Category: Publications

van Hazel I, Santini F, Müller J, Chang BS

BMC Evol. Biol. 2006;6:97

PubMed PMID: 17107620

Abstract

BACKGROUND: Vertebrate SWS1 visual pigments mediate visual transduction in response to light at short wavelengths. Due to their importance in vision, SWS1 genes have been isolated from a surprisingly wide range of vertebrates, including lampreys, teleosts, amphibians, reptiles, birds, and mammals. The SWS1 genes exhibit many of the characteristics of genes typically targeted for phylogenetic analyses. This study investigates both the utility of SWS1 as a marker for inferring vertebrate phylogenetic relationships, and the characteristics of the gene that contribute to its phylogenetic utility.

RESULTS: Phylogenetic analyses of vertebrate SWS1 genes produced topologies that were remarkably congruent with generally accepted hypotheses of vertebrate evolution at both higher and lower taxonomic levels. The few exceptions were generally associated with areas of poor taxonomic sampling, or relationships that have been difficult to resolve using other molecular markers. The SWS1 data set was characterized by a substantial amount of among-site rate variation, and a relatively unskewed substitution rate matrix, even when the data were partitioned into different codon sites and individual taxonomic groups. Although there were nucleotide biases in some groups at third positions, these biases were not convergent across different taxonomic groups.

CONCLUSION: Our results suggest that SWS1 may be a good marker for vertebrate phylogenetics due to the variable yet consistent patterns of sequence evolution exhibited across fairly wide taxonomic groups. This may result from constraints imposed by the functional role of SWS1 pigments in visual transduction.

Vencato M, Tian F, Alfano JR, Buell CR, Cartinhour S, DeClerck GA, Guttman DS, Stavrinides J, Joardar V, Lindeberg M, Bronstein PA, Mansfield JW, Myers CR, Collmer A, Schneider DJ

Mol. Plant Microbe Interact. 2006 Nov;19(11):1193-206

PubMed PMID: 17073302

Abstract

The ability of Pseudomonas syringae pv. phaseolicola to cause halo blight of bean is dependent on its ability to translocate effector proteins into host cells via the hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS). To identify genes encoding type III effectors and other potential virulence factors that are regulated by the HrpL alternative sigma factor, we used a hidden Markov model, weight matrix model, and type III targeting-associated patterns to search the genome of P. syringae pv. phaseolicola 1448A, which recently was sequenced to completion. We identified 44 high-probability putative Hrp promoters upstream of genes encoding the core T3SS machinery, 27 candidate effectors and related T3SS substrates, and 10 factors unrelated to the Hrp system. The expression of 13 of these candidate HrpL regulon genes was analyzed by real-time polymerase chain reaction, and all were found to be upregulated by HrpL. Six of the candidate type III effectors were assayed for T3SS-dependent translocation into plant cells using the Bordetella pertussis calmodulin-dependent adenylate cyclase (Cya) translocation reporter, and all were translocated. PSPPH1855 (ApbE-family protein) and PSPPH3759 (alcohol dehydrogenase) have no apparent T3SS-related function; however, they do have homologs in the model strain P. syringae pv. tomato DC3000 (PSPTO2105 and PSPTO0834, respectively) that are similarly upregulated by HrpL. Mutations were constructed in the DC3000 homologs and found to reduce bacterial growth in host Arabidopsis leaves. These results establish the utility of the bioinformatic or candidate gene approach to identifying effectors and other genes relevant to pathogenesis in P. syringae genomes.

Stavrinides J, Ma W, Guttman DS

PLoS Pathog. 2006 Oct;2(10):e104

PubMed PMID: 17040127

Abstract

Many bacterial pathogens employ a type III secretion system to deliver type III secreted effectors (T3SEs) into host cells, where they interact directly with host substrates to modulate defense pathways and promote disease. This interaction creates intense selective pressures on these secreted effectors, necessitating rapid evolution to overcome host surveillance systems and defenses. Using computational and evolutionary approaches, we have identified numerous mosaic and truncated T3SEs among animal and plant pathogens. We propose that these secreted virulence genes have evolved through a shuffling process we have called “terminal reassortment.” In terminal reassortment, existing T3SE termini are mobilized within the genome, creating random genetic fusions that result in chimeric genes. Up to 32% of T3SE families in species with relatively large and well-characterized T3SE repertoires show evidence of terminal reassortment, as compared to only 7% of non-T3SE families. Terminal reassortment may permit the near instantaneous evolution of new T3SEs and appears responsible for major modifications to effector activity and function. Because this process plays a more significant role in the evolution of T3SEs than non-effectors, it provides insight into the evolutionary origins of T3SEs and may also help explain the rapid emergence of new infectious agents.

