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.

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Ehrlichia chaffeensis, a tick-transmitted rickettsial agent, causes human monocyte/macrophage-tropic ehrlichiosis. In this study, proteomic approaches were used to demonstrate host cell-specific antigenic expression by E. chaffeensis. The differentially expressed antigens include those from the 28-kDa outer membrane protein (p28-Omp) multigene locus. The proteins expressed in infected macrophages are the products of p28-Omp19 and p28-Omp20 genes, whereas in tick cells, the protein expressed is the p28-Omp14 gene product. The differentially expressed proteins are posttranslationally modified by phosphorylation and glycosylation to generate multiple expressed forms. Host cell-specific protein expression is not influenced by growth temperatures and is reversible. Host cell-specific protein expression coupled with posttranslational modifications may be a hallmark for the pathogen's adaptation to a dual-host life cycle and its persistence.

Diverse gene products including phytotoxins, pathogen-associated molecular patterns, and type III secreted effectors influence interactions between Pseudomonas syringae strains and plants, with additional yet uncharacterized factors likely contributing as well. Of particular interest are those interactions governing pathogen-host specificity. Comparative genomics of closely related pathogens with different host specificity represents an excellent approach for identification of genes contributing to host-range determination. A draft genome sequence of Pseudomonas syringae pv. tomato T1, which is pathogenic on tomato but nonpathogenic on Arabidopsis thaliana, was obtained for this purpose and compared with the genome of the closely related A. thaliana and tomato model pathogen P. syringae pv. tomato DC3000. Although the overall genetic content of each of the two genomes appears to be highly similar, the repertoire of effectors was found to diverge significantly. Several P. syringae pv. tomato T1 effectors absent from strain DC3000 were confirmed to be translocated into plants, with the well-studied effector AvrRpt2 representing a likely candidate for host-range determination. However, the presence of avrRpt2 was not found sufficient to explain A. thaliana resistance to P. syringae pv. tomato T1, suggesting that other effectors and possibly type III secretion system-independent factors also play a role in this interaction.

SummaryWhile the existence of environmental reservoirs of human pathogens is well established, less is known about the role of nonagricultural environments in emergence, evolution, and spread of crop pathogens.Here, we analyzed phylogeny, virulence genes, host range, and aggressiveness of Pseudomonas syringae strains closely related to the tomato pathogen P. syringae pv. tomato (Pto), including strains isolated from snowpack and streams.The population of Pto relatives in nonagricultural environments was estimated to be large and its diversity to be higher than that of the population of Pto and its relatives on crops. Ancestors of environmental strains, Pto, and other genetically monomorphic crop pathogens were inferred to have frequently recombined, suggesting an epidemic population structure for P. syringae. Some environmental strains have repertoires of type III-secreted effectors very similar to Pto, are almost as aggressive on tomato as Pto, but have a wider host range than typical Pto strains.We conclude that crop pathogens may have evolved through a small number of evolutionary events from a population of less aggressive ancestors with a wider host range present in nonagricultural environments.

