The Ptr1 Locus of Solanum lycopersicoides Confers Resistance to Race 1 Strains of Pseudomonas syringae pv. tomato and to Ralstonia pseudosolanacearum by Recognizing the Type III Effectors AvrRpt2 and RipBN.

Race 1 strains of Pseudomonas syringae pv. tomato, which cause bacterial speck disease of tomato, are becoming increasingly common and no simply inherited genetic resistance to such strains is known. We discovered that a locus in Solanum lycopersicoides, termed Pseudomonas tomato race 1 (Ptr1), confers resistance to race 1 P. syringae pv. tomato strains by detecting the activity of type III effector AvrRpt2. In Arabidopsis, AvrRpt2 degrades the RIN4 protein, thereby activating RPS2-mediated immunity. Using site-directed mutagenesis of AvrRpt2, we found that, like RPS2, activation of Ptr1 requires AvrRpt2 proteolytic activity. Ptr1 also detected the activity of AvrRpt2 homologs from diverse bacteria, including one in Ralstonia pseudosolanacearum. The genome sequence of S. lycopersicoides revealed no RPS2 homolog in the Ptr1 region. Ptr1 could play an important role in controlling bacterial speck disease and its future cloning may shed light on an example of convergent evolution for recognition of a widespread type III effector.

Genome-Assisted Development of a Diagnostic Protocol for Distinguishing High Virulence Pseudomonas syringae pv. tomato Strains.

A severe outbreak of bacterial speck of tomato, caused by Pseudomonas syringae pv. tomato, occurred in central New York in 2009. Isolate 09150, collected from this outbreak and subsequently named NYS-T1, was found to be highly virulent on tomato. To better understand the relationship of 09150 to other P. syringae strains and develop a diagnostic assay for aggressive strains of this pathogen, the 09150 genome was sequenced. Genome comparison revealed it to be highly similar to a previously sequenced isolate, T1. Genetic factors linked to host interaction including type III effectors, toxin biosynthetic genes, and elicitors of host innate immunity were identified. Type III effector repertoires were compared with other strains in the high virulence T1-like subgroup and lower virulence DC3000/P. syringae pv. maculicola subgroup within P. syringae phylogenetic Group I. Primers for conventional PCR were developed using sequences for avrA, hopW, conserved in the former subgroup and hopN, present in the latter. These were tested on isolates in the two subgroups, other pseudomonads, and other bacterial pathogens of tomato. Primers developed for avaA and hopW were diagnostic for more virulent strains of P. syringae pv. tomato while primers for hopN were diagnostic for P. syringae pv. tomato DC3000 and related P. syringe pv. maculicola strains. Primers designed against hopR distinguished both of these P. syringae subgroups from other P. syringae strains.

Characterization of the Asian Citrus Psyllid Transcriptome.

The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is a vector for the causative agents of Huanglongbing, which threatens citrus production worldwide. This study reports and discusses the first D. citri transcriptomes, encompassing the three main life stages of D. citri, egg, nymph and adult. The transcriptomes were annotated using Gene Ontology (GO) and insecticide-related genes within each life stage were identified to aid the development of future D. citri insecticides. Transcriptome assemblies and other sequence data are available for download at the International Asian Citrus Psyllid Genome Consortium website [http://psyllid.org/download] and at NCBI [http://www.ncbi.nlm.nih.gov/bioproject/29447].

Bound to Succeed: transcription factor binding-site prediction and its contribution to understanding virulence and environmental adaptation in bacterial plant pathogens.

Bacterial plant pathogens rely on a battalion of transcription factors to fine-tune their response to changing environmental conditions and to marshal the genetic resources required for successful pathogenesis. Prediction of transcription factor binding sites (TFBS) represents an important tool for elucidating regulatory networks and has been conducted in multiple genera of plant-pathogenic bacteria for the purpose of better understanding mechanisms of survival and pathogenesis. The major categories of TFBS that have been characterized are reviewed here, with emphasis on in silico methods used for site identification and challenges therein, their applicability to different types of sequence datasets, and insights into mechanisms of virulence and survival that have been gained through binding-site mapping. An improved strategy for establishing E-value cutoffs when using existing models to screen uncharacterized genomes is also discussed.

Genome Sequences of Pseudomonas spp. Isolated from Cereal Crops.

