Syringae Species Complex Research Articles

Development of Tools to Detect and Identify Strains Belonging to the Pseudomonas syringae Species Complex Responsible for Vein Clearing of Zucchini

Vein clearing of zucchini (VCZ) is a disease caused by seedborne bacteria that affects young plants of Cucurbita pepo subsp. pepo. VCZ agents are distributed into four phylogenetic clusters within clades 2a and 2ba of phylogroup 2 of the Pseudomonas syringae species complex. All these strains are pathogenic to squash, but only certain strains can also attack melon and cucumber. Strains belonging to clades 2b and 2d are sometimes isolated from zucchini seeds but have not been associated with VCZ epidemics. Identification tools for VCZ agents are required to improve disease control. Primers were designed to implement a seven-gene multilocus sequence analysis (MLSA) scheme for a collection of 60 strains isolated from zucchini seeds. The MLSA showed a clear predominance of strains of cluster 2ba-A and the presence of VCZ strains in a fifth cluster (2ba-C). PCR tests were designed to characterize strains in the VCZ clusters, and a multiplex qPCR test was proposed to distinguish strains with a cucurbit host range extended to melon and cucumber, harboring hopZ5 and sylC, from other strains harboring avrRpt2 and sylC. Additional qPCR tests were also designed to gain insights into clade-2b and -2d strains that can be isolated from cucurbits. These tools evaluated in silico with the NCBI database and experimentally with a collection of 112 strains detected all target strains, except for the test dedicated to clade-2b strains, and excluded 96.7 to 100% of nontarget strains. These tools are intended to serve phylogenetic studies, epidemiological monitoring, and seed testing. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Novel phages of Pseudomonas syringae unveil numerous potential auxiliary metabolic genes.

Relatively few phages that infect plant pathogens have been isolated and investigated. The Pseudomonas syringae species complex is present in various environments, including plants. It can cause major crop diseases, such as bacterial canker on apricot trees. This study presents a collection of 25 unique phage genomes that infect P. syringae. These phages were isolated from apricot orchards with bacterial canker symptoms after enrichment with 21 strains of P. syringae. This collection comprises mostly virulent phages, with only three being temperate. They belong to 14 genera, 11 of which are newly discovered, and 18 new species, revealing great genetic diversity within this collection. Novel DNA packaging systems have been identified bioinformatically in one of the new phage species, but experimental confirmation is required to define the precise mechanism. Additionally, many phage genomes contain numerous potential auxiliary metabolic genes with diversified putative functions. At least three phages encode genes involved in bacterial tellurite resistance, a toxic metalloid. This suggests that viruses could play a role in bacterial stress tolerance. This research emphasizes the significance of continuing the search for new phages in the agricultural ecosystem to unravel novel ecological diversity and new gene functions. This work contributes to the foundation for future fundamental and applied research on phages infecting phytopathogenic bacteria.

Genetic dissection of the tissue-specific roles of type III effectors and phytotoxins in the pathogenicity of Pseudomonas syringae pv. syringae to cherry.

When compared with other phylogroups (PGs) of the Pseudomonas syringae species complex, P. syringae pv. syringae (Pss) strains within PG2 have a reduced repertoire of type III effectors (T3Es) but produce several phytotoxins. Effectors within the cherry pathogen Pss 9644 were grouped based on their frequency in strains from Prunus as the conserved effector locus (CEL) common to most P. syringae pathogens; a core of effectors common to PG2; a set of PRUNUS effectors common to cherry pathogens; and a FLEXIBLE set of T3Es. Pss 9644 also contains gene clusters for biosynthesis of toxins syringomycin, syringopeptin and syringolin A. After confirmation of virulence gene expression, mutants with a sequential series of T3E and toxin deletions were pathogenicity tested on wood, leaves and fruits of sweet cherry (Prunus avium) and leaves of ornamental cherry (Prunus incisa). The toxins had a key role in disease development in fruits but were less important in leaves and wood. An effectorless mutant retained some pathogenicity to fruit but not wood or leaves. Striking redundancy was observed amongst effector groups. The CEL effectors have important roles during the early stages of leaf infection and possibly acted synergistically with toxins in all tissues. Deletion of separate groups of T3Es had more effect in P. incisa than in P. avium. Mixed inocula were used to complement the toxin mutations in trans and indicated that strain mixtures may be important in the field. Our results highlight the niche-specific role of toxins in P. avium tissues and the complexity of effector redundancy in the pathogen Pss 9644.

