Publications

Holbrook-Smith D, Toh S, Tsuchiya Y, McCourt P.

Nat Chem Biol. 2016 Sep;12(9):724-9.

PMID: 27428512

Abstract

Striga spp. (witchweed) is an obligate parasitic plant that attaches to host roots to deplete them of nutrients. In Sub-Saharan Africa, the most destructive Striga species, Striga hermonthica, parasitizes major food crops affecting two-thirds of the arable land and over 100 million people. One potential weakness in the Striga infection process is the way it senses the presence of a host crop. Striga only germinates in the presence of the plant hormone strigolactone, which exudes from a host root. Hence small molecules that perturb strigolactone signaling may be useful tools for disrupting the Striga lifecycle. Here we developed a chemical screen to suppress strigolactone signaling in the model plant Arabidopsis. One compound, soporidine, specifically inhibited a S. hermonthica strigolactone receptor and inhibited the parasite’s germination. This indicates that strigolactone-based screens using Arabidopsis are useful in identifying lead compounds to combat Striga infestations.

Moorthy SD, Davidson S, Shchuka VM, Singh G, Malek-Gilani N, Langroudi L, Martchenko A, So V, Macpherson NN, Mitchell JA.

Genome Res. 2017 Feb;27(2):246-258.

PMID: 27895109

Abstract

Transcriptional enhancers are critical for maintaining cell-type-specific gene expression and driving cell fate changes during development. Highly transcribed genes are often associated with a cluster of individual enhancers such as those found in locus control regions. Recently, these have been termed stretch enhancers or super-enhancers, which have been predicted to regulate critical cell identity genes. We employed a CRISPR/Cas9-mediated deletion approach to study the function of several enhancer clusters (ECs) and isolated enhancers in mouse embryonic stem (ES) cells. Our results reveal that the effect of deleting ECs, also classified as ES cell super-enhancers, is highly variable, resulting in target gene expression reductions ranging from 12% to as much as 92%. Partial deletions of these ECs which removed only one enhancer or a subcluster of enhancers revealed partially redundant control of the regulated gene by multiple enhancers within the larger cluster. Many highly transcribed genes in ES cells are not associated with a super-enhancer; furthermore, super-enhancer predictions ignore 81% of the potentially active regulatory elements predicted by cobinding of five or more pluripotency-associated transcription factors. Deletion of these additional enhancer regions revealed their robust regulatory role in gene transcription. In addition, select super-enhancers and enhancers were identified that regulated clusters of paralogous genes. We conclude that, whereas robust transcriptional output can be achieved by an isolated enhancer, clusters of enhancers acting on a common target gene act in a partially redundant manner to fine tune transcriptional output of their target genes.

Waese J, Provart NJ

Methods Mol Biol. 2017;1533:119-148

PMID: 27987167

Abstract

Bioinformatic tools have become part of the way plant researchers undertake investigations. Large data sets encompassing genomes, transcriptomes, proteomes, epigenomes, and other “-omes” that have been generated in the past decade may be easily accessed with such tools, such that hypotheses may be generated at the click of a mouse. In this chapter, we’ll cover the use of bioinformatic tools available at the Bio-Analytic Resource for Plant Biology at http://bar.utoronto.ca for exploring gene expression and coexpression patterns, undertaking promoter analyses, performing functional classification enrichment analyses for sets of genes, and examining protein-protein interactions. We also touch on some newer bioinformatic tools that allow integration of data from several sources for improved hypothesis generation, both for Arabidopsis and translationally. Most of the data sets come from Arabidopsis, but useful BAR tools for other species will be mentioned where appropriate.

Thakur S, Guttman DS

BMC Bioinformatics 2016;17(1):260

PMID: 27363390

Abstract

BACKGROUND: Comparative analysis of whole genome sequence data from closely related prokaryotic species or strains is becoming an increasingly important and accessible approach for addressing both fundamental and applied biological questions. While there are number of excellent tools developed for performing this task, most scale poorly when faced with hundreds of genome sequences, and many require extensive manual curation.

