Publications

McCann HC, Rikkerink EH, Bertels F, Fiers M, Lu A, Rees-George J, Andersen MT, Gleave AP, Haubold B, Wohlers MW, Guttman DS, Wang PW, Straub C, Vanneste JL, Vanneste J, Rainey PB, Templeton MD

PLoS Pathog. 2013;9(7):e1003503

PMID: 23935484

Abstract

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.

Austin RS, Chatfield SP, Desveaux D, Guttman DS

Methods Mol. Biol. 2014;1062:301-15

PMID: 24057374

Abstract

Next-generation sequencing platforms have made it possible to very rapidly map genetic mutations in Arabidopsis using whole-genome resequencing against pooled members of an F2 mapping population. In the case of recessive mutations, all individuals expressing the phenotype will be homozygous for the mutant genome at the locus responsible for the phenotype, while all other loci segregate roughly equally for both parental lines due to recombination. Importantly, genomic regions flanking the recessive mutation will be in linkage disequilibrium and therefore also be homozygous due to genetic hitchhiking. This information can be exploited to quickly and effectively identify the causal mutation. To this end, sequence data generated from members of the pooled population exhibiting the mutant phenotype are first aligned to the reference genome. Polymorphisms between the mutant and mapping line are then identified and used to determine the homozygous, nonrecombinant region harboring the mutation. Polymorphisms in the identified region are filtered to provide a short list of markers potentially responsible for the phenotype of interest, which is followed by validation at the bench. Although the focus of recent studies has been on the mapping of point mutations exhibiting recessive phenotypes, the techniques employed can be extended to incorporate more complicated scenarios such as dominant mutations and those caused by insertions or deletions in genomic sequence. This chapter describes detailed procedures for performing next-generation mapping against an Arabidopsis mutant and discusses how different mutations might be approached.

Tyler AD, Knox N, Kabakchiev B, Milgrom R, Kirsch R, Cohen Z, McLeod RS, Guttman DS, Krause DO, Silverberg MS

PLoS ONE 2013;8(9):e66934

PMID: 24086242

Abstract

INTRODUCTION: Inflammatory complications following ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC) are common and thought to arise through mechanisms similar to de novo onset inflammatory bowel disease. The aim of this study was to determine whether specific organisms in the tissue-associated microbiota are associated with inflammatory pouch complications.

METHODS: Patients having previously undergone IPAA were recruited from Mount Sinai Hospital. Clinical and demographic information were collected and a pouchoscopy with biopsy of both the pouch and afferent limb was performed. Patients were classified based on post-surgical phenotype into four outcome groups: familial adenomatous polyposis controls (FAP), no pouchitis, pouchitis, and Crohn’s disease-like (CDL). Pyrosequencing of the 16S rRNA V1-V3 hypervariable region, and quantitative PCR for bacteria of interest, were used to identify organisms present in the afferent limb and pouch. Associations with outcomes were evaluated using exact and non-parametric tests of significance.

RESULTS: Analysis at the phylum level indicated that Bacteroidetes were detected significantly less frequently (P<0.0001) in the inflammatory outcome groups (pouchitis and CDL) compared to both FAP and no pouchitis. Conversely, Proteobacteria were detected more frequently in the inflammatory groups (P=0.01). At the genus level, organisms associated with outcome were detected less frequently among the inflammatory groups compared to those without inflammation. Several of these organisms, including Bacteroides (P<0.0001), Parabacteroides (P≤2.2×10(-3)), Blautia (P≤3.0×10(-3)) and Sutterella (P≤2.5×10(-3)), were associated with outcome in both the pouch and afferent limb. These associations remained significant even following adjustment for antibiotic use, smoking, country of birth and gender. Individuals with quiescent disease receiving antibiotic therapy displayed similar reductions in these organisms as those with active pouch inflammation.

CONCLUSIONS: Specific genera are associated with inflammation of the ileal pouch, with a reduction of typically ubiquitous organisms characterizing the inflammatory phenotypes.

