Pseudomonas syringae exchangeable effector loci: sequence diversity in representative pathovars and virulence function in P. syringae pv. syringae B728a

Abstract

Pseudomonas syringae is a plant pathogen whose pathogenicity and host specificity are thought to be determined by Hop/Avr effector proteins injected into plant cells by a type III secretion system. P. syringae pv. syringae B728a, which causes brown spot of bean, is a particularly well-studied strain. The type III secretion system in P. syringae is encoded by hrp (hypersensitive response and pathogenicity) and hrc (hrp conserved) genes, which are clustered in a pathogenicity island with a tripartite structure such that the hrp/hrc genes are flanked by a conserved effector locus and an exchangeable effector locus (EEL). The EELs of P. syringae pv. syringae B728a, P. syringae strain 61, and P. syringae pv. tomato DC3000 differ in size and effector gene composition; the EEL of P. syringae pv. syringae B728a is the largest and most complex. The three putative effector proteins encoded by the P. syringae pv. syringae B728a EEL--HopPsyC, HopPsyE, and HopPsyV--were demonstrated to be secreted in an Hrp-dependent manner in culture. Heterologous expression of hopPsyC, hopPsyE, and hopPsyV in P. syringae pv. tabaci induced the hypersensitive response in tobacco leaves, demonstrating avirulence activity in a nonhost plant. Deletion of the P. syringae pv. syringae B728a EEL strongly reduced virulence in host bean leaves. EELs from nine additional strains representing nine P. syringae pathovars were isolated and sequenced. Homologs of avrPphE (e.g., hopPsyE) and hopPsyA were particularly common. Comparative analyses of these effector genes and hrpK (which flanks the EEL) suggest that the EEL effector genes were acquired by horizontal transfer after the acquisition of the hrp/hrc gene cluster but before the divergence of modern pathovars and that some EELs underwent transpositions yielding effector exchanges or point mutations producing effector pseudogenes after their acquisition.

FIG. 1.

Construction of the P. syringae pv. syringae B728a EEL deletion mutation and its corresponding complementation clone. (A) Physical map of the P. syringae pv. syringae B728a EEL. Pointed boxes indicate the predicted ORFs and the direction of transcription, with black triangles representing the presence of an Hrp box promoter. Hatched regions represent mobile genetic elements, and open boxes are putative effector or chaperone genes. (B) EEL deletion mutant CUCPB5111, with the dotted line representing the internal deletion that is replaced by an ΩSp^r cassette. (C) The 10,488-bp XhoI fragment shown contains hrpK to tRNA^Leu in pCPP3092.

FIG. 2.

Immunoblot analysis of HopPsyC-FLAG, HopPsyE-FLAG, and HopPsyV-FLAG secretion by E. coli carrying an intact (pCPP3042) or defective (pCPP3127) E. chrysanthemi type III secretion system. The supernatant fraction (S) was concentrated 7.5 times more than the cell-bound fraction (C). The upper panel was immunostained with anti-FLAG monoclonal antibody, and the bottom panel was stained with anti-β-galactosidase serum.

FIG. 3.

Hypersensitive response of tobacco leaves to infiltration with P. syringae pv. tabaci 11528 transformants expressing Hops encoded by the P. syringae pv. syringae B728a EEL. Leaves were inoculated with P. syringae pv. tabaci 11528 harboring pCPP3153 (hopPsyC⁺), pCPP3154 (hopPsyE⁺), or pCPP3155 (hopPsyV⁺). The leaf on the left was infiltrated with 10⁸ CFU ml of inoculum⁻¹, and the one on the right was infiltrated with 10⁵ CFU ml⁻¹. Photographs were taken 1 and 5 days postinfiltration, respectively.

FIG. 4.

Growth of P. syringae pv. syringae B728a, its mutant derivatives CUCPB5102 (ΔhrcC::nptII) and CUCPB5111 (ΔEEL::ΩSp^r), and complemented strain CUCPB5111(pCPP3092) in bean leaves. Bacteria were inoculated by (A) infiltration with 10⁴ CFU ml⁻¹ or (B) dipping with 10⁵ CFU ml of inoculum⁻¹. Each point represents the mean and standard error of three samples, and each sample is composed of 10 7-mm leaf disks.

FIG. 5.

Physical maps of the EELs of P. syringae pv. syringae B728a and other strains. Pointed boxes indicate the predicted ORFs and the direction of transcription, black triangles indicate the presence of the Hrp box, and black boxes indicate the position of tRNA^Leu. Stippled regions are conserved among different pathovars and were used for primer design. Hatched regions represent mobile genetic elements. Open boxes with solid lines are putative effector genes, whereas boxes with dotted lines are apparent pseudogenes.

FIG. 6.

Phylograms of optimal trees of EELs in P. syringae strains based on 250 bp of hrpK (A) and full-length avrPphE (B) sequences. Horizontal branch length is proportional to the estimated number of nucleotide substitutions, and bootstrap probabilities (as percentages) are indicated above or below the internal branches. The trees are rooted at the midpoint. Abbreviations for strains used in the figure follow: Psy, pathovar syringae; Pdp, pathovar delphinii; Pto, pathovar tomato; Pau, pathovar angulata; Pmo, pathovar mori; Pph, pathovar phaseolicola; Pgy, pathovar glycinea. In panel A, strains with EELs encoding ShcA-HopPsyA homologs are shown in shaded boxes. In panel B, the open box indicates that the avrPphE homolog used in phylogenetic analysis is not associated with the EEL.