The plant pathogen Pseudomonas syringae pv. tomato is genetically monomorphic and under strong selection to evade tomato immunity

Abstract

Recently, genome sequencing of many isolates of genetically monomorphic bacterial human pathogens has given new insights into pathogen microevolution and phylogeography. Here, we report a genome-based micro-evolutionary study of a bacterial plant pathogen, Pseudomonas syringae pv. tomato. Only 267 mutations were identified between five sequenced isolates in 3,543,009 nt of analyzed genome sequence, which suggests a recent evolutionary origin of this pathogen. Further analysis with genome-derived markers of 89 world-wide isolates showed that several genotypes exist in North America and in Europe indicating frequent pathogen movement between these world regions. Genome-derived markers and molecular analyses of key pathogen loci important for virulence and motility both suggest ongoing adaptation to the tomato host. A mutational hotspot was found in the type III-secreted effector gene hopM1. These mutations abolish the cell death triggering activity of the full-length protein indicating strong selection for loss of function of this effector, which was previously considered a virulence factor. Two non-synonymous mutations in the flagellin-encoding gene fliC allowed identifying a new microbe associated molecular pattern (MAMP) in a region distinct from the known MAMP flg22. Interestingly, the ancestral allele of this MAMP induces a stronger tomato immune response than the derived alleles. The ancestral allele has largely disappeared from today's Pto populations suggesting that flagellin-triggered immunity limits pathogen fitness even in highly virulent pathogens. An additional non-synonymous mutation was identified in flg22 in South American isolates. Therefore, MAMPs are more variable than expected differing even between otherwise almost identical isolates of the same pathogen strain.

Figure 1. Strains of the T1-lineage have been the most common Pto strains since the 1960s and are present in all continents from which Pto strains were isolated.

(A) The lines indicate the frequency of T1-, JL1065-, and DC3000-like strains over time using a 10-year sliding window with a one-year step. Circles represent individual isolates and are placed in the graph in correspondence to the exact year at which isolates were collected. Full circles indicate isolates of which the genomes have been sequenced. (B) World map with pie charts showing ratio of T1-, JL1065-, and DC3000-like strains for the continents from which Pto strains have been analyzed. Pie size is proportional to the total number of strains considered per continent.

Figure 2. Phylogenetic trees based on SNPs reveal the evolutionary relationship between T1-like Pto strains.

(A) Maximum likelihood tree based on 157 high quality SNPs identified between five genomes of T1-like strains by aligning Illumina sequencing reads against the DC3000 genome (which was used as an outgroup). The number of SNPs/branch are indicated underneath each branch and bootstrap values are indicated above each branch. A neighbor-joining tree and maximum parsimony tree were also constructed and had identical topology. (B) Maximum likelihood tree based on twenty-four SNPs identified between DC3000-like, JL1065, and T1-like strains in the housekeeping genes rpoD, pgi, and gapA and based on 16 SNPs identified between T1-like strains in 11 fragments of P. syringae core genome genes (highlighted in Table S2). Bootstrap values are indicated above each branch and number of strains that belong to each genotype are indicated in parenthesis. Clade-specific fliC and hopM1 alleles are indicated below branches. The clade corresponding to strains called “T1-proper” in the main text is labeled as such. A maximum parsimony tree was also constructed and had identical topology. Since branch lengths of the tree are influenced by our selection of SNP loci, branch lengths are not scaled to evolutionary changes. Table 1 lists strains belonging to each genotype and Table S4 lists DNA sequences of each genotype.

Figure 3. T1 genotypes change in frequency over time and genetic distances from the outgroup strain DC3000 increase over time.

