Dynamic Evolution of Pathogenicity Revealed by Sequencing and Comparative Genomics of 19 Pseudomonas syringae Isolates David A. Baltrus1.¤a, Marc T. Nishimura1., Artur Romanchuk1, Jeff H. Chang1¤b, M. Shahid Mukhtar1¤c, Karen Cherkis1, Jeff Roach2, Sarah R. Grant1,3, Corbin D. Jones1,3,4*, Jeffery L. Dangl1,3,4,5* 1 Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America, 2 Research Computing Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America, 3 Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America, 4 Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America, 5 Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America Abstract Closely related pathogens may differ dramatically in host range, but the molecular, genetic, and evolutionary basis for these differences remains unclear. In many Gram- negative bacteria, including the phytopathogen Pseudomonas syringae, type III effectors (TTEs) are essential for pathogenicity, instrumental in structuring host range, and exhibit wide diversity between strains. To capture the dynamic nature of virulence gene repertoires across P. syringae,wescreened11diverse strains for novel TTE families and coupled this nearly saturating screen with the sequencing and assembly of 14 phylogenetically diverse isolates from a broad collection of diseased host plants. TTE repertoires vary dramatically in size and content across all P. syringae clades; surprisingly few TTEs are conserved and present in all strains. Those that are likely provide basal requirements for pathogenicity. We demonstrate that functional divergence within one conserved locus, hopM1, leads to dramatic differences in pathogenicity, and we demonstrate that phylogenetics-informed mutagenesis can be used to identify functionally critical residues of TTEs. The dynamism of the TTE repertoire is mirrored by diversity in pathways affecting the synthesis of secreted phytotoxins, highlighting the likely role of both types of virulence factors in determination of host range. We used these 14 draft genome sequences, plus five additional genome sequences previously reported, to identify the core genome for P. syringae and we compared this core to that of two closely related non-pathogenic pseudomonad species. These data revealed the recent acquisition of a 1 Mb megaplasmid by a sub-clade of cucumber pathogens. This megaplasmid encodes a type IV secretion system and a diverse set of unknown proteins, which dramatically increases both the genomic content of these strains and the pan- genome of the species. Citation: Baltrus DA, Nishimura MT, Romanchuk A, Chang JH, Mukhtar MS, et al. (2011) Dynamic Evolution of Pathogenicity Revealed by Sequencing and Comparative Genomics of 19 Pseudomonas syringae Isolates. PLoS Pathog 7(7): e1002132. doi:10.1371/journal.ppat.1002132 Editor: David S. Guttman, University of Toronto, Canada Received March 22, 2011; Accepted May 6, 2011; Published July 14, 2011 Copyright: ß 2011 Baltrus et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Supported by an NIH Ruth Kirschstein NRSA postdoctoral fellowship GM082279-03 (D.A.B.), by NIH grant 1-R01-GM066025 (J.L.D. and C.D.J.) and by the University Cancer Research Fund for providing support to the UNC High-Throughput Sequencing Facility. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (CDJ, computational queries); [email protected] (JLD, biological queries) ¤a Current address: School of Plant Sciences, The University of Arizona, Tucson, Arizona, United States of America ¤b Current address: Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon, United States of America ¤c Current address: Department of Biology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America . These authors contributed equally to this work. Introduction evolutionary forces shape the pan-genome of this species, especially virulence-related genes. Pseudomonas syringae is a Gram-negative bacterial phytopathogen The host ranges of many P. syringae isolates or pathovars have responsible for worldwide disease on many crop species [1]. not been thoroughly characterized. Research has largely focused Despite a collectively broad pathogenic range for the species, on identifying the molecular basis of pathogenesis across three individual isolates of P. syringae display pathogenic potential on a divergent strains with finished genome sequences and investigation limited set of plant species and either elicit immune responses, or of virulence mechanisms for a smattering of strains on a limited simply fail to thrive on alternative species [2–4]. In addition to number of hosts [7]. These studies have shown that a Type III disease outbreaks, strains can be isolated as epiphytes from non- secretion system (TTSS), which acts like a molecular syringe to diseased plants as well as from multiple phases of the water cycle translocate a suite of type III effector (TTE) proteins into plant [5,6]. How species with varied lifestyles like P. syringae maintain the cells, is a key virulence determinant [7–9]. Once inside the plant genomic flexibility required to survive across this broad range of cell, TTEs promote pathogenesis by disrupting and suppressing ecologies is not known. It remains particularly unclear how host defense responses at multiple levels [10–13]. TTEs can also PLoS Pathogens | www.plospathogens.org 1 July 2011 | Volume 7 | Issue 7 | e1002132 Evolution of Pathogenicity in P. syringae Author Summary when combined with orthogonal functional methods to validate candidates as TTEs. Breakthroughs in genomics have unleashed a new suite of The TTSS is not the sole determinant of virulence and host tools for studying the genetic bases of phenotypic range for P. syringae; coordination of host physiological responses differences across diverse bacterial isolates. Here, we and metabolic pathways is also necessary for pathogen growth analyze 19 genomes of P. syringae, a pathogen of many within host tissue [32]. Phytotoxins, which can be coordinately crop species, to reveal the genetic changes underlying regulated with the TTSS, but secreted independently from the differences in virulence across host plants ranging from TTEs [33], can disrupt host metabolism or act as mimics of plant rice to maple trees. Surprisingly, a pair of strains diverged hormones. Hence, they may replace or complement virulence dramatically via the acquisition of a 1 Mb megaplasmid, functions of TTEs [34]. Indeed, manipulation of stomatal function which constitutes roughly 14% of the genome. Novel by coronatine, a structural mimic of the plant hormone jasmonic plasmids and horizontal genetic exchange have contrib- acid, is essential for invasion of A. thaliana leaves by P. syringae pv. uted extensively to species-wide diversification. Type III effector proteins are essential for pathogenicity, exhibit tomato (Pto) DC3000. However, coronatine also possesses indepen- wide diversity between strains and are present in distinct dent virulence functions during the colonization of roots [35]. higher-level patterns across the species. Furthermore, we Therefore, pathogenesis of P. syringae on any given plant host use sequence comparisons within an evolutionary context species, results from both the absence of avirulence factors (an to identify functional changes in multiple virulence genes. operational definition of TTEs that activate a host immune Overall, our data provide a unique overview of evolution- receptor) and the presence of multiple virulence factors acting ary pressures within P. syringae and an important resource coordinately to promote disease and to suppress host immune for the phytopathogen research community. responses [4,7,13]. To date, genomics studies in P. syringae suggest that virulence mechanisms within this species are evolutionarily dynamic and be recognized by plant disease resistance proteins and recognition have experienced strong selective pressures [3,36]. Complete of a single effector is sufficient to trigger successful host immune genome sequences exist for three phylogenetically diverse P. response. However, the virulence functions of many TTEs are syringae isolates representing MLST groups I, II, and III (Pto redundant, making these phenotypes potentially robust to host- DC3000, P. syringae pv. syringae B728a (Psy), and P. syringae pv. mediated selection against single TTE genes [14]. Thus, host phaseolicola 1448a (Pph), respectively; [26–28]. Recently, additional range is structured by the totality of a strain’s TTE repertoire. draft genome sequences were generated by either Roche/454 or A high degree of divergence among commonly investigated Illumina sequencing technologies (for Pto T1, group I; Pta isolates makes it nearly impossible to pinpoint all the changes that ATCC11528, pathovar tabaci; Psv NCPP3335,