Pauline W. Wang

Next-generation mapping of Arabidopsis genes

Next-generation genomic sequencing technologies have made it possible to directly map mutations responsible for phenotypes of interest via direct sequencing. However, most mapping strategies proposed to date require some prior genetic analysis, which can be very time-consuming even in genetically tractable organisms. Here we present a de novo method for rapidly and robustly mapping the physical location of EMS mutations by sequencing a small pooled F₂ population. This method, called Next Generation Mapping (NGM), uses a chastity statistic to quantify the relative contribution of the parental mutant and mapping lines to each SNP in the pooled F₂ population. It then uses this information to objectively localize the candidate mutation based on its exclusive segregation with the mutant parental line. A user-friendly, web-based tool for performing NGM analysis is available at http://bar.utoronto.ca/NGM. We used NGM to identify three genes involved in cell-wall biology in Arabidopsis thaliana, and, in a power analysis, demonstrate success in test mappings using as few as ten F₂ lines and a single channel of Illumina Genome Analyzer data. This strategy can easily be applied to other model organisms, and we expect that it will also have utility in crops and any other eukaryote with a completed genome sequence.

Phylogenetic Characterization of Virulence and Resistance Phenotypes of Pseudomonas syringae

ABSTRACT Individual strains of the plant pathogenic bacterium Pseudomonas syringae vary in their ability to produce toxins, nucleate ice, and resist antimicrobial compounds. These phenotypes enhance virulence, but it is not clear whether they play a dominant role in specific pathogen-host interactions. To investigate the evolution of these virulence-associated phenotypes, we used functional assays to survey for the distribution of these phenotypes among a collection of 95 P. syringae strains. All of these strains were phylogenetically characterized via multilocus sequence typing (MLST). We surveyed for the production of coronatine, phaseolotoxin, syringomycin, and tabtoxin; for resistance to ampicillin, chloramphenicol, rifampin, streptomycin, tetracycline, kanamycin, and copper; and for the ability to nucleate ice at high temperatures via the ice-nucleating protein INA. We found that fewer than 50% of the strains produced toxins and significantly fewer strains than expected produced multiple toxins, leading to the speculation that there is a cost associated with the production of multiple toxins. None of these toxins was associated with host of isolation, and their distribution, relative to core genome phylogeny, indicated extensive horizontal genetic exchange. Most strains were resistant to ampicillin and copper and had the ability to nucleate ice, and yet very few strains were resistant to the other antibiotics. The distribution of the rare resistance phenotypes was also inconsistent with the clonal history of the species and did not associate with host of isolation. The present study provides a robust phylogenetic foundation for the study of these important virulence-associated phenotypes in P. syringae host colonization and pathogenesis.

Genomic Analysis of the Kiwifruit Pathogen Pseudomonas syringae pv. actinidiae Provides Insight into the Origins of an Emergent Plant Disease

The origins of crop diseases are linked to domestication of plants. Most crops were domesticated centuries – even millennia – ago, thus limiting opportunity to understand the concomitant emergence of disease. Kiwifruit (Actinidia spp.) is an exception: domestication began in the 1930s with outbreaks of canker disease caused by P. syringae pv. actinidiae (Psa) first recorded in the 1980s. Based on SNP analyses of two circularized and 34 draft genomes, we show that Psa is comprised of distinct clades exhibiting negligible within-clade diversity, consistent with disease arising by independent samplings from a source population. Three clades correspond to their geographical source of isolation; a fourth, encompassing the Psa-V lineage responsible for the 2008 outbreak, is now globally distributed. Psa has an overall clonal population structure, however, genomes carry a marked signature of within-pathovar recombination. SNP analysis of Psa-V reveals hundreds of polymorphisms; however, most reside within PPHGI-1-like conjugative elements whose evolution is unlinked to the core genome. Removal of SNPs due to recombination yields an uninformative (star-like) phylogeny consistent with diversification of Psa-V from a single clone within the last ten years. Growth assays provide evidence of cultivar specificity, with rapid systemic movement of Psa-V in Actinidia chinensis. Genomic comparisons show a dynamic genome with evidence of positive selection on type III effectors and other candidate virulence genes. Each clade has highly varied complements of accessory genes encoding effectors and toxins with evidence of gain and loss via multiple genetic routes. Genes with orthologs in vascular pathogens were found exclusively within Psa-V. Our analyses capture a pathogen in the early stages of emergence from a predicted source population associated with wild Actinidia species. In addition to candidate genes as targets for resistance breeding programs, our findings highlight the importance of the source population as a reservoir of new disease.

