Syringae Strains Research Articles

Genetic and pathogenic diversity of Pseudomonas syringae strains isolated from cucurbits

This work was done to determine the diversity of pathogenic Pseudomonas syringae pv. lachrymans and pv. syringae strains, isolated from cucurbit plants. Pathogenicity tests performed in this work on cucumber indicated that the 22 tested strains differed dramatically in virulence level. Genetic characterization using MLST loci, as well as ITS1, ERIC and REP fingerprinting, allowed the grouping of strains into four phylogenetic groups. After amplicon sequencing and BLAST alignment the collected strains comply with P. syringae pathovars: lachrymans and syringae and with saprophytic P. fluorescens. The strains of Pseudomonas syringae pv. lachrymans may be categorized into at least two types differing in both disease symptoms and molecular characteristics. We confirmed that MLST-loci-based grouping of strains corresponds well to grouping based on pathogenicity tests.

A framework to gauge the epidemic potential of plant pathogens in environmental reservoirs: the example of kiwifruit canker.

New economically important diseases on crops and forest trees emerge recurrently. An understanding of where new pathogenic lines come from and how they evolve is fundamental for the deployment of accurate surveillance methods. We used kiwifruit bacterial canker as a model to assess the importance of potential reservoirs of new pathogenic lineages. The current kiwifruit canker epidemic is at least the fourth outbreak of the disease on kiwifruit caused by Pseudomonas syringae in the mere 50 years in which this crop has been cultivated worldwide, with each outbreak being caused by different genetic lines of the bacterium. Here, we ask whether strains in natural (non-agricultural) environments could cause future epidemics of canker on kiwifruit. To answer this question, we evaluated the pathogenicity, endophytic colonization capacity and competitiveness on kiwifruit of P. syringae strains genetically similar to epidemic strains and originally isolated from aquatic and subalpine habitats. All environmental strains possessing an operon involved in the degradation of aromatic compounds via the catechol pathway grew endophytically and caused symptoms in kiwifruit vascular tissue. Environmental and epidemic strains showed a wide host range, revealing their potential as future pathogens of a variety of hosts. Environmental strains co-existed endophytically with CFBP 7286, an epidemic strain, and shared about 20 virulence genes, but were missing six virulence genes found in all epidemic strains. By identifying the specific gene content in genetic backgrounds similar to known epidemic strains, we developed criteria to assess the epidemic potential and to survey for such strains as a means of forecasting and managing disease emergence.

Differential control of pre-invasive and post-invasive antibacterial defense by the Arabidopsis circadian clock.

Plants show a suite of inducible defense responses against bacterial pathogens. Here we investigate in detail the effect of the circadian clock on these reactions in Arabidopsis thaliana. The magnitude of immune responses elicited by flg22, by virulent and by avirulent Pseudomonas syringae strains depends on the time of day of inoculation. The oxidative burst is stronger when flg22 is infiltrated in the morning in wild-type plants but not in the arrhythmic clock mutant lux arrhythmo/phytoclock1 (pcl1), and thus is controlled by the endogenous clock. Similarly, when bacteria are syringe-infiltrated into the leaf, defense gene induction is higher and bacterial growth is suppressed more strongly after morning inoculation in wild-type but not in pcl1 plants. Furthermore, cell death associated with the hypersensitive response was found to be under clock control. Notably, the clock effect depends on the mode of infection: upon spray inoculation onto the leaf surface, defense gene induction is higher and bacterial growth is suppressed more strongly upon evening inoculation. This different phasing of pre-invasive and post-invasive defense relates to clock-regulated stomatal movement. In particular, TIME FOR COFFEE may impact pathogen defense via clock-regulated stomata movement apart from its known role in time-of-day-dependent jasmonate responses. Taken together, these data highlight the importance of the circadian clock for the control of different immune responses at distinct times of the day.