RESULTS: We have developed a de-novo genome analysis pipeline (DeNoGAP) for the automated, iterative and high-throughput analysis of data from comparative genomics projects involving hundreds of whole genome sequences. The pipeline is designed to perform reference-assisted and de novo gene prediction, homolog protein family assignment, ortholog prediction, functional annotation, and pan-genome analysis using a range of proven tools and databases. While most existing methods scale quadratically with the number of genomes since they rely on pairwise comparisons among predicted protein sequences, DeNoGAP scales linearly since the homology assignment is based on iteratively refined hidden Markov models. This iterative clustering strategy enables DeNoGAP to handle a very large number of genomes using minimal computational resources. Moreover, the modular structure of the pipeline permits easy updates as new analysis programs become available.

CONCLUSION: DeNoGAP integrates bioinformatics tools and databases for comparative analysis of a large number of genomes. The pipeline offers tools and algorithms for annotation and analysis of completed and draft genome sequences. The pipeline is developed using Perl, BioPerl and SQLite on Ubuntu Linux version 12.04 LTS. Currently, the software package accompanies script for automated installation of necessary external programs on Ubuntu Linux; however, the pipeline should be also compatible with other Linux and Unix systems after necessary external programs are installed. DeNoGAP is freely available at https://sourceforge.net/projects/denogap/ .

Mott GA, Thakur S, Smakowska E, Wang PW, Belkhadir Y, Desveaux D, Guttman DS

Genome Biol. 2016;17:98

PMID: 27160854

Abstract

BACKGROUND: The recognition of microbe-associated molecular patterns during infection is central to the mounting of an effective immune response. In spite of their importance, it remains difficult to identify these molecules and the host receptors required for their perception, ultimately limiting our understanding of the role of these molecules in the evolution of host-pathogen relationships.

RESULTS: We employ a comparative genomics screen to identify six new immune eliciting peptides from the phytopathogenic bacterium Pseudomonas syringae. We then perform a reverse genetic screen to identify Arabidopsis thaliana leucine-rich repeat receptor-like kinases required for the recognition of these elicitors. We test the six elicitors on 187 receptor-like kinase knock-down insertion lines using a high-throughput peroxidase-based immune assay and identify multiple lines that show decreased immune responses to specific peptides. From this primary screen data, we focused on the interaction between the xup25 peptide from a bacterial xanthine/uracil permease and the Arabidopsis receptor-like kinase xanthine/uracil permease sensing 1; a family XII protein closely related to two well-characterized receptor-like kinases. We show that xup25 treatment increases pathogenesis-related gene induction, callose deposition, seedling growth inhibition, and resistance to virulent bacteria, all in a xanthine/uracil permease sensing 1-dependent manner. Finally, we show that this kinase-like receptor can bind the xup25 peptide directly. These results identify xup25 as a P. syringae microbe-associated molecular pattern and xanthine/uracil permease sensing 1 as a receptor-like kinase that detects the xup25 epitope to activate immune responses.

CONCLUSIONS: The present study demonstrates an efficient method to identify immune elicitors and the plant receptors responsible for their perception. Further exploration of these molecules will increase our understanding of plant-pathogen interactions and the basis for host specificity.

Krogan NT, Marcos D, Weiner AI, Berleth T

New Phytol. 2016 Jul;

PMID: 27441727

Abstract

The regulatory effect auxin has on its own transport is critical in numerous self-organizing plant patterning processes. However, our understanding of the molecular mechanisms linking auxin signal transduction and auxin transport is still fragmentary, and important regulatory genes remain to be identified. To track a key link between auxin signaling and auxin transport in development, we established an Arabidopsis thaliana genetic background in which fundamental patterning processes in both shoot and root were essentially abolished and the expression of PIN FORMED (PIN) auxin efflux facilitators was dramatically reduced. In this background, we demonstrate that activating a steroid-inducible variant of the auxin response factor (ARF) MONOPTEROS (MP) is sufficient to restore patterning and PIN gene expression. Further, we show that MP binds to distinct promoter elements of multiple genetically defined PIN genes. Our work identifies a direct regulatory link between central, well-characterized genes involved in auxin signal transduction and auxin transport. The steroid-inducible MP system directly demonstrates the importance of this molecular link in multiple patterning events in embryos, shoots and roots, and provides novel options for interrogating the properties of self-regulated auxin-based patterning in planta.