Pseudomonas syringae causes plant diseases, and the main virulence mechanism is a type III secretion system (T3SS) that translocates dozens of effector proteins into plant cells. Here we report the existence of a subgroup of P. syringae isolates that do not cause disease on any plant species tested. This group is monophyletic and most likely evolved from a pathogenic P. syringae ancestor through loss of the T3SS. In the nonpathogenic isolate P. syringae 508 the genomic region that in pathogenic P. syringae strains contains the hrp-hrc cluster coding for the T3SS and flanking effector genes is absent. P. syringae 508 was also surveyed for the presence of effector orthologues from the closely related pathogenic strain P. syringae pv. syringae B728a, but none were detected. The absence of the hrp-hrc cluster and effector orthologues was confirmed for other nonpathogenic isolates. Using the AvrRpt2 effector as reporter revealed the inability of P. syringae 508 to translocate effectors into plant cells. Adding a plasmid-encoded T3SS and the P. syringae pv. syringae 61 effector gene hopA1 increased in planta growth almost 10-fold. This suggests that P. syringae 508 supplemented with a T3SS could be used to determine functions of individual effectors in the context of a plant infection, avoiding the confounding effect of other effectors with similar functions present in effector mutants of pathogenic isolates.Pseudomonas syringae is probably the most intensively studied bacterial plant pathogen for which molecular interactions with host and nonhost plants have been dissected in great detail (16,43,50). P. syringae is a member of the Gammaproteobacteria and comprises strains isolated from dozens of cultivated, ornamental, and wild plants. According to the current taxonomy, isolates are grouped into different pathovars based on the plant host from which they were isolated (65). The diseases that P. syringae strains cause range from foliar spot diseases to blights, stripes, and cankers (1). Bacteria are transmitted mainly by rain and wind, can survive for periods of time on leaf surfaces as epiphytes without causing disease, and then enter leaves either through natural openings like stomata or through wounds and finally reach high population densities in the intercellular plant spaces and cause visible disease symptoms (26). Recently, P. syringae isolates have been found in clouds, rain, snow, and river epilithion, suggesting that these environments are important inoculum sources (44).P. syringae is able to cause diseases in its hosts because of its ability to suppress plant defenses elicited by microbe-associated molecular patterns (MAMPs) or pathogen-associated molecular patterns (PAMPs) like flagellin (23). These defenses are called PAMP-triggered immunity (PTI) (11). Suppression of PTI is accomplished by effector proteins that are translocated from P. syringae into plant cells by means of a type III secretion system (T3SS) and by toxins (for example, coronatine) (38, 43). However, on some plants, effectors are directl...

Pseudomonas syringae pv. tomato strain DC3000 (PtoDC3000) is one of the most intensively studied bacterial plant pathogens today. Here we report a thorough investigation into PtoDC3000 and close relatives isolated from Antirrhinum majus (snapdragon), Apium graveolens (celery), and Solanaceae and Brassicaceae species. Multilocus sequence typing (MLST) was used to resolve the precise phylogenetic relationship between isolates and to determine the importance of recombination in their evolution. MLST data were correlated with an analysis of the locus coding for the type III secreted (T3S) effector AvrPto1 to investigate the role of recombination in the evolution of effector repertoires. Host range tests were performed to determine if closely related isolates from different plants have different host ranges. It was found that PtoDC3000 is located in the same phylogenetic cluster as isolates from several Brassicaceae and Solanaceae species and that these isolates have a relatively wide host range that includes tomato, Arabidopsis thaliana, and cauliflower. All other analyzed tomato isolates from three different continents form a distinct cluster and are pathogenic only on tomato. Therefore, PtoDC3000 is a very unusual tomato isolate. Several recombination breakpoints were detected within sequenced gene fragments, and population genetic tests indicate that recombination contributed more than mutation to the variation between isolates. Moreover, recombination may play an important role in the reassortment of T3S effectors between strains. The data are finally discussed from a taxonomic standpoint, and P. syringae pv. tomato is proposed to be divided into two pathovars.Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) is one of the most intensively studied plant pathogen isolates today. It was completely sequenced (6), and a large part of what is known about the plant immune system has been learned by studying the interaction of PtoDC3000 with its hosts Arabidopsis thaliana and tomato (Solanum lycopersicum), as can be seen from many recent high-profile publications (see references 39 and 47 for examples). However, much less is known about how PtoDC3000 relates to other P. syringae strains. Although PtoDC3000 is a rifampin-resistant derivative of the type strain of P. syringae pv. tomato (9; D. Cuppels, personal communication), its host range (which includes tomato, cauliflower [Brassica oleracea var. botrytis], and A. thaliana) was found to be more similar to that of pathovar maculicola isolates from Brassicaceae species than to the host range of other P. syringae pv. tomato strains (which are limited to tomato) (10, 58). Also, based on physiological (10) and molecular analyses (10, 63), PtoDC3000 was suggested to be more similar to pathovar maculicola strains than to other pathovar tomato strains. However, since strains of pathovars tomato, maculicola, antirrhini (isolated from ornamental snapdragon, Antirrhinum majus), and apii (isolated from celery, Apium graveolens) were all found to be closely related (18, 25), the...

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