Lung microbiota across age and disease stage in cystic fibrosis

Understanding the significance of bacterial species that colonize and persist in cystic fibrosis (CF) airways requires a detailed examination of bacterial community structure across a broad range of age and disease stage. We used 16S ribosomal RNA sequencing to characterize the lung microbiota in 269 CF patients spanning a 60 year age range, including 76 pediatric samples from patients of age 4–17, and a broad cross-section of disease status to identify features of bacterial community structure and their relationship to disease stage and age. The CF lung microbiota shows significant inter-individual variability in community structure, composition and diversity. The core microbiota consists of five genera - Streptococcus, Prevotella, Rothia, Veillonella and Actinomyces. CF-associated pathogens such as Pseudomonas, Burkholderia, Stenotrophomonas and Achromobacter are less prevalent than core genera, but have a strong tendency to dominate the bacterial community when present. Community diversity and lung function are greatest in patients less than 10 years of age and lower in older age groups, plateauing at approximately age 25. Lower community diversity correlates with worse lung function in a multivariate regression model. Infection by Pseudomonas correlates with age-associated trends in community diversity and lung function.

A bacterial acetyltransferase destroys plant microtubule networks and blocks secretion.

The eukaryotic cytoskeleton is essential for structural support and intracellular transport, and is therefore a common target of animal pathogens. However, no phytopathogenic effector has yet been demonstrated to specifically target the plant cytoskeleton. Here we show that the Pseudomonas syringae type III secreted effector HopZ1a interacts with tubulin and polymerized microtubules. We demonstrate that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor phytic acid. Activated HopZ1a acetylates itself and tubulin. The conserved autoacetylation site of the YopJ / HopZ superfamily, K289, plays a critical role in both the avirulence and virulence function of HopZ1a. Furthermore, HopZ1a requires its acetyltransferase activity to cause a dramatic decrease in Arabidopsis thaliana microtubule networks, disrupt the plant secretory pathway and suppress cell wall-mediated defense. Together, this study supports the hypothesis that HopZ1a promotes virulence through cytoskeletal and secretory disruption.

Complete Sequence and Evolutionary Genomic Analysis of the Pseudomonas aeruginosa Transposable Bacteriophage D3112

Bacteriophage D3112 represents one of two distinct groups of transposable phage found in the clinically relevant, opportunistic pathogen Pseudomonas aeruginosa. To further our understanding of transposable phage in P. aeruginosa, we have sequenced the complete genome of D3112. The genome is 37,611 bp, with an overall G+C content of 65%. We have identified 53 potential open reading frames, including three genes (the c repressor gene and early genes A and B) that have been previously characterized and sequenced. The organization of the putative coding regions corresponds to published genetic and transcriptional maps and is very similar to that of enterobacteriophage Mu. In contrast, the International Committee on Taxonomy of Viruses has classified D3112 as a lambda-like phage on the basis of its morphology. Similarity-based analyses identified 27 open reading frames with significant matches to proteins in the NCBI databases. Forty-eight percent of these were similar to Mu-like phage and prophage sequences, including proteins responsible for transposition, transcriptional regulation, virion morphogenesis, and capsid formation. The tail proteins were highly similar to prophage sequences in Escherichia coli and phage Phi12 from Staphylococcus aureus, while proteins at the right end were highly similar to proteins in Xylella fastidiosa. We performed phylogenetic analyses to understand the evolutionary relationships of D3112 with respect to Mu-like versus lambda-like bacteriophages. Different results were obtained from similarity-based versus phylogenetic analyses in some instances. Overall, our findings reveal a highly mosaic structure and suggest that extensive horizontal exchange of genetic material played an important role in the evolution of D3112.

Using a Commercial DiversiLab Semiautomated Repetitive Sequence-Based PCR Typing Technique for Identification of Escherichia coli Clone ST131 Producing CTX-M-15

ABSTRACT A study was designed to evaluate the ability of the DiversiLab fingerprinting kit, a type of repetitive element PCR (rep-PCR), to identify Escherichia coli clone ST131 producing β-lactamase CTX-M-15. A set of 53 nonduplicate isolates of extended-spectrum β-lactamase-producing E. coli underwent rep-PCR, pulsed-field gel electrophoresis, and multilocus sequence typing. The DiversiLab system successfully identified E. coli clone ST131 producing CTX-M-15 and provides a simple standardized typing protocol for monitoring the spread of this clone.

The Arabidopsis ZED1 pseudokinase is required for ZAR1-mediated immunity induced by the Pseudomonas syringae type III effector HopZ1a.

