Pseudomonas syringae coffee blight is associated with the horizontal transfer of plasmid-encoded type III effectors.

The emergence of new pathogens is an ongoing threat to human health and agriculture. While zoonotic spillovers received considerable attention, the emergence of crop diseases is less well studied. Here, we identify genomic factors associated with the emergence of Pseudomonas syringae bacterial blight of coffee. Fifty-three P. syringae strains from diseased Brazilian coffee plants were sequenced. Comparative and evolutionary analyses were used to identify loci associated with coffee blight. Growth and symptomology assays were performed to validate the findings. Coffee isolates clustered in three lineages, including primary phylogroups PG3 and PG4, and secondary phylogroup PG11. Genome-wide association study of the primary PG strains identified 37 loci, including five effectors, most of which were encoded on a plasmid unique to the PG3 and PG4 coffee strains. Evolutionary analyses support the emergence of coffee blight in PG4 when the coffee-associated plasmid and associated effectors derived from a divergent plasmid carried by strains associated with other hosts. This plasmid was only recently transferred into PG3. Natural diversity and CRISPR-Cas9 plasmid curing were used to show that strains with the coffee-associated plasmid grow to higher densities and cause more severe disease symptoms in coffee. This work identifies possible evolutionary mechanisms underlying the emergence of a new lineage of coffee pathogens.

Predictive modeling of Pseudomonas syringae virulence on bean using gradient boosted decision trees.

Pseudomonas syringae is a genetically diverse bacterial species complex responsible for numerous agronomically important crop diseases. Individual P. syringae isolates are assigned pathovar designations based on their host of isolation and the associated disease symptoms, and these pathovar designations are often assumed to reflect host specificity although this assumption has rarely been rigorously tested. Here we developed a rapid seed infection assay to measure the virulence of 121 diverse P. syringae isolates on common bean (Phaseolus vulgaris). This collection includes P. syringae phylogroup 2 (PG2) bean isolates (pathovar syringae) that cause bacterial spot disease and P. syringae phylogroup 3 (PG3) bean isolates (pathovar phaseolicola) that cause the more serious halo blight disease. We found that bean isolates in general were significantly more virulent on bean than non-bean isolates and observed no significant virulence difference between the PG2 and PG3 bean isolates. However, when we compared virulence within PGs we found that PG3 bean isolates were significantly more virulent than PG3 non-bean isolates, while there was no significant difference in virulence between PG2 bean and non-bean isolates. These results indicate that PG3 strains have a higher level of host specificity than PG2 strains. We then used gradient boosting machine learning to predict each strain's virulence on bean based on whole genome k-mers, type III secreted effector k-mers, and the presence/absence of type III effectors and phytotoxins. Our model performed best using whole genome data and was able to predict virulence with high accuracy (mean absolute error = 0.05). Finally, we functionally validated the model by predicting virulence for 16 strains and found that 15 (94%) had virulence levels within the bounds of estimated predictions. This study strengthens the hypothesis that P. syringae PG2 strains have evolved a different lifestyle than other P. syringae strains as reflected in their lower level of host specificity. It also acts as a proof-of-principle to demonstrate the power of machine learning for predicting host specific adaptation.

Immunodiversity of the Arabidopsis ZAR1 NLR Is Conveyed by Receptor-Like Cytoplasmic Kinase Sensors.

The Arabidopsis nucleotide-binding leucine-rich repeat protein ZAR1 can recognize at least six distinct families of pathogenic effector proteins to mount an effector-triggered immune response. This remarkable immunodiversity appears to be conveyed by receptor-like cytoplasmic kinase (RLCK) complexes, which associate with ZAR1 to sense several effector-induced kinase perturbations. Here we show that the recently identified ZAR1-mediated immune responses against the HopX1, HopO1, and HopBA1 effector families of Pseudomonas syringae rely on an expanded diversity of RLCK sensors. We show that individual sensors can recognize distinct effector families, thereby contributing to the expanded surveillance potential of ZAR1 and supporting its role as a guardian of the plant kinome.