Konya T, Koster B, Maughan H, Escobar M, Azad MB, Guttman DS, Sears MR, Becker AB, Brook JR, Takaro TK, Kozyrskyj AL, Scott JA,

Environ. Res. 2014 May;131:25-30

PMID: 24637181

Abstract

The human gut is host to a diverse and abundant community of bacteria that influence health and disease susceptibility. This community develops in infancy, and its composition is strongly influenced by environmental factors, notably perinatal anthropogenic exposures such as delivery mode (Cesarean vs. vaginal) and feeding method (breast vs. formula); however, the built environment as a possible source of exposure has not been considered. Here we report on a preliminary investigation of the associations between bacteria in house dust and the nascent fecal microbiota from 20 subjects from the Canadian Healthy Infant Longitudinal Development (CHILD) Study using high-throughput sequence analysis of portions of the 16S rRNA gene. Despite significant differences between the dust and fecal microbiota revealed by Nonmetric Multidimensional Scaling (NMDS) analysis, permutation analysis confirmed that 14 bacterial OTUs representing the classes Actinobacteria (3), Bacilli (3), Clostridia (6) and Gammaproteobacteria (2) co-occurred at a significantly higher frequency in matched dust-stool pairs than in randomly permuted pairs, indicating an association between these dust and stool communities. These associations could indicate a role for the indoor environment in shaping the nascent gut microbiota, but future studies will be needed to confirm that our findings do not solely reflect a reverse pathway. Although pet ownership was strongly associated with the presence of certain genera in the dust for dogs (Agrococcus, Carnobacterium, Exiguobacterium, Herbaspirillum, Leifsonia and Neisseria) and cats (Escherichia), no clear patterns were observed in the NMDS-resolved stool community profiles as a function of pet ownership.

Prouse MB, Campbell MM

PLoS ONE 2013;8(5):e65132

PMID: 23741471

Abstract

Despite the prominent roles played by R2R3-MYB transcription factors in the regulation of plant gene expression, little is known about the details of how these proteins interact with their DNA targets. For example, while Arabidopsis thaliana R2R3-MYB protein AtMYB61 is known to alter transcript abundance of a specific set of target genes, little is known about the specific DNA sequences to which AtMYB61 binds. To address this gap in knowledge, DNA sequences bound by AtMYB61 were identified using cyclic amplification and selection of targets (CASTing). The DNA targets identified using this approach corresponded to AC elements, sequences enriched in adenosine and cytosine nucleotides. The preferred target sequence that bound with the greatest affinity to AtMYB61 recombinant protein was ACCTAC, the AC-I element. Mutational analyses based on the AC-I element showed that ACC nucleotides in the AC-I element served as the core recognition motif, critical for AtMYB61 binding. Molecular modelling predicted interactions between AtMYB61 amino acid residues and corresponding nucleotides in the DNA targets. The affinity between AtMYB61 and specific target DNA sequences did not correlate with AtMYB61-driven transcriptional activation with each of the target sequences. CASTing-selected motifs were found in the regulatory regions of genes previously shown to be regulated by AtMYB61. Taken together, these findings are consistent with the hypothesis that AtMYB61 regulates transcription from specific cis-acting AC elements in vivo. The results shed light on the specifics of DNA binding by an important family of plant-specific transcriptional regulators.

Lewis JD, Lee AH, Hassan JA, Wan J, Hurley B, Jhingree JR, Wang PW, Lo T, Youn JY, Guttman DS, Desveaux D

Proc. Natl. Acad. Sci. U.S.A. 2013 Nov;110(46):18722-7

PMID: 24170858

Abstract

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.

Yu CG, Tonikian R, Felsensteiner C, Jhingree JR, Desveaux D, Sidhu SS, Harris TJ

PLoS ONE 2014;9(1):e86412

PMID: 24466078

Abstract

The Par complex is a conserved cell polarity regulator. Bazooka/Par-3 is scaffold for the complex and contains three PDZ domains in tandem. PDZ domains can act singly or synergistically to bind the C-termini of interacting proteins. Sequence comparisons among Drosophila Baz and its human and C. elegans Par-3 counterparts indicate a divergence of the peptide binding pocket of PDZ1 and greater conservation for the pockets of PDZ2 and PDZ3. However, it is unclear whether the domains from different species share peptide binding preferences, or if their tandem organization affects their peptide binding properties. To investigate these questions, we first used phage display screens to identify unique peptide binding profiles for each single PDZ domain of Baz. Comparisons with published phage display screens indicate that Baz and C. elegans PDZ2 bind to similar peptides, and that the peptide binding preferences of Baz PDZ3 are more similar to C. elegans versus human PDZ3. Next we quantified the peptide binding preferences of each Baz PDZ domain using single identified peptides in surface plasmon resonance assays. In these direct binding studies, each peptide had a binding preference for a single PDZ domain (although the peptide binding of PDZ2 was weakest and the least specific). PDZ1 and PDZ3 bound their peptides with dissociation constants in the nM range, whereas PDZ2-peptide binding was in the µM range. To test whether tandem PDZ domain organization affects peptide binding, we examined a fusion protein containing all three PDZ domains and their normal linker regions. The binding strengths of the PDZ-specific peptides to single PDZ domains and to the PDZ domain tandem were indistinguishable. Thus, the peptide binding pockets of each PDZ domain in Baz are not obviously affected by the presence of neighbouring PDZ domains, but act as isolated modules with specific in vitro peptide binding preferences.