Compared to those of dicot-infecting bacteria, the available genome sequences of bacteria that infect wheat and barley are limited. Herein, we report the draft genome sequences of four pseudomonads originally isolated from these cereals. These genome sequences provide a useful resource for comparative analyses within the genus and for cross-kingdom analyses of plant pathogenesis.

Survey of endosymbionts in the Diaphorina citri metagenome and assembly of a Wolbachia wDi draft genome.

Diaphorina citri (Hemiptera: Psyllidae), the Asian citrus psyllid, is the insect vector of Ca. Liberibacter asiaticus, the causal agent of citrus greening disease. Sequencing of the D. citri metagenome has been initiated to gain better understanding of the biology of this organism and the potential roles of its bacterial endosymbionts. To corroborate candidate endosymbionts previously identified by rDNA amplification, raw reads from the D. citri metagenome sequence were mapped to reference genome sequences. Results of the read mapping provided the most support for Wolbachia and an enteric bacterium most similar to Salmonella. Wolbachia-derived reads were extracted using the complete genome sequences for four Wolbachia strains. Reads were assembled into a draft genome sequence, and the annotation assessed for the presence of features potentially involved in host interaction. Genome alignment with the complete sequences reveals membership of Wolbachia wDi in supergroup B, further supported by phylogenetic analysis of FtsZ. FtsZ and Wsp phylogenies additionally indicate that the Wolbachia strain in the Florida D. citri isolate falls into a sub-clade of supergroup B, distinct from Wolbachia present in Chinese D. citri isolates, supporting the hypothesis that the D. citri introduced into Florida did not originate from China.

Genome-enabled perspectives on the composition, evolution, and expression of virulence determinants in bacterial plant pathogens.

Genome sequence analyses of bacterial plant pathogens are revealing important insights into the molecular determinants of pathogenicity and, through transcript characterization, responses to environmental conditions, evidence for small RNAs, and validation of uncharacterized genes. Genome comparison sheds further light on the processes impacting pathogen evolution and differences in gene repertoire among isolates contributing to niche specialization. Information derived from pathogen genome analysis is providing tools for use in diagnosis and interference with host-pathogen interactions for the purpose of disease control. However, the existing information infrastructure fails to adequately integrate the increasing numbers of sequence data sets, bioinformatic analyses, and experimental characterization, as required for effective systems-level analysis. Enhanced standardization of data formats at the point of publication is proposed as a possible solution.

Pseudomonas syringae type III effector repertoires: last words in endless arguments.

Many plant pathogens subvert host immunity by injecting compositionally diverse but functionally similar repertoires of cytoplasmic effector proteins. The bacterial pathogen Pseudomonas syringae is a model for exploring the functional structure of such repertoires. The pangenome of P. syringae encodes 57 families of effectors injected by the type III secretion system. Distribution of effector genes among phylogenetically diverse strains reveals a small set of core effectors targeting antimicrobial vesicle trafficking and a much larger set of variable effectors targeting kinase-based recognition processes. Complete disassembly of the 28-effector repertoire of a model strain and reassembly of a minimal functional repertoire reveals the importance of simultaneously attacking both processes. These observations, coupled with growing knowledge of effector targets in plants, support a model for coevolving molecular dialogs between effector repertoires and plant immune systems that emphasizes mutually-driven expansion of the components governing recognition.

The plant pathogen Pseudomonas syringae pv. tomato is genetically monomorphic and under strong selection to evade tomato immunity.

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.

Genome sequence of the plant-pathogenic bacterium Dickeya dadantii 3937.

Dickeya dadantii is a plant-pathogenic enterobacterium responsible for the soft rot disease of many plants of economic importance. We present here the sequence of strain 3937, a strain widely used as a model system for research on the molecular biology and pathogenicity of this group of bacteria.

Information Management of Genome Enabled Data Streams for Pseudomonas syringae on the Pseudomonas-Plant Interaction (PPI) Website.

Genome enabled research has led to a large and ever-growing body of data on Pseudomonas syringae genome variation and characteristics, though systematic capture of this information to maximize access by the research community remains a significant challenge. Major P. syringae data streams include genome sequence data, newly identified type III effectors, biological characterization data for type III effectors, and regulatory feature characterization. To maximize data access, the Pseudomonas-Plant Interaction (PPI) website [1] is primarily focused on categorization of type III effectors and curation of effector functional data represented in the Hop database and Pseudomonas-Plant Interaction Resource, respectively. The PPI website further serves as a conduit for incorporation of new genome characterization data into the annotation records at NCBI and other data repositories, and clearinghouse for additional data sets and updates in response to the evolving needs of the research community.

