Genomic analysis of the Kiwifruit pathogen Pseudomonas syringae pv. actinidiae provides insight into the origins of an emergent plant disease

Abstract

The origins of crop diseases are linked to domestication of plants. Most crops were domesticated centuries--even millennia--ago, thus limiting opportunity to understand the concomitant emergence of disease. Kiwifruit (Actinidia spp.) is an exception: domestication began in the 1930s with outbreaks of canker disease caused by P. syringae pv. actinidiae (Psa) first recorded in the 1980s. Based on SNP analyses of two circularized and 34 draft genomes, we show that Psa is comprised of distinct clades exhibiting negligible within-clade diversity, consistent with disease arising by independent samplings from a source population. Three clades correspond to their geographical source of isolation; a fourth, encompassing the Psa-V lineage responsible for the 2008 outbreak, is now globally distributed. Psa has an overall clonal population structure, however, genomes carry a marked signature of within-pathovar recombination. SNP analysis of Psa-V reveals hundreds of polymorphisms; however, most reside within PPHGI-1-like conjugative elements whose evolution is unlinked to the core genome. Removal of SNPs due to recombination yields an uninformative (star-like) phylogeny consistent with diversification of Psa-V from a single clone within the last ten years. Growth assays provide evidence of cultivar specificity, with rapid systemic movement of Psa-V in Actinidia chinensis. Genomic comparisons show a dynamic genome with evidence of positive selection on type III effectors and other candidate virulence genes. Each clade has highly varied complements of accessory genes encoding effectors and toxins with evidence of gain and loss via multiple genetic routes. Genes with orthologs in vascular pathogens were found exclusively within Psa-V. Our analyses capture a pathogen in the early stages of emergence from a predicted source population associated with wild Actinidia species. In addition to candidate genes as targets for resistance breeding programs, our findings highlight the importance of the source population as a reservoir of new disease.

Figure 1. Synteny plot of Psa NZ V-13 and Psa J-35.

MUMmer dotplot displaying stretches of conserved sequence between the genomes of Psa NZ V-13 and J-35 as lines with slope = 1. Inverted and translocated stretches of conserved sequence are displayed as lines with slope = −1.

Figure 2. Phylogeny of Psa and recombination between canker-causing Psa clades.

RAxML Maximum likelihood phylogenetic analysis of 32 draft and complete genome sequences based on 15,329 SNPs and 463,396 invariant sites (A). Each phylogenetic group is assigned its own color. With the exception of a single Italian strain (*) isolated in 1992 grouping with the Japanese clade, the canker-causing Japanese, Korean and low-virulent foliar NZ isolates form monophyletic clades reflecting their geographic origin, while global isolates from the 2008–2010 outbreak form a single clade. Bootstrap scores shown are based on 100 replicates. A Splitstree analysis of recombination predicts recombination between canker-causing clades of Psa (B). All bootstrap scores are 100 (shown and otherwise).

Figure 3. Shared and unique SNPs in Psa K-26 and J-31 compared to Psa NZ V-13.

Sample view from Artemis showing SNPs from Psa J-31 and K-26 aligned against a ∼30 kb region of the NZ V-13 genome. Each line represents a SNP that distinguishes J-31 and/or K-26 from NZ V-13. Over this region, K-26 differs from NZ V-13 by 155 SNPs; J-31 differs by 65 SNPs. In the blue region there are 50 SNPs that distinguish J-31 and K-26 from NZ V-13. Of these, J-31 and K-26 are identical at 42 positions. GENECONV predicts that this region represents a gene conversion event between J-31 and K-26. The purple region denotes a gene conversion event into K-26 from a strain outside the set analyzed here. The set of Artemis input files allowing representation of the full set of SNPs, coverage of SNPs and regions identified by GENECONV as statistically supported regions involved in gene conversion events are available as Supplementary Dataset 1.

Figure 4. Phylogeny of Psa-V isolates and the divergent Chinese isolate C-9.

Neighbor joining tree of Psa-V and C-9 isolates built in PHYLIP using SNPs due to mutation alone. These distances are also displayed in the upper right section of Table 3.

Figure 5. Unique and shared ortholog groups between clades.

Numbers outside the rainbow plot show the number of ortholog groups with at least one representative ORFs per strain per clade.

Figure 6. Structure of the Pacific, Mediterranean, Andean and PPHGI-1 islands.

Grey genes are orthologs with ∼75% nucleotide identity. Blue genes are variable accessory genes. Purple genes are accessory genes with complete conservation across the Pacific, Andean and Mediterranean islands. Red genes have translocated via transposon-mediated insertion events. Each island is bounded by 52 bp att sequences overlapping tRNA-Lys. Primer sites for the confirmation of excision and chromosomal integration are shown in green (Figure S4).

Figure 7. Type 3 secreted effector and toxin distribution in Psa clades.

The numbers inside each region of the Venn diagram represent T3SEs with orthologs present in the low-virulent (LV), Korean (K), Japanese (J) and recent global outbreak (V) clades (A). The outer boxes in B reflect the clade-specific distribution of T3SEs (italicized) and toxins, while the color of the internal text boxes refers to their occurrence on genomic islands. ¹premature terminations, partial translocations, frame shifts, and out of frame indels in some strains. ²variation between alleles (>2% variation, in frame fusions/indels). ³indicates the T3SE may not be in all Psa K strains. ⁴indicates the T3SE may not be in all Psa J strains. ⁵T3SE that occur within a transposon region. ⁶T3SE is present in the conserved effector locus. ⁷LV has two effectors in the HopAF1 group, HopAF1-2 is most closely related to HopAF1-1 in the Korean clade while HopAF1-1 is most closely related to HopAF1-1 in the outbreak and Japanese clades.

Figure 8. Rearrangements, insertions and deletions in the hopA1 locus.

The deletion of the schA chaperone and N-terminus of hopA1 in K-28 and deletion of hopA1 in J-35 are displayed with grey triangles. The transposon-mediated excision of a region in the hopA1 locus and its reintegration 500 kb away in the Psa NZ V-13 genome is shown with a blue triangle. Arrows at the 5′ end of the coding sequence indicate which alleles are functional. Stars above hopA1 indicate the presence of non-synonymous (NS) mutations. The white bar in NZ V-13 refers to a deletion and the black bar in Psa K-28 indicates the position of a nonsense mutation in hopA1. The deletion in NZ V-13 includes one of the non-synonymous mutations in K-28.

Figure 9. Pathogenicity assay of Psa and Pmp strains on kiwifruit.

The growth of the canker-causing Psa J-35 (blue), NZ V-13 (red), and K-26 (purple) isolates was assayed on the ‘Hort16A’ (A) and ‘Hayward’ (B) cultivars of kiwifruit, along with the low-virulent NZ LV-5 (green) and a strain of P. syringae pv. morsprunorum (Pmp, yellow),that causes canker disease in Prunus spp. The average bacterial density (cfu ± SE) was assayed in the stem tissue at day 0 immediately following stab inoculation, as well as in the base of the first leaf above the inoculation site (no Psa or Pmp observed, data not shown). The bacterial density was quantified in the base of the first leaf above the inoculation site (dark colored bar), the center of the leaf along the mid-vein (medium colored bar), and at the leaf tip and periphery (light colored bar) 4, 8 and 14 days after inoculation. A mock inoculation with MgSO₄ buffer was also performed, no Psa or Pmp growth was observed (not shown).