Rapid dissemination of host metabolism–manipulating genes via integrative and conjugative elements

Integrative and conjugative elements (ICEs) are self-transmissible mobile elements that transfer functional genetic units across broad phylogenetic distances. Accessory genes shuttled by ICEs can make significant contributions to bacterial fitness. Most ICEs characterized to date encode readily observable phenotypes contributing to symbiosis, pathogenicity, and antimicrobial resistance, yet the majority of ICEs carry genes of unknown function. Recent observations of rapid acquisition of ICEs in a pandemic lineage of Pseudomonas syringae pv. actinidae led to investigation of the structural and functional diversity of these elements. Fifty-three unique ICE types were identified across the P. syringae species complex. Together they form a distinct family of ICEs (PsICEs) that share a distant relationship to ICEs found in Pseudomonas aeruginosa. PsICEs are defined by conserved backbone genes punctuated by an array of accessory cargo genes, are highly recombinogenic, and display distinct evolutionary histories compared to their bacterial hosts. The most common cargo is a recently disseminated 16-kb mobile genetic element designated Tn6212. Deletion of Tn6212 did not alter pathogen growth in planta, but mutants displayed fitness defects when grown on tricarboxylic acid (TCA) cycle intermediates. RNA-seq analysis of a set of nested deletion mutants showed that a Tn6212-encoded LysR regulator has global effects on chromosomal gene expression. We show that Tn6212 responds to preferred carbon sources and manipulates bacterial metabolism to maximize growth.

Genomic characterization of Pseudomonas syringae pv. syringae from Callery pear and the efficiency of associated phages in disease protection.

The Pseudomonas syringae species complex is a heterogeneous group of plant pathogenic bacteria associated with a wide distribution of plant species. Advances in genomics are revealing the complex evolutionary history of this species complex and the wide array of genetic adaptations underpinning their diverse lifestyles. Here, we genomically characterize two P. syringae isolates collected from diseased Callery pears (Pyrus calleryana) in Berkeley, California in 2019 and 2022. We also isolated a lytic bacteriophage, which we characterized and evaluated for biocontrol efficiency. Using a multilocus sequence analysis and core genome alignment, we classified the P. syringae isolates as members of phylogroup 2, related to other strains previously isolated from Pyrus and Prunus. An analysis of effector proteins demonstrated an evolutionary conservation of effectoromes across isolates classified in PG2 and yet uncovered unique effector profiles for each, including the two newly identified isolates. Whole-genome sequencing of the associated phage uncovered a novel phage genus related to Pseudomonas syringae pv. actinidiae phage PHB09 and the Flaumdravirus genus. Finally, using in planta infection assays, we demonstrate that the phage was equally useful in symptom mitigation of immature pear fruit regardless of the Pss strain tested. Overall, this study demonstrates the diversity of P. syringae and their viruses associated with ornamental pear trees, posing spill-over risks to commercial pear trees and the possibility of using phages as biocontrol agents to reduce the impact of disease.IMPORTANCEGlobal change exacerbates the spread and impact of pathogens, especially in agricultural settings. There is a clear need to better monitor the spread and diversity of plant pathogens, including in potential spillover hosts, and for the development of novel and sustainable control strategies. In this study, we characterize the first described strains of Pseudomonas syringae pv. syringae isolated from Callery pear in Berkeley, California from diseased tissues in an urban environment. We show that these strains have divergent virulence profiles from previously described strains and that they can cause disease in commercial pears. Additionally, we describe a novel bacteriophage that is associated with these strains and explore its potential to act as a biocontrol agent. Together, the data presented here demonstrate that ornamental pear trees harbor novel P. syringae pv. syringae isolates that potentially pose a risk to local fruit production, or vice versa-but also provide us with novel associated phages, effective in disease mitigation.