Significance Bacterial pathogens can use a syringe-like structure to inject virulence proteins (effectors) directly into host cells. The YopJ/HopZ superfamily of effectors found in animal and plant pathogens can modify host kinase proteins to suppress host immunity. In the model plant Arabidopsis, HopZ1a is recognized by the resistance protein ZAR1 to induce a robust immune response that blocks pathogen growth. Here, we show that the HopZ1a effector from the plant pathogen Pseudomonas syringae targets the Arabidopsis nonfunctional pseudokinase ZED1 and that ZED1 is required for recognition of HopZ1a by ZAR1. We hypothesize that HopZ1a targets kinases to promote pathogen virulence, and Arabidopsis ZED1 evolved as a decoy to trap HopZ1a in the ZAR1 complex for recognition by the plant immune system. Plant and animal pathogenic bacteria can suppress host immunity by injecting type III secreted effector (T3SE) proteins into host cells. However, T3SEs can also elicit host immunity if the host has evolved a means to recognize the presence or activity of specific T3SEs. The diverse YopJ/HopZ/AvrRxv T3SE superfamily, which is found in both animal and plant pathogens, provides examples of T3SEs playing this dual role. The T3SE HopZ1a is an acetyltransferase carried by the phytopathogen Pseudomonas syringae that elicits effector-triggered immunity (ETI) when recognized in Arabidopsis thaliana by the nucleotide-binding leucine-rich repeat (NB-LRR) protein ZAR1. However, recognition of HopZ1a does not require any known ETI-related genes. Using a forward genetics approach, we identify a unique ETI-associated gene that is essential for ZAR1-mediated immunity. The hopZ-ETI-deficient1 (zed1) mutant is specifically impaired in the recognition of HopZ1a, but not the recognition of other unrelated T3SEs or in pattern recognition receptor (PRR)-triggered immunity. ZED1 directly interacts with both HopZ1a and ZAR1 and is acetylated on threonines 125 and 177 by HopZ1a. ZED1 is a nonfunctional kinase that forms part of small genomic cluster of kinases in Arabidopsis. We hypothesize that ZED1 acts as a decoy to lure HopZ1a to the ZAR1–resistance complex, resulting in ETI activation.

Phenotypic diversity within a Pseudomonas aeruginosa population infecting an adult with cystic fibrosis

Chronic airway infections caused by Pseudomonas aeruginosa contribute to the progression of pulmonary disease in individuals with cystic fibrosis (CF). In the setting of CF, within-patient adaptation of a P. aeruginosa strain generates phenotypic diversity that can complicate microbiological analysis of patient samples. We investigated within- and between- sample diversity of 34 phenotypes among 235 P. aeruginosa isolates cultured from sputum samples collected from a single CF patient over the span of one year, and assessed colony morphology as a screening tool for predicting phenotypes, including antimicrobial susceptibilities. We identified 15 distinct colony morphotypes that varied significantly in abundance both within and between sputum samples. Substantial within sample phenotypic heterogeneity was also noted in other phenotypes, with morphotypes being unreliable predictors of antimicrobial susceptibility and other phenotypes. Emergence of isolates with reduced susceptibility to β-lactams was observed during periods of clinical therapy with aztreonam. Our findings confirm that the P. aeruginosa population in chronic CF lung infections is highly dynamic, and that intra-sample phenotypic diversity is underestimated if only one or few colonies are analyzed per sample.

Quantitative Interactor Screening with next- generation Sequencing (QIS-Seq) identifies Arabidopsis thaliana MLO2 as a target of the Pseudomonas syringae type III effector HopZ2

BackgroundIdentification of protein-protein interactions is a fundamental aspect of understanding protein function. A commonly used method for identifying protein interactions is the yeast two-hybrid system.ResultsHere we describe the application of next-generation sequencing to yeast two-hybrid interaction screens and develop Quantitative Interactor Screen Sequencing (QIS-Seq). QIS-Seq provides a quantitative measurement of enrichment for each interactor relative to its frequency in the library as well as its general stickiness (non-specific binding). The QIS-Seq approach is scalable and can be used with any yeast two-hybrid screen and with any next-generation sequencing platform. The quantitative nature of QIS-Seq data make it amenable to statistical evaluation, and importantly, facilitates the standardization of experimental design, data collection, and data analysis. We applied QIS-Seq to identify the Arabidopsis thaliana MLO2 protein as a target of the Pseudomonas syringae type III secreted effector protein HopZ2. We validate the interaction between HopZ2 and MLO2 in planta and show that the interaction is required for HopZ2-associated virulence.ConclusionsWe demonstrate that QIS-Seq is a high-throughput quantitative interactor screen and validate MLO2 as an interactor and novel virulence target of the P. syringae type III secreted effector HopZ2.