Guttman DS, Gropp SJ, Morgan RL, Wang PW

Mol. Biol. Evol. 2006 Dec;23(12):2342-54

PubMed PMID: 16950758

Abstract

The plant pathogenic bacterium Pseudomonas syringae uses a type III secretion system to inject virulence proteins directly into the cytoplasm of its hosts. The P. syringae type III secretion apparatus is encoded, in part, by the HrpZ operon, which carries the hrpA gene encoding the pilin subunit of the pilus, various components of the structural apparatus, and the HrpZ harpin protein that is believed to produce pores in the host cell membrane. The pilus of the type III system comes into direct contact with the host cell and is, therefore, a likely target of the host’s pathogen surveillance systems. We sequenced and analyzed 22 HrpZ operons from P. syringae strains spanning the diversity of the species. Selection analyses, including K(a)/K(s) tests and Tajima’s D, revealed strong diversifying selection acting on the hrpA gene. This form of selection enables pathogens to maintain genetic diversity within their populations and is often driven by selection imposed by host defense systems. The HrpZ operon also revealed a single significant recombination event that dramatically changed the evolutionary relationships among P. syringae strains from 2 quite distinct phylogroups. This recombination event appears to have introduced genetic diversity into a clade of strains that may now be undergoing positive selection. The identification of diversifying selection acting on the Hrp pilus across the whole population sample and positive selection within one P. syringae lineage supports a trench warfare coevolutionary model between P. syringae and its plant hosts.

Taylor J, Provart NJ

BMC Genet. 2006;7:27

PubMed PMID: 16686952

Abstract

BACKGROUND: Genotyping may be carried out by a number of different methods including direct sequencing and polymorphism analysis. For a number of reasons, PCR-based polymorphism analysis may be desirable, owing to the fact that only small amounts of genetic material are required, and that the costs are low. One popular and cheap method for detecting polymorphisms is by using cleaved amplified polymorphic sequence, or CAPS, molecular markers. These are also known as PCR-RFLP markers.

RESULTS: We have developed a program, called CapsID, that identifies snip-SNPs (single nucleotide polymorphisms that alter restriction endonuclease cut sites) within a set or sets of reference sequences, designs PCR primers around these, and then suggests the most parsimonious combination of markers for genotyping any individual who is not a member of the reference set. The output page includes biologist-friendly features, such as images of virtual gels to assist in genotyping efforts. CapsID is freely available at http://bbc.botany.utoronto.ca/capsid.

CONCLUSION: CapsID is a tool that can rapidly provide minimal sets of CAPS markers for molecular identification purposes for any biologist working in genetics, community genetics, plant and animal breeding, forensics and other fields.

Toufighi K, Brady SM, Austin R, Ly E, Provart NJ

Plant J. 2005 Jul;43(1):153-63

PubMed PMID: 15960624

Abstract

The Botany Array Resource provides the means for obtaining and archiving microarray data for Arabidopsis thaliana as well as biologist-friendly tools for viewing and mining both our own and other’s data, for example, from the AtGenExpress Consortium. All the data produced are publicly available through the web interface of the database at http://bbc.botany.utoronto.ca. The database has been designed in accordance with the Minimum Information About a Microarray Experiment convention — all expression data are associated with the corresponding experimental details. The database is searchable and it also provides a set of useful and easy-to-use web-based data-mining tools for researchers with sophisticated yet understandable output graphics. These include Expression Browser for performing ‘electronic Northerns’, Expression Angler for identifying genes that are co-regulated with a gene of interest, and Promomer for identifying potential cis-elements in the promoters of individual or co-regulated genes.

Becraft PW

Annu. Rev. Cell Dev. Biol. 2002;18:163-92

PubMed PMID: 12142267

Abstract

The Arabidopsis genome sequence has revealed that plants contain a much larger complement of receptor kinase genes than other organisms. Early analysis of these genes revealed involvement in a diverse array of developmental and defense functions that included gametophyte development, pollen-pistil interactions, shoot apical meristem equilibrium, hormone perception, and cell morphogenesis. Amino acid sequence motifs and binding studies indicate that the ectodomains are capable of binding, either directly or indirectly, various classes of molecules including proteins, carbohydrates, and steroids. Genetic and biochemical approaches have begun to identify other components of several signal transduction pathways. Some receptor-like kinases (RLKs) appear to function with coreceptors lacking kinase domains, and genome analysis suggests this might be true for many RLKs. The KAPP protein phosphatase functions as a negative regulator of at least two RLK systems, and in vitro studies suggest it could be a common component of more. Whether plant signaling systems display a modularity similar to animal systems remains to be determined. Future efforts will reveal unknown functions of other RLKs and elucidate the relationships among their signaling networks.