Translocation and Functional Analysis ofPseudomonas savastanoipv.savastanoiNCPPB 3335 Type III Secretion System Effectors Reveals Two Novel Effector Families of thePseudomonas syringaeComplex

Pseudomonas savastanoi pv. savastanoi NCPPB 3335 causes olive knot disease and is a model pathogen for exploring bacterial infection of woody hosts. The type III secretion system (T3SS) effector repertoire of this strain includes 31 effector candidates plus two novel candidates identified in this study which have not been reported to translocate into plant cells. In this work, we demonstrate the delivery of seven NCPPB 3335 effectors into Nicotiana tabacum leaves, including three proteins from two novel families of the P. syringae complex effector super-repertoire (HopBK and HopBL), one of which comprises two proteins (HopBL1 and HopBL2) that harbor a SUMO protease domain. When delivered by P. fluorescens heterologously expressing a P. syringae T3SS, all seven effectors were found to suppress the production of defense-associated reactive oxygen species. Moreover, six of these effectors, including the truncated versions of HopAA1 and HopAZ1 encoded by NCPPB 3335, suppressed callose deposition. The expression of HopAZ1 and HopBL1 by functionally effectorless P. syringae pv. tomato DC3000D28E inhibited the hypersensitive response in tobacco and, additionally, expression of HopBL2 by this strain significantly increased its competitiveness in N. benthamiana. DNA sequences encoding HopBL1 and HopBL2 were uniquely detected in a collection of 31 P. savastanoi pv. savastanoi strains and other P. syringae strains isolated from woody hosts, suggesting a relevant role of these two effectors in bacterial interactions with olive and other woody plants.

Mangotoxin production of Pseudomonas syringae pv. syringae is regulated by MgoA.

BackgroundThe antimetabolite mangotoxin is a key factor in virulence of Pseudomonas syringae pv. syringae strains which cause apical necrosis of mango trees. Previous studies showed that mangotoxin biosynthesis is governed by the mbo operon. Random mutagenesis led to the identification of two other gene clusters that affect mangotoxin biosynthesis. These are the gacS/gacA genes and mgo operon which harbors the four genes mgoBCAD.ResultsThe current study shows that disruption of the nonribosomal peptide synthetase (NRPS) gene mgoA resulted in loss of mangotoxin production and reduced virulence on tomato leaves. Transcriptional analyses by qPCR and promoter reporter fusions revealed that mbo expression is regulated by both gacS/gacA and mgo genes. Also, expression of the mgo operon was shown to be regulated by gacS/gacA. Heterologous expression under the native promoter of the mbo operon resulted in mangotoxin production in non-producing P. syringae strains, but not in other Pseudomonas species. Also introduction of the mbo and mgo operons in nonproducing P. protegens Pf-5 did not confer mangotoxin production but did enhance transcription of the mbo promoter.ConclusionsFrom the data obtained in this study, we conclude that both mbo and mgo operons are under the control of the gacS/gacA two-component system and that the MgoA product acts as a positive regulator of mangotoxin biosynthesis.

Genomics-Based Exploration of Virulence Determinants and Host-Specific Adaptations of Pseudomonas syringae Strains Isolated from Grasses.

The Pseudomonas syringae species complex has recently been named the number one plant pathogen, due to its economic and environmental impacts, as well as for its role in scientific research. The bacterium has been repeatedly reported to cause outbreaks on bean, cucumber, stone fruit, kiwi and olive tree, as well as on other crop and non-crop plants. It also serves as a model organism for research on the Type III secretion system (T3SS) and plant-pathogen interactions. While most of the current work on this pathogen is either carried out on one of three model strains found on dicot plants with completely sequenced genomes or on isolates obtained from recent outbreaks, not much is known about strains isolated from grasses (Poaceae). Here, we use comparative genomics in order to identify putative virulence-associated genes and other Poaceae-specific adaptations in several newly available genome sequences of strains isolated from grass species. All strains possess only a small number of known Type III effectors, therefore pointing to the importance of non-Type III secreted virulence factors. The implications of this finding are discussed.

Draft Genome Sequences of a Phylogenetically Diverse Suite of Pseudomonas syringae Strains from Multiple Source Populations

Here, we report the draft genome sequences for 7 phylogenetically diverse isolates of Pseudomonas syringae, obtained from numerous environmental sources and geographically proximate crop species. Overall, these sequences provide a wealth of information about the differences (or lack thereof) between isolates from disease outbreaks and those from other sources.