Unifying themes in microbial associations with animal and plant hosts described using the gene ontology.

Microbes form intimate relationships with hosts (symbioses) that range from mutualism to parasitism. Common microbial mechanisms involved in a successful host association include adhesion, entry of the microbe or its effector proteins into the host cell, mitigation of host defenses, and nutrient acquisition. Genes associated with these microbial mechanisms are known for a broad range of symbioses, revealing both divergent and convergent strategies. Effective comparisons among these symbioses, however, are hampered by inconsistent descriptive terms in the literature for functionally similar genes. Bioinformatic approaches that use homology-based tools are limited to identifying functionally similar genes based on similarities in their sequences. An effective solution to these limitations is provided by the Gene Ontology (GO), which provides a standardized language to describe gene products from all organisms. The GO comprises three ontologies that enable one to describe the molecular function(s) of gene products, the biological processes to which they contribute, and their cellular locations. Beginning in 2004, the Plant-Associated Microbe Gene Ontology (PAMGO) interest group collaborated with the GO consortium to extend the GO to accommodate terms for describing gene products associated with microbe-host interactions. Currently, over 900 terms that describe biological processes common to diverse plant- and animal-associated microbes are incorporated into the GO database. Here we review some unifying themes common to diverse host-microbe associations and illustrate how the new GO terms facilitate a standardized description of the gene products involved. We also highlight areas where new terms need to be developed, an ongoing process that should involve the whole community.

Transcriptome analysis of Pseudomonas syringae identifies new genes, noncoding RNAs, and antisense activity.

To fully understand how bacteria respond to their environment, it is essential to assess genome-wide transcriptional activity. New high-throughput sequencing technologies make it possible to query the transcriptome of an organism in an efficient unbiased manner. We applied a strand-specific method to sequence bacterial transcripts using Illumina's high-throughput sequencing technology. The resulting sequences were used to construct genome-wide transcriptional profiles. Novel bioinformatics analyses were developed and used in combination with proteomics data for the qualitative classification of transcriptional activity in defined regions. As expected, most transcriptional activity was consistent with predictions from the genome annotation. Importantly, we identified and confirmed transcriptional activity in areas of the genome inconsistent with the annotation and in unannotated regions. Further analyses revealed potential RpoN-dependent promoter sequences upstream of several noncoding RNAs (ncRNAs), suggesting a role for these ncRNAs in RpoN-dependent phenotypes. We were also able to validate a number of transcriptional start sites, many of which were consistent with predicted promoter motifs. Overall, our approach provides an efficient way to survey global transcriptional activity in bacteria and enables rapid discovery of specific areas in the genome that merit further investigation.

The evolution of Pseudomonas syringae host specificity and type III effector repertoires.

The discovery 45 years ago that many Pseudomonas syringae pathovars elicit the hypersensitive response in plant species other than their hosts fostered the use of these bacteria as experimental models. However, the basis for host specificity and the corresponding resistance of nonhosts remain unclear. Pseudomonas syringae is now known to inject into the host cytoplasm, via the type III secretion system, effector proteins that suppress basal innate immunity, but may be recognized by cognate resistance (R) proteins in a second level of defence. The identification and manipulation of complete repertoires of type III effectors have revealed the highly polymorphic nature of effector repertoires and their potential to limit the host range. However, the maintenance of compatible effector repertoires may be driven by adaptations to life in a given plant species involving many factors. Tools are now available to test several hypotheses for the nature and evolution of P. syringae host specificity and nonhost resistance.

Gene Ontology for type III effectors: capturing processes at the host-pathogen interface.

Disease development is determined by the interplay of host defense processes and pathogen factors that subvert defenses and remodel the host for parasitic benefit. The goal of the Plant-Associated Microbe Gene Ontology (PAMGO) interest group is the development of Gene Ontology (GO) terms that capture the range of biological processes occurring between hosts and symbionts (from mutualists to pathogens). Here, the application of the new GO terms to type III effector proteins (T3Es) from the plant pathogen Pseudomonas syringae serves as an example to systematically document the available extensive data and to reveal shared aspects of interactions with various host plants. Extending the comparison to T3Es deployed by animal pathogens further highlights how GO can uncover the common strategies employed by diverse symbionts as they exploit the host niche. Future application of GO terms to gene products mediating pathogenic or mutualistic interactions involving other microbes will enhance researchers' abilities to identify fundamental patterns among diverse systems and generate new hypotheses based on associations among annotations.

