Erwinia amylovora novel plasmid pEI70: complete sequence, biogeography, and role in aggressiveness in the fire blight phytopathogen

Abstract

Comparative genomics of several strains of Erwinia amylovora, a plant pathogenic bacterium causal agent of fire blight disease, revealed that its diversity is primarily attributable to the flexible genome comprised of plasmids. We recently identified and sequenced in full a novel 65.8 kb plasmid, called pEI70. Annotation revealed a lack of known virulence-related genes, but found evidence for a unique integrative conjugative element related to that of other plant and human pathogens. Comparative analyses using BLASTN showed that pEI70 is almost entirely included in plasmid pEB102 from E. billingiae, an epiphytic Erwinia of pome fruits, with sequence identities superior to 98%. A duplex PCR assay was developed to survey the prevalence of plasmid pEI70 and also that of pEA29, which had previously been described in several E. amylovora strains. Plasmid pEI70 was found widely dispersed across Europe with frequencies of 5-92%, but it was absent in E. amylovora analyzed populations from outside of Europe. Restriction analysis and hybridization demonstrated that this plasmid was identical in at least 13 strains. Curing E. amylovora strains of pEI70 reduced their aggressiveness on pear, and introducing pEI70 into low-aggressiveness strains lacking this plasmid increased symptoms development in this host. Discovery of this novel plasmid offers new insights into the biogeography, evolution and virulence determinants in E. amylovora.

Figure 1. Circular representation of plasmid pEI70.

Locus tags (EaACW_pEI700xx) are indicated in the graph. Genes in dark blue are genes with orthologs in ICE elements. Genes that were also identified in the ICE element of E. pyrifoliae, but which are not related to a function in the ICE element are highlighted in light blue. Genes in grey are unrelated to the ICE element.

Figure 2. Comparison of plasmid pEB102 of E. billingiae Eb661 (A) with E. amylovora ACW 56400 plasmid pEI70 (B), and the conserved region of GAI-2 of Pectobacterium atrosepticum SCRI 1043 (C).

Orthologous genes are indicated by blue shading (conserved ICE element genes) and shading. Genes in white do not have orthologs in these regions.

Figure 3. Biogeographic distribution map of strains harboring pEI70 in different European countries obtained by duplex-PCR.

Figure 4. Analysis of several strains by restriction digestion with BamHI (A) and hybridisation analyses using pEA29 as probe (B) or pEI70 as probe (C).

Isolate E 70 (lane 5) from Ireland shows a slightly different profile, with two extra bands that do not belong either to pEA29 or pEI70. Lane 1: strain IVIA 1614-2a; lane 2: CFBP 1430; lane 3: IVIA 1596; lane 4: NCPPB 3299; lane 5: E70; lane 6: Ea 273. M λ: marker Lambda (Invitrogen).

Figure 5. Severity and infection incidence in immature pear fruits by inoculation of strains of E. amylovora before and after receiving plasmid pEA29 or after curing of pEI70.

The severity and incidence of infection were measured 5 days after inoculation. The experiment was performed at 1×10⁵ cfu/ml (white columns) and 1×10⁶ cfu/ml (black columns). Means with the same letters (lower-case letters for low pathogen dosages, upper-case letters for high pathogen dosages) do not differ significantly according to Tukey's test (P≤0.05). Asterisks ‘*’ indicate assays that were not performed.

Figure 6. Severity and infection incidence in immature pear fruits by inoculation of strains of E. amylovora with or without pEI70.

The severity and incidence of infection were measured 7 days after inoculation. The experiment was performed at 1×10⁵ cfu/ml (white columns) and 5×10⁵ cfu/ml (black columns). Means with the same letters (lower-case letters for low dosages, upper-case letters for high dosages) do not differ significantly according to Tukey's test (P≤0.05).