Peek J, Castiglione G, Shi T, Christendat D

Mol. Biochem. Parasitol. 2014 Mar-Apr;194(1-2):16-9

PMID: 24731949

Abstract

The apicomplexan parasite Toxoplasma gondii, the etiologic agent of toxoplasmosis, is estimated to infect 10-80% of different human populations. T. gondii encodes a large pentafunctional polypeptide known as the AROM complex which catalyzes five reactions in the shikimate pathway, a metabolic pathway required for the biosynthesis of the aromatic amino acids and a promising target for anti-parasitic agents. Here, we present the isolation, cloning and kinetic characterization of the shikimate dehydrogenase domain (TgSDH) from the T. gondii AROM complex. Recombinant TgSDH catalyzed the NADP(+)-dependent oxidation of shikimate in the absence of the remaining AROM domains and was sensitive to inhibition by a previously identified SDH inhibitor. Analysis of the TgSDH amino acid sequence revealed a number of novel insertions not found in SDH homologs from other organisms. Nevertheless, a three-dimensional structural model of TgSDH predicts a high level of conservation in the ‘core’ structure of the enzyme.

Lumba S, Toh S, Handfield LF, Swan M, Liu R, Youn JY, Cutler SR, Subramaniam R, Provart N, Moses A, Desveaux D, McCourt P

Dev. Cell 2014 May;29(3):360-72

PMID: 24823379

Abstract

The sesquiterpenoid abscisic acid (ABA) mediates an assortment of responses across a variety of kingdoms including both higher plants and animals. In plants, where most is known, a linear core ABA signaling pathway has been identified. However, the complexity of ABA-dependent gene expression suggests that ABA functions through an intricate network. Here, using systems biology approaches that focused on genes transcriptionally regulated by ABA, we defined an ABA signaling network of over 500 interactions among 138 proteins. This map greatly expanded ABA core signaling but was still manageable for systematic analysis. For example, functional analysis was used to identify an ABA module centered on two sucrose nonfermenting (SNF)-like kinases. We also used coexpression analysis of interacting partners within the network to uncover dynamic subnetwork structures in response to different abiotic stresses. This comprehensive ABA resource allows for application of approaches to understanding ABA functions in higher plants.

Stock AJ, Campitelli BE, Stinchcombe JR

Philos. Trans. R. Soc. Lond., B, Biol. Sci. 2014 Aug;369(1649)

PMID: 25002704

Abstract

Clinal variation is commonly interpreted as evidence of adaptive differentiation, although clines can also be produced by stochastic forces. Understanding whether clines are adaptive therefore requires comparing clinal variation to background patterns of genetic differentiation at presumably neutral markers. Although this approach has frequently been applied to single traits at a time, we have comparatively fewer examples of how multiple correlated traits vary clinally. Here, we characterize multivariate clines in the Ivyleaf morning glory, examining how suites of traits vary with latitude, with the goal of testing for divergence in trait means that would indicate past evolutionary responses. We couple this with analysis of genetic variance in clinally varying traits in 20 populations to test whether past evolutionary responses have depleted genetic variance, or whether genetic variance declines approaching the range margin. We find evidence of clinal differentiation in five quantitative traits, with little evidence of isolation by distance at neutral loci that would suggest non-adaptive or stochastic mechanisms. Within and across populations, the traits that contribute most to population differentiation and clinal trends in the multivariate phenotype are genetically variable as well, suggesting that a lack of genetic variance will not cause absolute evolutionary constraints. Our data are broadly consistent theoretical predictions of polygenic clines in response to shallow environmental gradients. Ecologically, our results are consistent with past findings of natural selection on flowering phenology, presumably due to season-length variation across the range.