Bridgman SL, Konya T, Azad MB, Guttman DS, Sears MR, Becker AB, Turvey SE, Mandhane PJ, Subbarao P, , Scott JA, Field CJ, Kozyrskyj AL

Microbes Infect. 2016 May;

PMID: 27235197

Abstract

Colonization of infants with Clostridium difficile is on the rise. Although better tolerated by infants than adults, it is a risk factor for future allergic disease. The present study describes associations between infant fecal immunoglobulin A (IgA) and colonization with C. difficile in 47 infants enrolled in the Canadian Healthy Infant Longitudinal Development (CHILD) study. C. difficile colonization was observed in over half (53%) of the infants. Median IgA was lower in infants colonized with C. difficile (10.9 μg versus 25.5 μg per g protein; p = 0.18). A smaller proportion of infants with IgA in the highest tertile were colonized with C. difficile compared to the other tertiles (31.3% versus 64.5%, p = 0.03). In unadjusted analysis, odds of colonization with C. difficile was reduced by 75% (OR 0.25 95% CI 0.07, 0.91 p = 0.04) among infants with IgA in the highest tertile compared to those in the other tertiles. Following adjustment for parity, birth mode and breastfeeding, this association was even stronger (aOR 0.17, 95% CI 0.03, 0.94, p = 0.04). Our study provides evidence that high fecal IgA, independent of breastfeeding, is associated with reduced likelihood of C. difficile colonization in infancy.

Bondy-Denomy J, Qian J, Westra ER, Buckling A, Guttman DS, Davidson AR, Maxwell KL

ISME J 2016 Jun;

PMID: 27258950

Abstract

The activity of bacteriophages poses a major threat to bacterial survival. Upon infection, a temperate phage can either kill the host cell or be maintained as a prophage. In this state, the bacteria carrying the prophage is at risk of superinfection, where another phage injects its genetic material and competes for host cell resources. To avoid this, many phages have evolved mechanisms that alter the bacteria and make it resistant to phage superinfection. The mechanisms underlying these phentoypic conversions and the fitness consequences for the host are poorly understood, and systematic studies of superinfection exclusion mechanisms are lacking. In this study, we examined a wide range of Pseudomonas aeruginosa phages and found that they mediate superinfection exclusion through a variety of mechanisms, some of which affected the type IV pilus and O-antigen, and others that functioned inside the cell. The strongest resistance mechanism was a surface modification that we showed is cost-free for the bacterial host in a natural soil environment and in a Caenorhabditis. elegans infection model. This study represents the first systematic approach to address how a population of prophages influences phage resistance and bacterial behavior in P. aeruginosa.The ISME Journal advance online publication, 3 June 2016; doi:10.1038/ismej.2016.79.

Gould BA, Stinchcombe JR

Mol. Ecol. 2016 Apr;

PMID: 27064998

Abstract

A long-standing question in evolutionary biology is whether the evolution of convergent phenotypes results from selection on the same heritable genetic components. Using whole genome sequencing and genome scans, we tested whether the evolution of parallel longitudinal flowering time clines in the native and introduced ranges of Arabidopsis thaliana has a similar genetic basis. We found that common variants of large effect on flowering time in the native range do not appear to have been under recent strong selection in the introduced range. We identified a set of 38 new candidate genes that are putatively linked to the evolution of flowering time. A high degree of conditional neutrality of flowering time variants between the native and introduced range may preclude parallel evolution at the level of genes. Overall, neither gene pleiotropy nor available standing genetic variation appears to have restricted the evolution of flowering time to high frequency variants from the native range or to known flowering time pathway genes. This article is protected by copyright. All rights reserved.