Host specificity, pathogenicity and the presence of virulence genes in Iranian strains of Pseudomonas syringae pv. syringae from different hosts

Pseudomonas syringae pv. syringae (Pss) strains were isolated from almond, apricot, peach, pear, sweet cheery and wheat in Kohgiluye and Boyer-Ahmad, Kordestan, Fras and Chaharmahal and Bakhtiari provinces of Iran. The strains were examined for host specificity, the presence of virulence genes and pathogenicity on different hosts. After inoculation of isolates, in compatible reactions bacterial populations increased within six days of inoculation and final cell numbers increased several-fold over initial inoculum levels, but in incompatible reactions, bacterial populations declined within four days of inoculation. Almond, sweet cherry and wheat isolates induced progressive necrotic symptoms on almond leaves and stems. Apricot, peach and sweet cherry isolates induced necrotic lesions when inoculated on apricot leaves. On pear leaves and stems, only the pear isolate incited pathogenic reaction and isolates from other hosts did not. The syrB gene was detected in all of the tested isolates. Almond and pear isolates did not have the syrD gene. The sypA gene was detected in the almond, peach, pear and sweet cherry isolates while the sypB gene was detected in the apricot, peach, sweet cherry and wheat isolates. Almond, apricot, pear and wheat isolates gave negative results for the detection of nit gene. The gene Ach, was detected only in the peach isolate and gene hrmA, was detected only in the wheat isolate. This study indicates that host specificity exists among different Pss strains, and genes responsible for syringomycin and syringopeptin production contribute to the virulence of Pss strains.

Incongruence between multi‐locus sequence analysis (MLSA) and whole‐genome‐based phylogenies: Pseudomonas syringae pathovar pisi as a cautionary tale

Previous phylogenies, built using a subset of genomic loci, split Pseudomonas syringae pv. pisi into two well-supported clades and implied convergence in host range for these lineages. The analysis of phenotypic and genotypic data within the context of this phylogenetic relationship implied further convergence at the level of virulence gene loss and acquisition. We generate draft genome assemblies for two additional P. syringae strains, isolated from diseased pea plants, and demonstrate incongruence between phylogenies created from a subset of the data compared with the whole genomes. Our whole-genome analysis demonstrates that strains classified as pv. pisi actually form a coherent monophyletic clade, so that apparent convergence is actually the product of shared ancestry. We use this example to urge caution when making evolutionary inferences across closely related strains of P. syringae.

ThePseudomonas syringaetypeIIIeffectorHopD1 suppresses effector‐triggered immunity, localizes to the endoplasmic reticulum, and targets theArabidopsis transcription factorNTL9

• Pseudomonas syringae type III effectors are known to suppress plant immunity to promote bacterial virulence. However, the activities and targets of these effectors are not well understood. • We used genetic, molecular, and cell biology methods to characterize the activities, localization, and target of the HopD1 type III effector in Arabidopsis. • HopD1 contributes to P. syringae virulence in Arabidopsis and reduces effector-triggered immunity (ETI) responses but not pathogen-associated molecular pattern-triggered immunity (PTI) responses. Plants expressing HopD1 supported increased growth of ETI-inducing P. syringae strains compared with wild-type Arabidopsis. We show that HopD1 interacts with the membrane-tethered Arabidopsis transcription factor NTL9 and demonstrate that this interaction occurs at the endoplasmic reticulum (ER). A P. syringae hopD1 mutant and ETI-inducing P. syringae strains exhibited enhanced growth on Arabidopsis ntl9 mutant plants. Conversely, growth of P. syringae strains was reduced in plants expressing a constitutively active NTL9 derivative, indicating that NTL9 is a positive regulator of plant immunity. Furthermore, HopD1 inhibited the induction of NTL9-regulated genes during ETI but not PTI. • HopD1 contributes to P. syringae virulence in part by targeting NTL9, resulting in the suppression of ETI responses but not PTI responses and the promotion of plant pathogenicity.

