Comparative genomics of Pseudomonas syringae pv. syringae strains B301D and HS191 and insights into intrapathovar traits associated with plant pathogenesis

Abstract

Pseudomonas syringae pv. syringae is a common plant-associated bacterium that causes diseases of both monocot and dicot plants worldwide. To help delineate traits critical to adaptation and survival in the plant environment, we generated complete genome sequences of P. syringae pv. syringae strains B301D and HS191, which represent dicot and monocot strains with distinct host specificities. Intrapathovar comparisons of the B301D (6.09 Mb) and HS191 (5.95 Mb plus a 52 kb pCG131 plasmid) genomes to the previously sequenced B728a genome demonstrated that the shared genes encompass about 83% of each genome, and include genes for siderophore biosynthesis, osmotolerance, and extracellular polysaccharide production. Between 7% and 12% of the genes are unique among the genomes, and most of the unique gene regions carry transposons, phage elements, or IS elements associated with horizontal gene transfer. Differences are observed in the type III effector composition for the three strains that likely influences host range. The HS191 genome had the largest number at 25 of effector genes, and seven effector genes are specific to this monocot strain. Toxin production is another major trait associated with virulence of P. syringae pv. syringae, and HS191 is distinguished by genes for production of syringopeptin SP25 and mangotoxin.

Keywords: Comparative genomics; Pseudomonas syringae; effector gene; genome sequence; plant pathogen.

Figure 1

Phylogenetic relationships of the genomes of strains B301D and HS191 to other Pseudomonas species and pathovars. The maximum likelihood tree was constructed using concatenated alignments of 10 housekeeping genes: acsA, aroE, dnaE, guaA, gyrB, mutL, ppsA, pyrC, recA, and rpoB. The MrBayes package (Ronquist and Huelsenbeck 2003) was used to calculate the clade credibility values shown as values within interior nodes. Strains of Pseudomonas syringae in genomospecies 1 are indicated by the outside bracket and the strains originating from monocot and dicot hosts are as indicated by two inside brackets. Strains B301D and HS191 that were sequenced in this study are shown in red, enlarged lettering. The outgroup genome sequence used was Escherichia coli K-12 substrain MG1655.

Figure 2

Pairwise alignment between the chromosomes of Pseudomonas syringae pv. syringae strains (A) B728a, (B) B301D, and (C) HS191 using MAUVE software (Darling et al. 2010). Colors depict conserved and highly related genomic regions and white areas identify unique or low identity regions. Blocks shifted below the center line indicate segments that align in the reverse orientation as inversions relative to reference strain B728a.

Figure 3

Circular representation of three genome comparisons using the BLAST Ring Image Generator (BRIG) software (Alikhan et al. 2011). The inner scales designate the coordinates in kilobase pairs (kbp). White spaces indicate regions with no identity to the reference genome. The gene clusters absent in B301D are indicated by a red arrow, clusters absent in HS191 are indicated by a green arrow, clusters absent in B728a are indicated by a purple arrow, and clusters absent in both the query genomes are indicated by a black arrow. (A) B728a genome (center) compared against the genomes of B301D (Ring 1 in red) and HS191 (Ring 2 in green). (B) B301D genome (center) compared against the genomes of B3728a (Ring 1 in purple) and HS191 (Ring 2 in green). (C) HS191 genome (center) compared against the genomes of B728a (Ring 1 in purple) and B301D (Ring 2 in red). Relative shading density (from darker to lighter) within each circle represents relative levels of nucleotide homology.

Figure 4

Venn diagram comparing the unique and shared genes between the genomes of B728a (top), B301D (left), and HS191 (right) using BLASTp. The numbers below the strain names identify the total number of protein coding genes within each genome. The strain HS191 genome has 645 unique genes, including 61 unique genes on plasmid pCG131. Genes that are conserved among all three strains are shown in the center of the diagram as the pangenome.

Figure 5

Comparison of the organization of the syringopeptin nonribosomal peptide synthetase genes sypA, sypB, sypC in strains B301D (top), B728a (center), and HS191 (bottom). Strains B301D and B728a produce SP22 (Grgurina et al. 2002) and HS191 produces SP25 (Ballio et al. 1991). The amino composition for SP22 and SP25 are shown below the respective synthetase genes; the color of the amino acid corresponds to the specific syp synthetase gene. The sypA gene of HS191 encodes NRPS modules for three additional amino acids in SP25 as shown in blue; B728a compared to B301D is modified in amino acids at positions four and seven (underlined). The size of the bar approximately corresponds to gene size.

Figure 6

Comparison of the region in strain HS191 carrying the mbo operon (mboA to mboF) responsible for mangotoxin production with the corresponding regions in strains B728a and B301D (blue arrows indicate predicted location) that do not produce mangotoxin and lack the mbo operon. Colors indicate ortholog groups and the size of the bar approximately corresponds to gene size. This comparison was done using JGI-IMG/ER (https://img.jgi.doe.gov/cgi-bin/w/main.cgi).

Figure 7

Circular representation of plasmids from pathovars of Pseudomonas syringae against HS191 plasmid pCG131 using the BLAST Ring Image Generator (BRIG) software (Alikhan et al. 2011). The inner scales designate the coordinates in kilobase pairs (kbp). Ring 1 = P. syringae pv. syringae plasmid pSM1 (purple); Ring 2 = P. syringae pv. syringae plasmid pPSR1 (blue); Ring 3 = P. syringae pv. phaseolicola pPph1448A-B (red); Ring 4 = P. syringae pv. maculicola pPMA4326A (green); Ring 5 = Pseudomonas fluorescens pA506 (black); Ring 6 = coding DNA sequence (CDS) of HS191 plasmid (yellow). The areas in pCG131 not occurring in the four plasmids used for comparison are indicated by red brackets. Relative shading density (from darker to lighter) within each circle represents relative levels of nucleotide homology.