Several genotypes are present in both North America and Europe. (A) The lines indicate the frequency of T1 genotypes over time using a 10-year sliding window with a one-year step. Circles represent individual isolates and are placed in the graph in correspondence to the exact year at which isolates were collected. Full circles indicate those isolates for which genomes have been sequenced. (B) Genetic distance of strains from the out-group strain DC3000 plotted over time. Genetic distance was calculated based on the 24 MLST SNPs and the 16 genome SNPs that were analyzed in all strains. When more than one isolate with the same genotype was collected during the same year, the total number of isolates is indicated next to the genotype symbol. (C) World map with pie charts showing ratio of T1 genotypes for the continents from which T1-like strains have been analyzed. Pie size is proportional to the total number of strains considered per continent.

Figure 4. The hopM1 gene is disrupted in all T1-like and JL1065-like strains.

The encoded truncated proteins do not trigger cell death in tomato while the full-length protein encoded by the DC3000 hopM1 gene does. (A) Graphical presentation of Pto hopM1 alleles. The stars indicate the position of deletions causing frameshifts in the PT21 and NCPPB1108 alleles. The PT21 allele is present in four strains of SNP genotype NCPPB1108 and in the only strain with SNP genotype CA315 while the NCPPB1108 allele is only present in strain NCPPB1108 (SNP genotype NCPPB1108). The T1 allele is present in all other T1-like strains, which are referred to as T1-proper in the text. (B) Agrobacterium-mediated transient expression of hopM1 alleles fused to gfp in the tomato cultivar “Chico III”. Only the hopM1 _DC3000 allele triggered cell death. Similar results were obtained on the tomato cultivars “Rio Grande” and “Sunpride” in at least two independent experiments/cultivar. Leaf areas infiltrated with Agrobacterium tumefaciens strains are traced in black. Strain names indicate which hopM1::gfp fusion construct was expressed in which leaf area. Agro EV: Agrobacterium carrying an empty vector control, T1-HA: in this leaf area the hopM1_T1 allele was expressed with an HA tag, CD: cell death. (C) Western Blot analysis with GFP antibody of HopM1::GFP fusion proteins from extracts of Nicotiana benthamiana leaf disks infiltrated with the same Agrobacterium tumefaciens strains used in panel B. * indicate the bands of the expected size based on the sequence of the hopM1 alleles in panel A. The Rubisco large subunit band from the Coomassie-stained gel is shown as loading control underneath the Western Blot.

Figure 5. The flagellin epitope flgII-28 triggers reactive oxygen species (ROS) in tomato leaves whereby derived alleles - typical of today's Pto strains - induce less ROS than the ancestral alleles - typical of strains isolated before 1985.

Alleles of flgII-28 also induce stomatal closure and interfere with leaf invasion. (A) Amino acid sequences of flg22 and flgII-28 alleles. The T1 alleles are identical to the DC3000 alleles and thus represent the ancestral states. The derived alleles are named after one of the genotypes in which they are present. (B) Induction of reactive oxygen species (ROS) in tomato leaf disks of cultivar ‘Chico III’ after incubation with flg22 and flgII-28 peptides at a 1 µM concentration. ROS induction was significantly different at the 2 minutes time point in an unpaired Student's t-test at the 0.05 level between flg22_T1 and flg22_Colombia338 and between flgII-28_T1 on one hand and flgII-28_LNPV17.41 and flgII-28_Colombia198 on the other. flgII-28_T1 and flgII-28_Colombia198 were also significantly different from each other at the 5 minutes time point. Similar results were obtained with three different tomato cultivars whereby experiments on each cultivar were repeated at least twice. (C) Stomatal closure induced in tomato leaves of cultivar ‘Chico III’ after infiltration with flg22 and flgII-28 peptides at a 5 µM concentration or mock infiltration with sterile water. Similar results were obtained in three independent experiments. Different letters indicate significance at the 0.05 level in an unpaired Student's t-test. (D) Leaves of tomato cultivar ‘Chico III” were infiltrated with flg22 and flgII-28 peptides at a 1 µM concentration. Strain NCPPB1108 (flgII-28_T1) was then sprayed on leaf surfaces 24 hours later and apoplastic population sizes were measured another 24 hours later. Different letters indicate significance at the 0.05 level in an unpaired Student's t-test.