Naïve Bayes Classifiers and accompanying dataset for Pseudomonas syringae isolate characterization

The Pseudomonas syringae species complex (PSSC) is a diverse group of plant pathogens with a collective host range encompassing almost every food crop grown today. As a threat to global food security, rapid detection and characterization of epidemic and emerging pathogenic lineages is essential. However, phylogenetic identification is often complicated by an unclarified and ever-changing taxonomy, making practical use of available databases and the proper training of classifiers difficult. As such, while amplicon sequencing is a common method for routine identification of PSSC isolates, there is no efficient method for accurate classification based on this data. Here we present a suite of five Naïve bayes classifiers for PCR primer sets widely used for PSSC identification, trained on in-silico amplicon data from 2,161 published PSSC genomes using the life identification number (LIN) hierarchical clustering algorithm in place of traditional Linnaean taxonomy. Additionally, we include a dataset for translating classification results back into traditional taxonomic nomenclature (i.e. species, phylogroup, pathovar), and for predicting virulence factor repertoires.

Unexpected Diversity of Pseudomonads Associated with Bacterial Leaf Spot of Cucurbits in the Southeastern United States.

Bacterial leaf spot of cucurbits (BLS) is an emerging disease in the southeastern United States that is capable of causing widespread outbreaks under conducive conditions. Historically attributed solely to the bacterium Pseudomonas syringae pv. lachrymans, recent studies have identified additional P. syringae pathovars as causal agents of the disease. To further investigate the identity and diversity of P. syringae strains associated with BLS in the southeastern United States, 47 bacterial isolates were recovered from symptomatic cucurbits from Florida, Alabama, and Georgia. Strains were characterized using the LOPAT testing scheme, fluorescence, and pathogenicity to watermelon and squash seedlings. Thirty-eight fluorescent isolates underwent whole-genome sequencing and were further characterized with 16S rRNA, four gene multilocus sequence analysis (MLSA) phylogeny, and average nucleotide identity analysis. Thirty-four isolates were identified as members of the P. syringae species complex, including P. syringae sensu stricto (12), P. alliivorans (12), P. capsici (nine), and P. viridiflava (one). An additional four isolates were found to belong to the Pseudomonas genus outside of the syringae species complex, though they did not share 95% or greater average nucleotide identity to any validly published species and are believed to belong to three novel Pseudomonas species. These results reveal an unpredicted level of diversity of Pseudomonas strains associated with BLS in the region and show the benefits of whole-genome sequencing for strain identification. Identification of P. capsici, which is capable of causing disease at higher temperatures than P. syringae, as a causal agent of BLS may also affect management strategies in the future.

Genetic rearrangements in Pseudomonas amygdali pathovar aesculi shape coronatine plasmids.

Plant pathogenic Pseudomonas species use multiple classes of toxins and virulence factors during host infection. The genes encoding these pathogenicity factors are often located on plasmids and other mobile genetic elements, suggesting that they are acquired through horizontal gene transfer to confer an evolutionary advantage for successful adaptation to host infection. However, the genetic rearrangements that have led to mobilization of the pathogenicity genes are not fully understood. In this study, we have sequenced and analyzed the complete genome sequences of four Pseudomonas amygdali pv. aesculi (Pae), which infect European horse chestnut trees (Aesculus hippocastanum) and belong to phylogroup 3 of the P. syringae species complex. The four investigated genomes contain six groups of plasmids that all encode pathogenicity factors. Effector genes were found to be mostly associated with insertion sequence elements, suggesting that virulence genes are generally mobilized and potentially undergo horizontal gene transfer after transfer to a conjugative plasmid. We show that the biosynthetic gene cluster encoding the phytotoxin coronatine was recently transferred from a chromosomal location to a mobilizable plasmid that subsequently formed a co-integrate with a conjugative plasmid.

Genetic characterization and prevalence of Pseudomonas syringae strains from sweet cherry orchards in New Zealand

AbstractBacterial canker of cherry, caused by Pseudomonas syringae pathovars, is a major constraint to cherry growing in New Zealand. The prevalence of strains from cherry orchards in Central Otago, the main growing area for cherries in New Zealand, was studied, to better understand the epidemiology of the disease. Pseudomonas spp. isolates were collected from symptomatic and asymptomatic cherry tissue from 23 commercial cherry orchards in 2015. Isolates were classified into strains belonging to three different taxonomic groups by determining their phylogeny using the gltA gene sequence for all the strains and multilocus sequence analysis (MLSA) of four housekeeping genes for 35 strains. Pathogenicity of all Central Otago strains was tested on immature cherry fruit to support the phylogenetic classification. The two main taxonomic groups were P. syringae pv. syringae (Pss) and P. syringae pv. morsprunorum race 1 (Psm1), in Phylogroup 2 (PG2) and Phylogroup 3 (PG3), respectively. The third group comprised nonpathogenic strains classified as Pseudomonas spp. Strains of Psm1 formed a monophyletic group, representing an almost clonal population. There was more variation detected within strains of Pss, although they were restricted to group PG2d. Nonpathogenic Pseudomonas spp. and pathogenic Pss and Psm1 strains coexisted in the same orchard. It was concluded that Pss is the predominant pathovar in Central Otago. This is the first detailed study of the P. syringae species complex in cherry orchards in New Zealand and provides the basis for future epidemiology studies.

TaqMan qPCR assays improve Pseudomonas syringae pv. actinidiae biovar 3 and P. viridiflava (PG07) detection within the Pseudomonas sp. community of kiwifruit

TaqMan qPCR assays improve Pseudomonas syringae pv. actinidiae biovar 3 and P. viridiflava (PG07) detection within the Pseudomonas sp. community of kiwifruit

Evaluation of the taxonomic accuracy and pathogenicity prediction power of 16 primer sets amplifying single copy marker genes in the Pseudomonas syringae species complex.

The Pseudomonas syringae species complex is composed of several closely related species of bacterial plant pathogens. Here, we used in silico methods to assess 16 PCR primer sets designed for broad identification of isolates throughout the species complex. We evaluated their in silico amplification rate in 2161 publicly available genomes, the correlation between pairwise amplicon sequence distance and whole genome average nucleotide identity, and trained naive Bayes classification models to quantify classification resolution. Furthermore, we show the potential for using single amplicon sequence data to predict type III effector protein repertoires, which are important determinants of host specificity and range.

First report of Pseudomonas viridiflava causing fruit rot on sweet cherry trees in Chile.

Chile is an important producers of sweet cherry (Prunus avium L.), with a total of 356,385 t exported in the 2021 to 2022 season. The production area includes most of the country's regions. Bacterial samples were isolated in 2017 and 2018 from 18 commercial sweet cherry orchards with canker disease. From one of this samples collected in the spring of 2018, was isolated the strain A2M176 from buds of trees that presented canker and gomosis in Malloa locality (34°23' 46'' S 71°01' 39'' W). The strain produced fluorescent pigment on King's B agar medium. Is oxidase and arginine dihydrolase negative, potato soft rot positive and showed a slight degree of tobacco hypersensitivity. It was able to growth up to 0.8 mM (200 ppm) of CuSO4·5H2O. The strain A2M176 was deposited in the Colección Chilena de Recursos Genéticos Microbianos (CChRGM) under the no. RGM 3342. The DNA of this strain was extracted from a pure culture using silica spin columns (Epoch Life Science Inc., Sugar Land, USA). The complete DNA was sequenced using HiSeq with 150 bp paired-end at GENEWIZ (New York, USA). Raw data was checked using FASTQC and trimmed with BBDuk. The genome was assembled using Unicycler v0.4.9 with defaulf settings and annotated with Prokaryotic Genome Annotation Pipeline (PGAP) v4.3. The reads and genomes were uploaded to GenBank under the BioProyect no. PRJNA750090, BioSample no. SAMN26870984 and assembly no. GCA_022936465.1. The sequenced genome was compared through Average Nucleotide Identity algorithm (ANI) using FastANI v1.33 to compare with closest complete genome available on NCBI. The strain A2M176 was identified as P. viridiflava with ANI value of 98.06% with the strain p22.E7 (GCF_900585495). Maximum likelihood phylogenetic estimation clustered strain A2M176 with other P. viridiflava strains with 95% bootstrap. The pathogenicity of the strain was tested inoculating immature cherry fruits with a needle with a bacterial suspension (1x108 CFU/ml). The inoculated fruits were placed at room temperature in a humid chamber for 10 d. Soft rot lesions were observed, which appeared at 6 days post-inoculation (DPI). The control fruits treated with sterile water did not show symptoms. Further analyses in the genome of strain A2M176 led to identify genes related to pathogenicity, such as the effector gene avrE and the regulator gen HrpL, suggesting the pathogenic capacity of the strain. Also, there were identify genes of two known Pseudomonas copper resistance mechanisms, the cus and cop operon. These genes were found part of the copABCDns cluster similar to what was observed in Pseudomonas from Mango. Presence of P. viridiflava strains causing fruit rot in P. avium is not surprising, since P. viridiflava has a wide host range and causes a variety of symptoms in different plant parts, including stems, leaves, and blossoms. P. viridiflava represents one of the multiple phylogroups found within the P. syringae species complex. To our knowledge, this is the first report of a strain of P. viridiflava copper resistant causing infection on sweet cherries in Chile.