The pan-genome effector-triggered immunity landscape of a host-pathogen interaction.

Effector-triggered immunity (ETI), induced by host immune receptors in response to microbial effectors, protects plants against virulent pathogens. However, a systematic study of ETI prevalence against species-wide pathogen diversity is lacking. We constructed the Pseudomonas syringae Type III Effector Compendium (PsyTEC) to reduce the pan-genome complexity of 5127 unique effector proteins, distributed among 70 families from 494 strains, to 529 representative alleles. We screened PsyTEC on the model plant Arabidopsis thaliana and identified 59 ETI-eliciting alleles (11.2%) from 19 families (27.1%), with orthologs distributed among 96.8% of P. syringae strains. We also identified two previously undescribed host immune receptors, including CAR1, which recognizes the conserved effectors AvrE and HopAA1, and found that 94.7% of strains harbor alleles predicted to be recognized by either CAR1 or ZAR1.

Molecular Evolution of Pseudomonas syringae Type III Secreted Effector Proteins.

Diverse Gram-negative pathogens like Pseudomonas syringae employ type III secreted effector (T3SE) proteins as primary virulence factors that combat host immunity and promote disease. T3SEs can also be recognized by plant hosts and activate an effector triggered immune (ETI) response that shifts the interaction back toward plant immunity. Consequently, T3SEs are pivotal in determining the virulence potential of individual P. syringae strains, and ultimately help to restrict P. syringae pathogens to a subset of potential hosts that are unable to recognize their repertoires of T3SEs. While a number of effector families are known to be present in the P. syringae species complex, one of the most persistent challenges has been documenting the complex variation in T3SE contents across a diverse collection of strains. Using the entire pan-genome of 494 P. syringae strains isolated from more than 100 hosts, we conducted a global analysis of all known and putative T3SEs. We identified a total of 14,613 putative T3SEs, 4,636 of which were unique at the amino acid level, and show that T3SE repertoires of different P. syringae strains vary dramatically, even among strains isolated from the same hosts. We also find substantial diversification within many T3SE families, and in many cases find strong signatures of positive selection. Furthermore, we identify multiple gene gain and loss events for several families, demonstrating an important role of horizontal gene transfer (HGT) in the evolution of P. syringae T3SEs. These analyses provide insight into the evolutionary history of P. syringae T3SEs as they co-evolve with the host immune system, and dramatically expand the database of P. syringae T3SEs alleles.

Recombination of ecologically and evolutionarily significant loci maintains genetic cohesion in the Pseudomonas syringae species complex.

BACKGROUND: Pseudomonas syringae is a highly diverse bacterial species complex capable of causing a wide range of serious diseases on numerous agronomically important crops. We examine the evolutionary relationships of 391 agricultural and environmental strains using whole-genome sequencing and evolutionary genomic analyses. RESULTS: We describe the phylogenetic distribution of all 77,728 orthologous gene families in the pan-genome, reconstruct the core genome phylogeny using the 2410 core genes, hierarchically cluster the accessory genome, identify the diversity and distribution of type III secretion systems and their effectors, predict ecologically and evolutionary relevant loci, and establish the molecular evolutionary processes operating on gene families. Phylogenetic and recombination analyses reveals that the species complex is subdivided into primary and secondary phylogroups, with the former primarily comprised of agricultural isolates, including all of the well-studied P. syringae strains. In contrast, the secondary phylogroups include numerous environmental isolates. These phylogroups also have levels of genetic diversity typically found among distinct species. An analysis of rates of recombination within and between phylogroups revealed a higher rate of recombination within primary phylogroups than between primary and secondary phylogroups. We also find that "ecologically significant" virulence-associated loci and "evolutionarily significant" loci under positive selection are over-represented among loci that undergo inter-phylogroup genetic exchange. CONCLUSIONS: While inter-phylogroup recombination occurs relatively rarely, it is an important force maintaining the genetic cohesion of the species complex, particularly among primary phylogroup strains. This level of genetic cohesion, and the shared plant-associated niche, argues for considering the primary phylogroups as a single biological species.