A Pseudomonas syringae Diversity Survey Reveals a Differentiated Phylotype of the Pathovar syringae Associated with the Mango Host and Mangotoxin Production

Pseudomonas syringae pv. syringae, the causal agent of bacterial apical necrosis (BAN) in mango crops, has been isolated in different mango-producing areas worldwide. An extensive collection of 87 P. syringae pv. syringae strains isolated from mango trees affected by BAN from different countries, but mainly from Southern Spain, were initially examined by repetitive sequence-based polymerase chain reaction (rep-PCR) to analyze the genetic diversity with an epidemiological aim. rep-PCR was powerful in assessing intrapathovar distribution and also allowing clustering of the P. syringae pv. syringae strains isolated from mango, depending on the isolation area. A clear pattern of clustering was observed for all the P. syringae pv. syringae strains isolated from mango distinct from strains from other hosts, including strains for the same geographical regions as the mango isolates. For this reason, a representative group of 51 P. syringae pv. syringae strains isolated from mango and other hosts, as well as some P. syringae strains from other pathovars, were further characterized to determine their possible genetic, phenotypic, and phylogenetic relationships. Similar to the rep-PCR results, the randomly amplified polymorphic DNA PCR (RAPD-PCR) and catabolic diversity analysis using the Biolog GN2 profile grouped 90% of the mango isolates together in a unique cluster. Interestingly, the majority of P. syringae pv. syringae strains isolated from mango produced mangotoxin. The analysis of the phylogenetic distribution using the multilocus sequence typing analysis strongly supports the existence of a differentiated phylotype of the pathovar syringae mainly associated with the mango host and characterized by the mangotoxin production.

Bacterial Effector Activates Jasmonate Signaling by Directly Targeting JAZ Transcriptional Repressors

Gram-negative bacterial pathogens deliver a variety of virulence proteins through the type III secretion system (T3SS) directly into the host cytoplasm. These type III secreted effectors (T3SEs) play an essential role in bacterial infection, mainly by targeting host immunity. However, the molecular basis of their functionalities remains largely enigmatic. Here, we show that the Pseudomonas syringae T3SE HopZ1a, a member of the widely distributed YopJ effector family, directly interacts with jasmonate ZIM-domain (JAZ) proteins through the conserved Jas domain in plant hosts. JAZs are transcription repressors of jasmonate (JA)-responsive genes and major components of the jasmonate receptor complex. Upon interaction, JAZs can be acetylated by HopZ1a through a putative acetyltransferase activity. Importantly, P. syringae producing the wild-type, but not a catalytic mutant of HopZ1a, promotes the degradation of HopZ1-interacting JAZs and activates JA signaling during bacterial infection. Furthermore, HopZ1a could partially rescue the virulence defect of a P. syringae mutant that lacks the production of coronatine, a JA-mimicking phytotoxin produced by a few P. syringae strains. These results highlight a novel example by which a bacterial effector directly manipulates the core regulators of phytohormone signaling to facilitate infection. The targeting of JAZ repressors by both coronatine toxin and HopZ1 effector suggests that the JA receptor complex is potentially a major hub of host targets for bacterial pathogens.

Influence of Biological Treatment and Ultraviolet Disinfection System on Pseudomonas spp. Diversity in Wastewater as Assessed by Denaturing Gradient Gel Electrophoresis

Biological treatment methods use natural processes of ubiquitous living organisms to improve or upgrade the quality of a wastewater. To investigate the dominance of Pseudomonas spp. community in untreated and treated wastewater from full‐scale bio‐discs treatment plant, PCR–denaturing gradient gel electrophoresis coupled with sequence analysis of 16S rRNA gene fragments from dominant bands, were performed. The Pseudomonas species distribution over the total treatment process and over seasons (summer and winter) was obtained by comparing the denaturing gradient gel electrophoresis (DGGE) patterns to a normalized mix of nine reference Pseudomonas strains. Results indicated that Pseudomonas putida and Pseudomonas fluorescens strains showed dominance in wastewater, remarkable stability in the biofilm and resistance to UV irradiation. A reduction of Pseudomonas aeruginosa in treated wastewater was also observed. In addition, climatic and operational conditions results in selection of microbial community. The high temperature and intense solar radiation contribute to the total absence of the Pseudomonas syringae strain. In this work, the effect of increasing UV254 germicidal dose on the Pseudomonas community in secondary treated wastewater effluent was also tested. At a dose of 1200 mWs/cm2, total disappearance of the band corresponding to P. aeruginosa, was noted. The DGGE approach can be used as an effective method to assess directly the Pseudomonas community shifts in studied wastewater treatment plant.