Genome Analysis of Two Pseudomonas syringae pv. aptata Strains with Different Virulence Capacity Isolated from Sugar Beet: Features of Successful Pathogenicity in the Phyllosphere Microbiome.

Members of the Pseudomonas syringae species complex are heterogeneous bacteria that are the most abundant bacterial plant pathogens in the plant phyllosphere, with strong abilities to exist on and infect different plant hosts and survive in/outside agroecosystems. In this study, the draft genome sequences of two pathogenic P. syringae pv. aptata strains with different in planta virulence capacities isolated from the phyllosphere of infected sugar beet were analyzed to evaluate putative features of survival strategies and to determine the pathogenic potential of the strains. The draft genomes of P. syringae pv. aptata strains P16 and P21 are 5,974,057 bp and 6,353,752 bp in size, have GC contents of 59.03% and 58.77%, respectively, and contain 3,439 and 3,536 protein-coding sequences, respectively. For both average nucleotide identity and pangenome analysis, P16 and P21 largely clustered with other pv. aptata strains from the same isolation source. We found differences in the repertoire of effectors of the type III secretion system among all 102 selected strains, suggesting that the type III secretion system is a critical factor in the different virulent phenotypes of P. syringae pv. aptata. During genome analysis of the highly virulent strain P21, we discovered genes for T3SS effectors (AvrRpm1, HopAW1, and HopAU1) that were not previously found in genomes of P. syringae pv. aptata. We also identified coding sequences for pantothenate kinase, VapC endonuclease, phospholipase, and pectate lyase in both genomes, which may represent novel effectors of the type III secretion system. IMPORTANCE Genome analysis has an enormous effect on understanding the life strategies of plant pathogens. Comparing similarities with pathogens involved in other epidemics could elucidate the pathogen life cycle when a new outbreak happens. This study represents the first in-depth genome analysis of Pseudomonas syringae pv. aptata, the causative agent of leaf spot disease of sugar beet. Despite the increasing number of disease reports in recent years worldwide, there is still a lack of information about the genomic features, epidemiology, and pathogenic life strategies of this particular pathogen. Our findings provide advances in disease etiology (especially T3SS effector repertoire) and elucidate the role of environmental adaptations required for prevalence in the pathobiome of the sugar beet. From the perspective of the very heterogeneous P. syringae species complex, this type of analysis has specific importance in reporting the characteristics of individual strains.

Characterization of Pseudomonas syringae Strains Associated with Shoot Blight of Raspberry and Blackberry in Serbia.

During May 2016, severe blight symptoms were observed in several raspberry and blackberry fields in Serbia. In total, 22 strains were isolated: 16 from symptomatic raspberry shoots, 2 from asymptomatic raspberry leaves, and 4 from symptomatic blackberry shoots. Additionally, eight raspberry strains, isolated earlier from two similar outbreaks, were included in the study. Pathogenicity of the strains was confirmed on detached raspberry and blackberry shoots by reproducing the symptoms of natural infection. The strains were Gram-negative, fluorescent on King's medium B, ice nucleation positive, and utilized glucose oxidatively. All strains were levan positive, oxidase negative, nonpectolytic, arginine dihydrolase negative, and induced hypersensitivity in tobacco leaves (LOPAT + - - - +, Pseudomonas group Ia). Furthermore, all strains liquefied gelatin and hydrolyzed aesculin but did not show tyrosinase activity or utilize tartrate (GATTa + + - -). Tentative identification using morphology, LOPAT, GATTa, and ice-nucleating ability tests suggested that isolated strains belong to Pseudomonas syringae. The syrB gene associated with syringomycin production was detected in all strains. DNA fingerprints with REP, ERIC, and BOX primers generated identical profiles for 29 strains, except for strain KBI 222, which showed a unique genomic fingerprint. In all, 9 of 10 selected strains exhibited identical sequences of four housekeeping genes: gyrB, rpoD, gapA, and gltA. Five nucleotide polymorphisms were found in strain KBI 222 at the rpoD gene locus only. In the phylogenetic tree based on a concatenated sequence of all four housekeeping genes, strains clustered within phylogroup 2 (i.e., genomospecies 1) of the P. syringae species complex, with pathotype strains of P. syringae pv. aceris and P. syringae pv. solidagae as their closest relatives. There was no correlation between genotype and geographic origin, particular outbreak, host, or cultivar.

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue.

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic bacteria of special relevance for a wide number of hosts. P. syringae enters the plant from the leaf surface and multiplies rapidly within the apoplast, forming microcolonies that occupy the intercellular space. The constitutive expression of fluorescent proteins by the bacteria allows for visualization of the microcolonies and monitoring of the development of the infection at the microscopic level. Recent advances in single-cell analysis have revealed the large complexity reached by clonal isogenic bacterial populations. This complexity, referred to as phenotypic heterogeneity, is the consequence of cell-to-cell differences in gene expression (not linked to genetic differences) among the bacterial community. To analyze the expression of individual loci at the single-cell level, transcriptional fusions to fluorescent proteins have been widely used. Under stress conditions, such as those occurring during colonization of the plant apoplast, P. syringae differentiates into distinct subpopulations based on the heterogeneous expression of key virulence genes (i.e., the Hrp type III secretion system). However, single-cell analysis of any given P. syringae population recovered from plant tissue is challenging due to the cellular debris released during the mechanical disruption intrinsic to the inoculation and bacterial extraction processes. The present report details a method developed to monitor the expression of P. syringae genes of interest at the single-cell level during the colonization of Arabidopsis and bean plants. The preparation of the plants and the bacterial suspensions used for inoculation using a vacuum chamber are described. The recovery of endophytic bacteria from infected leaves by apoplastic fluid extraction is also explained here. Both the bacterial inoculation and bacterial extraction methods are empirically optimized to minimize plant and bacterial cell damage, resulting in bacterial preparations optimal for microscopy and flow cytometry analysis.

Genome Sequence Resource for Strains of Pseudomonas syringae Phylogroup 2b and P. viridiflava Phylogroup 7a Causing Bacterial Stem Blight of Alfalfa.

Genome Sequence Resource for Strains of Pseudomonas syringae Phylogroup 2b and P. viridiflava Phylogroup 7a Causing Bacterial Stem Blight of Alfalfa.

Metaeffector interactions modulate the type III effector-triggered immunity load of Pseudomonas syringae.

The bacterial plant pathogen Pseudomonas syringae requires type III secreted effectors (T3SEs) for pathogenesis. However, a major facet of plant immunity entails the recognition of a subset of P. syringae’s T3SEs by intracellular host receptors in a process called Effector-Triggered Immunity (ETI). Prior work has shown that ETI-eliciting T3SEs are pervasive in the P. syringae species complex raising the question of how P. syringae mitigates its ETI load to become a successful pathogen. While pathogens can evade ETI by T3SE mutation, recombination, or loss, there is increasing evidence that effector-effector (a.k.a., metaeffector) interactions can suppress ETI. To study the ETI-suppression potential of P. syringae T3SE repertoires, we compared the ETI-elicitation profiles of two genetically divergent strains: P. syringae pv. tomato DC3000 (PtoDC3000) and P. syringae pv. maculicola ES4326 (PmaES4326), which are both virulent on Arabidopsis thaliana but harbour largely distinct effector repertoires. Of the 529 T3SE alleles screened on A. thaliana Col-0 from the P. syringae T3SE compendium (PsyTEC), 69 alleles from 21 T3SE families elicited ETI in at least one of the two strain backgrounds, while 50 elicited ETI in both backgrounds, resulting in 19 differential ETI responses including two novel ETI-eliciting families: AvrPto1 and HopT1. Although most of these differences were quantitative, three ETI responses were completely absent in one of the pathogenic backgrounds. We performed ETI suppression screens to test if metaeffector interactions contributed to these ETI differences, and found that HopQ1a suppressed AvrPto1m-mediated ETI, while HopG1c and HopF1g suppressed HopT1b-mediated ETI. Overall, these results show that P. syringae strains leverage metaeffector interactions and ETI suppression to overcome the ETI load associated with their native T3SE repertoires.