Common and contrasting themes in host cell-targeted effectors from bacterial, fungal, oomycete and nematode plant symbionts described using the Gene Ontology.

A wide diversity of plant-associated symbionts, including microbes, produce proteins that can enter host cells, or are injected into host cells in order to modify the physiology of the host to promote colonization. These molecules, termed effectors, commonly target the host defense signaling pathways in order to suppress the defense response. Others target the gene expression machinery or trigger specific modifications to host morphology or physiology that promote the nutrition and proliferation of the symbiont. When recognized by the host's surveillance machinery, which includes cognate resistance (R) gene products, defense responses are engaged to restrict pathogen proliferation. Effectors from diverse symbionts may be delivered into plant cells via varied mechanisms, including whole organism cellular entry (viruses, some bacteria and fungi), type III and IV secretion (in bacteria), physical injection (nematodes and insects) and protein translocation signal sequences (oomycetes and fungi). This mini-review will summarize both similarities and differences in effectors and effector delivery systems found in diverse plant-associated symbionts as well as how these are described with Plant-Associated Microbe Gene Ontology (PAMGO) terms.

Gene Ontology annotation highlights shared and divergent pathogenic strategies of type III effector proteins deployed by the plant pathogen Pseudomonas syringae pv tomato DC3000 and animal pathogenic Escherichia coli strains.

Genome-informed identification and characterization of Type III effector repertoires in various bacterial strains and species is revealing important insights into the critical roles that these proteins play in the pathogenic strategies of diverse bacteria. However, non-systematic discipline-specific approaches to their annotation impede analysis of the accumulating wealth of data and inhibit easy communication of findings among researchers working on different experimental systems. The development of Gene Ontology (GO) terms to capture biological processes occurring during the interaction between organisms creates a common language that facilitates cross-genome analyses. The application of these terms to annotate type III effector genes in different bacterial species - the plant pathogen Pseudomonas syringae pv tomato DC3000 and animal pathogenic strains of Escherichia coli - illustrates how GO can effectively describe fundamental similarities and differences among different gene products deployed as part of diverse pathogenic strategies. In depth descriptions of the GO annotations for P. syringae pv tomato DC3000 effector AvrPtoB and the E. coli effector Tir are described, with special emphasis given to GO capability for capturing information about interacting proteins and taxa. GO-highlighted similarities in biological process and molecular function for effectors from additional pathosystems are also discussed.

Programmed cell death in host-symbiont associations, viewed through the Gene Ontology.

Manipulation of programmed cell death (PCD) is central to many host microbe interactions. Both plant and animal cells use PCD as a powerful weapon against biotrophic pathogens, including viruses, which draw their nutrition from living tissue. Thus, diverse biotrophic pathogens have evolved many mechanisms to suppress programmed cell death, and mutualistic and commensal microbes may employ similar mechanisms. Necrotrophic pathogens derive their nutrition from dead tissue, and many produce toxins specifically to trigger programmed cell death in their hosts. Hemibiotrophic pathogens manipulate PCD in a most exquisite way, suppressing PCD during the biotrophic phase and stimulating it during the necrotrophic phase. This mini-review will summarize the mechanisms that have evolved in diverse microbes and hosts for controlling PCD and the Gene Ontology terms developed by the Plant-Associated Microbe Gene Ontology (PAMGO) Consortium for describing those mechanisms.

Pseudomonas syringae type III secretion system effectors: repertoires in search of functions.

The ability of Pseudomonas syringae to grow and cause diseases in plants is dependent on the injection of multiple effector proteins into plant cells via the type III secretion system (T3SS). Genome-enabled bioinformatic/experimental methods have comprehensively identified the repertoires of effectors and related T3SS substrates for P. syringae pv. tomato DC3000 and three other sequenced strains. The effector repertoires are diverse and internally redundant. Insights into effector functions are being gained through the construction of mutants lacking one or more effector genes, which may be reduced in growth in planta, and through gain-of-function assays for the ability of single effectors to suppress plant innate immune defenses, manipulate hormone signaling, elicit cell death, and/or display biochemical activities on plant protein targets.