Physiological and Transcriptional Responses to Osmotic Stress of Two Pseudomonas syringae Strains That Differ in Epiphytic Fitness and Osmotolerance

The foliar pathogen Pseudomonas syringae is a useful model for understanding the role of stress adaptation in leaf colonization. We investigated the mechanistic basis of differences in the osmotolerance of two P. syringae strains, B728a and DC3000. Consistent with its higher survival rates following inoculation onto leaves, B728a exhibited superior osmotolerance over DC3000 and higher rates of uptake of plant-derived osmoprotective compounds. A global transcriptome analysis of B728a and DC3000 following an osmotic upshift demonstrated markedly distinct responses between the strains; B728a showed primarily upregulation of genes, including components of the type VI secretion system (T6SS) and alginate biosynthetic pathways, whereas DC3000 showed no change or repression of orthologous genes, including downregulation of the T3SS. DC3000 uniquely exhibited improved growth upon deletion of the biosynthetic genes for the compatible solute N-acetylglutaminylglutamine amide (NAGGN) in a minimal medium, due possibly to NAGGN synthesis depleting the cellular glutamine pool. Both strains showed osmoreduction of glnA1 expression, suggesting that decreased glutamine synthetase activity contributes to glutamate accumulation as a compatible solute, and both strains showed osmoinduction of 5 of 12 predicted hydrophilins. Collectively, our results demonstrate that the superior epiphytic competence of B728a is consistent with its strong osmotolerance, a proactive response to an osmotic upshift, osmoinduction of alginate synthesis and the T6SS, and resiliency of the T3SS to water limitation, suggesting sustained T3SS expression under the water-limited conditions encountered during leaf colonization.

High-Quality Draft Genome Sequence of Pseudomonas syringae pv. Syringae Strain SM, Isolated from Wheat

Pseudomonas syringae is one of the most widespread plant pathogens that can cause significant damage to crop plantations. Here, we announce a noncontiguous finished genome sequence of Pseudomonas syringae pv. syringae strain SM, isolated from hexaploid wheat. The genome sequence revealed the smallest described complement of type III effectors.

Non contiguous-finished genome sequence of Pseudomonas syringae pathovar syringae strain B64 isolated from wheat.

The Gram-negative gammaproteobacterium Pseudomonas syringae is one of the most wide-spread plant pathogens and has been repeatedly reported to cause significant damage to crop plantations. Research on this pathogen is very intensive, but most of it is done on isolates that are pathogenic to Arabidopsis, tomato, and bean. Here, we announce a high-quality draft genome sequence of Pseudomonas syringae pv. syringae B64 which is the first published genome of a P. syringae strain isolated from wheat up to date. The genome sequence will assist in gaining insights into basic virulence mechanisms of this pathogen which has a relatively small complement of type III effectors.

Strains of Pseudomonas syringae pv. syringae from pea are phylogenetically and pathogenically diverse.