Lumba S, Tsuchiya Y, Delmas F, Hezky J, Provart NJ, Shi Lu Q, McCourt P, Gazzarrini S

BMC Biol. 2012 ;10():8

PubMed PMID: 22348746

Abstract

BACKGROUND:
The embryonic temporal regulator FUSCA3 (FUS3) plays major roles in the establishment of embryonic leaf identity and the regulation of developmental timing. Loss-of-function mutations of this B3 domain transcription factor result in replacement of cotyledons with leaves and precocious germination, whereas constitutive misexpression causes the conversion of leaves into cotyledon-like organs and delays vegetative and reproductive phase transitions.

RESULTS:
Herein we show that activation of FUS3 after germination dampens the expression of genes involved in the biosynthesis and response to the plant hormone ethylene, whereas a loss-of-function fus3 mutant shows many phenotypes consistent with increased ethylene signaling. This FUS3-dependent regulation of ethylene signaling also impinges on timing functions outside embryogenesis. Loss of FUS3 function results in accelerated vegetative phase change, and this is again partially dependent on functional ethylene signaling. This alteration in vegetative phase transition is dependent on both embryonic and vegetative FUS3 function, suggesting that this important transcriptional regulator controls both embryonic and vegetative developmental timing.

CONCLUSION:
The results of this study indicate that the embryonic regulator FUS3 not only controls the embryonic-to-vegetative phase transition through hormonal (ABA/GA) regulation but also functions postembryonically to delay vegetative phase transitions by negatively modulating ethylene-regulated gene expression.

Maughan H, Cunningham KS, Wang PW, Zhang Y, Cypel M, Chaparro C, Tullis DE, Waddell TK, Keshavjee S, Liu M, Guttman DS, Hwang DM

Pulm Med 2012 ;2012():746358

PubMed PMID: 22448327

Abstract

Background. Recurrent bacterial infections play a key role in the pathogenesis of bronchiectasis, but conventional microbiologic methods may fail to identify pathogens in many cases. We characterized and compared the pulmonary bacterial communities of cystic fibrosis (CF) and non-CF bronchiectasis patients using a culture-independent molecular approach.

Methods. Bacterial 16S rRNA gene libraries were constructed from lung tissue of 10 non-CF bronchiectasis and 21 CF patients, followed by DNA sequencing of isolates from each library. Community characteristics were analyzed and compared between the two groups.

Results. A wide range of bacterial diversity was detected in both groups, with between 1 and 21 bacterial taxa found in each patient. Pseudomonas was the most common genus in both groups, comprising 49% of sequences detected and dominating numerically in 13 patients. Although Pseudomonas appeared to be dominant more often in CF patients than in non-CF patients, analysis of entire bacterial communities did not identify significant differences between these two groups.

Conclusions. Our data indicate significant diversity in the pulmonary bacterial community of both CF and non-CF bronchiectasis patients and suggest that this community is similar in surgically resected lungs of CF and non-CF bronchiectasis patients.

Patel RV, Nahal HK, Breit R, Provart NJ

Plant J. 2012 Sep;71(6):1038-50

PubMed PMID: 22607031

Abstract

Large numbers of sequences are now readily available for many plant species, allowing easy identification of homologous genes. However, orthologous gene identification across multiple species is made difficult by evolutionary events such as whole-genome or segmental duplications. Several developmental atlases of gene expression have been produced in the past couple of years, and it may be possible to use these transcript abundance data to refine ortholog predictions. In this study, clusters of homologous genes between seven plant species – Arabidopsis, soybean, Medicago truncatula, poplar, barley, maize and rice – were identified. Following this, a pipeline to rank homologs within gene clusters by both sequence and expression profile similarity was devised by determining equivalent tissues between species, with the best expression profile match being termed the ‘expressolog’. Five electronic fluorescent pictograph (eFP) browsers were produced as part of this effort, to aid in visualization of gene expression data and to complement existing eFP browsers at the Bio-Array Resource (BAR). Within the eFP browser framework, these expression profile similarity rankings were incorporated into an Expressolog Tree Viewer to allow cross-species homolog browsing by both sequence and expression pattern similarity. Global analyses showed that orthologs with the highest sequence similarity do not necessarily exhibit the highest expression pattern similarity. Other orthologs may show different expression patterns, indicating that such genes may require re-annotation or more specific annotation. Ultimately, it is envisaged that this pipeline will aid in improvement of the functional annotation of genes and translational plant research.