Comparative Genomics of Prunus-Associated Members of the Pseudomonas syringae Species Complex Reveals Traits Supporting Co-evolution and Host Adaptation.

Members of the Pseudomonas syringae species complex cause symptoms that are ranging from leaf spots to cankers on a multitude of plant species, including some of the genus Prunus. To date, a total of two species of the P. syringae species complex and six different pathovars have been associated with diseases on Prunus spp., which were shown to belong to different phylogenetic units (phylogroups, PG) based on sequence similarity of housekeeping genes or whole genomes, suggesting that virulence to Prunus spp. may be the result of convergent pathoadaptation. In this study, a comparative genomics approach was used to determine genes significantly associated with strains isolated from Prunus spp. across a phylogeny of 97 strains belonging to the P. syringae species complex. Our study revealed the presence of a set of orthologous proteins which were significantly associated with strains isolated from Prunus spp. than in strains isolated from other hosts or from non-agricultural environments. Among them, the type III effector HopAY predicted to encode for a C58 cysteine protease was found to be highly associated with strains isolated from Prunus spp. and revealed patterns supporting co-evolution and host adaptation.

Genome Context Influences Evolutionary Flexibility of Nearly Identical Type III Effectors in Two Phytopathogenic Pseudomonads.

Integrative Conjugative Elements (ICEs) are replicons that can insert and excise from chromosomal locations in a site-specific manner, can conjugate across strains, and which often carry a variety of genes useful for bacterial growth and survival under specific conditions. Although ICEs have been identified and vetted within certain clades of the agricultural pathogen Pseudomonas syringae, the impact of ICE carriage and transfer across the entire P. syringae species complex remains underexplored. Here we identify and vet an ICE (PmaICE-DQ) from P. syringae pv. maculicola ES4326, a strain commonly used for laboratory virulence experiments, demonstrate that this element can excise and conjugate across strains, and highlight that this element contains loci encoding multiple type III effector proteins. Moreover, genome context suggests that another ICE (PmaICE-AOAB) is highly similar in comparison with and found immediately adjacent to PmaICE-DQ within the chromosome of strain ES4326, and also contains multiple type III effectors. Lastly, we present passage data from in planta experiments that suggests that genomic plasticity associated with ICEs may enable strains to more rapidly lose type III effectors that trigger R-gene mediated resistance in comparison to strains where nearly isogenic effectors are not present in active ICEs. Taken together, our study sheds light on a set of ICE elements from P. syringae pv. maculicola ES4326 and suggests how genomic context may lead to different evolutionary dynamics for shared virulence genes between strains.

Genome Mining Shows Ubiquitous Presence and Extensive Diversity of Toxin-Antitoxin Systems in Pseudomonas syringae.

Bacterial toxin-antitoxin (TA) systems consist of two or more adjacent genes, encoding a toxin and an antitoxin. TA systems are implicated in evolutionary and physiological functions including genome maintenance, antibiotics persistence, phage defense, and virulence. Eight classes of TA systems have been described, based on the mechanism of toxin neutralization by the antitoxin. Although studied well in model species of clinical significance, little is known about the TA system abundance and diversity, and their potential roles in stress tolerance and virulence of plant pathogens. In this study, we screened the genomes of 339 strains representing the genetic and lifestyle diversity of the Pseudomonas syringae species complex for TA systems. Using bioinformatic search and prediction tools, including SLING, BLAST, HMMER, TADB2.0, and T1TAdb, we show that P. syringae strains encode 26 different families of TA systems targeting diverse cellular functions. TA systems in this species are almost exclusively type II. We predicted a median of 15 TA systems per genome, and we identified six type II TA families that are found in more than 80% of strains, while others are more sporadic. The majority of predicted TA genes are chromosomally encoded. Further functional characterization of the predicted TA systems could reveal how these widely prevalent gene modules potentially impact P. syringae ecology, virulence, and disease management practices.