Pseudomonas syringae pv. syringae causes extensive yield losses in the pea crop worldwide, although there is little information on its host specialization and its interactions with pea. A collection of 88 putative P. syringae pv. syringae strains (including 39 strains isolated from pea) was characterized by repetitive polymerase chain reaction (rep-PCR), multilocus sequence typing (MLST), and syrB amplification and evaluated for pathogenicity and virulence. rep-PCR data grouped the strains from pea into two groups (1B and 1C) together with strains from other hosts; a third group (1A) was formed exclusively with strains isolated from non-legume species. MLST data included all strains from pea in the genomospecies 1 of P. syringae pathovars defined in previous studies; they were distributed in the same three groups defined by rep-PCR. The inoculations performed in two pea cultivars showed that P. syringae pv. syringae strains from groups 1A and 1C were less virulent than strains from group 1B, suggesting a possible pathogenic specialization in this group. This study shows the existence of genetically and pathogenically distinct P. syringae pv. syringae strain groups from pea, which will be useful for the diagnostic and epidemiology of this pathogen and for disease resistance breeding.

Effects of aqueous ozone on Pseudomonas syringae viability and ice nucleating activity

This study reports the impact of different ozone treatments on a Pseudomonas syringae strain known for its ice nucleation activity (INA). Ozone is a very powerful germicidal agent used for water treatment. The effect of ozone on viability and on cultivability of P. syringae was determined by flow cytometry analysis and by plate counting respectively. The impact of ozone on the outer membrane using the INA as marker was investigated by the drop freezing technique.The destruction curve followed a shoulder pattern with a slight reduction in population with CT values between 0 and 8min. For an initial population of 9.3logCFUmL−1, the cultivability was lost starting at 14min and a loss of viability was observed after 16min of ozone treatment at 0.45mgL−1. Microscopic observations at this point revealed whole but aggregated bacilli. INA decreased after 8min of ozone treatment but did not disappear. This decrease could be due to the progressive disruption of ice nucleating sites in the outer membrane. It was however partially restored after long storage at 4°C of dead cells treated for 16min.

Functional Interplay Between Arabidopsis NADPH Oxidases and Heterotrimeric G Protein

The plant NADPH oxidases produce reactive oxygen species (ROS) in response to pathogens that have diverse functions in different cellular contexts. Distinct phenotypic outcomes may derive from the interaction of NADPH oxidase-dependent ROS with other signaling components that mediate defense activation. We analyze the interaction between NADPH oxidases AtRbohD and AtRbohF and the Arabidopsis heterotrimeric G protein. The Gβ subunit (AGB1) of the heterotrimeric G protein is required for full disease resistance to different Pseudomonas syringae strains. Genetic studies reveal that, upon P. syringae infection, AGB1 and AtRbohD and AtRbohF can function in the same pathway, as the agb1 null allele is epistatic to the NADPH oxidase null alleles, combinatorial mutants display the agb1 phenotypes, and agb1 suppresses some of the atrbohD atrbohF double mutant phenotypes. In contrast, increased susceptibility to the necrotrophic fungus Plectosphaerella cucumerina displayed by agb1 and atrbohD atrbohF is enhanced in the agb1 atrbohD atrbohF triple mutant, suggesting that NADPH oxidase and heterotrimeric G proteins mediate different response pathways in response to this necrotrophic pathogen. The defense response mediated by AGB1 is independent of pathogen-dependent salicylic acid accumulation and signaling, as the agb1 sid2 (isochorismate synthase 2) double mutant showed enhanced disease susceptibility to P. syringae and Plectosphaerella cucumerina as compared with both single mutants. This study exemplifies the complex interplay between signaling events mediating defense activation, depending on the type of plant-pathogen interaction.

Manipulation of Host Proteasomes as a Virulence Mechanism of Plant Pathogens

The ubiquitin-26S proteasome degradation system (UPS) in plants is involved in the signal transduction of many cellular processes, including host immune responses triggered by pathogen attack. Attacking pathogens produce effectors that are translocated into host cells, where they interfere with the host's defense signaling in very specific ways. Perhaps not surprising in view of the broad involvement of the host proteasome in plant immunity, certain bacterial effectors exploit or require the host UPS for their action, as currently best studied in Pseudomonas syringae. Intriguingly, some P. syringae strains also secrete the virulence factor syringolin A, which irreversibly inhibits the proteasome by a novel mechanism. Here, the role of the UPS in plant defense and its exploitation by effectors are summarized, and the biology, taxonomic distribution, and emerging implications for virulence strategies of syringolin A and similar compounds are discussed.