Pathogenomics and Bioinformatics
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Pathogenomics and Bioinformatics
Novel methods to tease out the mechanisms involved in host ~ pathogen interactions.
Curated by Adam Taranto
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Leveraging transcript quantification for fast computation of alternative splicing profiles | RNA-Seq Blog

Leveraging transcript quantification for fast computation of alternative splicing profiles | RNA-Seq Blog | Pathogenomics and Bioinformatics | Scoop.it

"SUPPA calculates possible alternative splicing events with the operation generateEvents from an annotation, which can be obtained from a database or built from RNA-seq data using a transcript reconstruction method."

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The dangers of default parameters in bioinformatics: lessons from Bowtie and TopHat

The dangers of default parameters in bioinformatics: lessons from Bowtie and TopHat | Pathogenomics and Bioinformatics | Scoop.it

Most bioinformatics tools are equipped with a vast array of command-line options which let the user configure the inputs, outputs, and performance of the software. You may not wish to explore every possible option when using a particular piece of software, but you should always try to have a look at the manual. 


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Integrating alignment-based and alignment-free sequence similarity measures for biological sequence classification

Integrating alignment-based and alignment-free sequence similarity measures for biological sequence classification | Pathogenomics and Bioinformatics | Scoop.it

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A survey of tools for variant analysis of next-generation genome sequencing data

A survey of tools for variant analysis of next-generation genome sequencing data | Pathogenomics and Bioinformatics | Scoop.it

Here, [they] surveyed 205 tools for whole-genome/whole-exome sequencing data analysis supporting five distinct analytical steps: quality assessment, alignment, variant identification, variant annotation and visualization. [They] report an overview of the functionality, features and specific requirements of the individual tools. [They] then selected 32 programs for variant identification, variant annotation and visualization...


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New Phytologist: Virtual Special Issue on phytopathogen effector proteins (2014)

New Phytologist: Virtual Special Issue on phytopathogen effector proteins (2014) | Pathogenomics and Bioinformatics | Scoop.it

Upon analysis of phytopathogen genomes it turned out that phytopathogenic microbes typically express dozens (bacteria; