The Pseudomonas syringae Hrp pathogenicity island has a tripartite mosaic structure composed of a cluster of type III secretion genes bounded by exchangeable effector and conserved effector loci that contribute to parasitic fitness and pathogenicity in plants

Abstract

The plant pathogenic bacterium Pseudomonas syringae is divided into pathovars differing in host specificity, with P. syringae pv. syringae (Psy) and P. syringae pv. tomato (Pto) representing particularly divergent pathovars. P. syringae hrp/hrc genes encode a type III protein secretion system that appears to translocate Avr and Hop effector proteins into plant cells. DNA sequence analysis of the hrp/hrc regions in Psy 61, Psy B728a, and Pto DC3000 has revealed a Hrp pathogenicity island (Pai) with a tripartite mosaic structure. The hrp/hrc gene cluster is conserved in all three strains and is flanked by a unique exchangeable effector locus (EEL) and a conserved effector locus (CEL). The EELs begin 3 nt downstream of the stop codon of hrpK and end, after 2.5-7.3 kb of dissimilar intervening DNA with tRNA(Leu)-queA-tgt sequences that are also found in Pseudomonas aeruginosa but without linkage to any Hrp Pai sequences. The EELs encode diverse putative effectors, including HopPsyA (HrmA) in Psy 61 and proteins similar to AvrPphE and the AvrB/AvrC/AvrPphC and AvrBsT/AvrRxv/YopJ protein families in Psy B728a. The EELs also contain mobile genetic element sequences and have a G + C content significantly lower than the rest of the Hrp Pai or the P. syringae genome. The CEL carries at least seven ORFs that are conserved between Psy B728a and Pto DC3000. Deletion of the Pto DC3000 EEL slightly reduces bacterial growth in tomato, whereas deletion of a large portion of the CEL strongly reduces growth and abolishes pathogenicity in tomato.

Figure 1

The conserved arrangement of hrp/hrc genes within the Hrp Pais of Psy 61, Psy B728a, and Pto DC3000. Regions sequenced in B728a and DC3000 are indicated by lines beneath the strain 61 sequence. Known regulatory genes are shaded. Arrows indicate the direction of transcription, with small boxes denoting the presence of a Hrp box. The triangle denotes the 3.6-kb insert with phage genes in the B728a hrp/hrc region.

Figure 2

The exchangeable effector loci (EELs) of Pto DC3000, Psy B728a, and Psy 61, the tgt-queA-tRNA^Leu locus in P. aeruginosa, and EEL border sequences. (A) EELs of the three P. syringae strains are shown aligned by their hrpK sequences and are compared with the tgt-queA-tRNA^Leu locus in P. aeruginosa (Pa PA01). Arrows indicate the direction of transcription, with small boxes denoting the presence of a Hrp box. Shaded regions are conserved, hatched regions denote mobile genetic elements, and open boxes denote genes that are completely dissimilar from each other. (B) The sequences of the DC3000 (DC), B728a (B7), and 61 EELs at the border with tRNA^Leu are shown with conserved nucleotides in uppercase. (C) The sequences of the EELs at the border with hrpK are shown as above.

Figure 3

The Hrp Pai conserved effector locus (CEL) of P. syringae. The Pto DC3000 CEL is shown with the corresponding fragments of Psy B728a that were sequenced aligned below. The nucleotide identity of the sequenced fragments in coding regions ranged from 72% to 83%. Arrows indicate the direction of transcription, with small boxes denoting the presence of a Hrp box.

Figure 4

Plant interaction phenotypes of Pto mutants carrying deletions of the EEL (CUCPB5110) and CEL (CUCPB5115). (A) Growth in tomato of DC3000 and CUCPB5110 (mean and SE). (B) Growth in tomato of DC3000, CUCPB5115, and CUCPB5115(pCPP3016) (mean and SE). (C) HR collapse in tobacco leaf tissue 24 hr after infiltration with 10⁷ cfu/ml DC3000 and CUCPB5115. (D) Absence of disease symptoms in tomato leaf 4 days after inoculation with 10⁴ cfu/ml CUCPB5115. (E) Disease symptoms typical of wild type in tomato leaf 4 days after inoculation with 10⁴ cfu/ml CUCPB5115(pCPP3016).

Figure 5

Immunoblot analysis of AvrPto secretion by Pto DC3000 derivatives with deletions affecting the three major regions of the Hrp Pai. Bacteria were grown in Hrp-inducing minimal medium at pH 5.5 and 22°C to an OD₆₀₀ of 0.35 and then separated into cell-bound (C) and supernatant (S) fractions by centrifugation. Proteins were then resolved by SDS/PAGE, blotted, and immunostained with antibodies against AvrPto and β-lactamase as described (18), except that supernatant fractions were concentrated 3-fold relative to cell-bound fractions before loading. Pto DC3000, CUCPB5115 (CEL deletion), CUCPB5114 (hrp/hrc deletion), and CUCPB5110 (EEL deletion) all carried pCPP2318, which expresses β-lactamase without a signal peptide as a cytoplasmic marker.