, 73, 76, and R2-ORF8-14, 16-20, 24-36 (Tables 1 and 2). Shown are bootstrap probabilities with 1,000 replications. The clade and PAI [clade, PAI] are given for each P. viridiflava isolate, as in Fig. 1. PAIs in P. syringae DC3000 and in P. cichorii 83-1 are orthologous to T-PAI and S-PAI, respectively.

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 (D_xy) by using the Jukes and Cantor correction (22, 23) were plotted for the flanking regions of the T-PAI (A, Region 1) and of the S-PAI (B, Region 2) for the five P. viridiflava isolates in Figs. 1 and 2. Window size was 100 bp with 25-bp steps. The locations of ORFs are indicated by boxes; insertions found in only one isolate and the locations of PAIs are represented by open and closed triangles, respectively. Orthologous ORFs found in the Pto DC3000 genome (24) are indicated by filled bars with their accession ID. The PAI in Pto DC3000 was defined from Pspto1411 to Pspto1367 (16). Sharp, clear peaks of genetic divergence are observed around the indel-junction both of T-PAI and S-PAI. Fu and Li’s test of selective neutrality (D* and F*) (27) detected significant departure from neutrality around the peaks (P < 0.02, indicated by asterisks) based on a sample of 96 isolates.

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 Col-0 (x axis) and Tobacco cv. Burley (y axis). To minimize the effect of their genetic background, only isolates in clade A (19) were selected. [AT], LP23.1a, PNA3.3a, LU5.1a, LU9.1e, LU13.1a, LU18.1a, LU19.1a, SP3.1a, ME210.1b, and PT220.1a; [AS], RMX23.1a, ME3.1a, KNOX3.4a, BOG1.1a, BOG4.2a, BOR1.3d, KY5.1a, KY7.1d, SP1.1a, and SP12.1a.