Phylogenetic Characterization of Virulence and Resistance Phenotypes of Pseudomonas syringae

Abstract

Individual strains of the plant pathogenic bacterium Pseudomonas syringae vary in their ability to produce toxins, nucleate ice, and resist antimicrobial compounds. These phenotypes enhance virulence, but it is not clear whether they play a dominant role in specific pathogen-host interactions. To investigate the evolution of these virulence-associated phenotypes, we used functional assays to survey for the distribution of these phenotypes among a collection of 95 P. syringae strains. All of these strains were phylogenetically characterized via multilocus sequence typing (MLST). We surveyed for the production of coronatine, phaseolotoxin, syringomycin, and tabtoxin; for resistance to ampicillin, chloramphenicol, rifampin, streptomycin, tetracycline, kanamycin, and copper; and for the ability to nucleate ice at high temperatures via the ice-nucleating protein INA. We found that fewer than 50% of the strains produced toxins and significantly fewer strains than expected produced multiple toxins, leading to the speculation that there is a cost associated with the production of multiple toxins. None of these toxins was associated with host of isolation, and their distribution, relative to core genome phylogeny, indicated extensive horizontal genetic exchange. Most strains were resistant to ampicillin and copper and had the ability to nucleate ice, and yet very few strains were resistant to the other antibiotics. The distribution of the rare resistance phenotypes was also inconsistent with the clonal history of the species and did not associate with host of isolation. The present study provides a robust phylogenetic foundation for the study of these important virulence-associated phenotypes in P. syringae host colonization and pathogenesis.Pseudomonas syringae is one of the preeminent model systems for the study of host specificity and virulence. This gramnegative plant-pathogenic bacterium is the causal agent of a variety of bacterial spot, speck, and blight diseases on a wide range of plant hosts, including (but not limited to) apples, beets, beans, cabbage, cucumbers, flowers, oats, olives, peas, tobacco, tomato, and rice (25). Isolates of P. syringae are taxonomically subdivided into pathogenic varieties known as pathovars, based largely on their host of isolation. The tremendous diversity of hosts and disease symptomatology found in this species presents a unique opportunity to investigate the factors that determine host specificity.P. syringae uses an impressive variety of virulence-associated systems during the course of its host interactions. These systems produce toxins, ice nucleation proteins, antimicrobial resistance, and secreted effectors. The best-studied virulenceassociated factors are the effector proteins secreted through the type III secretion system, which both restrict and promote specific pathogen-host interactions (1,21,26,30,45). Also well studied, although perhaps less well understood, are the systems that produce toxins, nucleate ice, and confer antimicrobial resistance.

De novo assembly using low-coverage short read sequence data from the rice pathogen Pseudomonas syringae pv. oryzae

“…Addition of the Por 1_6 genome brings the number of P. syringae clades with a member sequenced to four out of five (Hwang et al 2005). We found that Por 1_6 contains homologs for 29 of the previously identified type III effector proteins and that some but not all of these are present in one or more or the previously sequenced P. syringae genomes (Fig.…”

“…The three sequenced reference genomes of P. syringae share less than 75% of their genes (Feil et al 2005;Joardar et al 2005). Further, multilocus sequence typing (MLST) analysis (Hwang et al 2005) suggests that Por 1_6 is likely to be highly diverged from any of the three sequenced strains, as it is a member of a fourth phylogenetic clade. We estimated the divergence between our Por 1_6 Illumina reads and the Pto DC3000 reference genome using SOAP (Li et al 2008).…”

“…oryzae 1_6 (Por 1_6 ), a rice (monocot) pathogen. This isolate also represents a fourth phylogenetic clade of P. syringae (Hwang et al 2005) for which a genome sequence is now available. It is significantly diverged from the three previously sequenced genomes, making this new sequence a valuable comparative resource.…”

Genetic and pathogenic diversity of Pseudomonas syringae strains isolated from cucurbits

“…These studies showed that the MLST technique and rep-PCR are useful as methods for identification and classification of strains belonging to Pseudomonas syringae (Hwang et al 2005;Louws et al 1994). Therefore we decided to use these methods to group Pseudomonas strains isolated primarily from cucumber and kept in our Department's collection.…”

“…lachrymans strains was done by PCR amplification using primers for the following housekeeping genes: aconitate hydratase B (acnB), citrate synthase (cts), glyceraldehyde-3-phosphate dehydrogenase (gapA), gyrase B (gyrB), phosphofructokinase (pfk), phosphoglucoisomerase (pgi) (Sarkar and Guttman 2004;Hwang et al 2005). In addition, we performed an amplification of harpin elicitor (hrpZ) (Inoue and Takikawa 2006) using the same conditions as for the MLST loci.…”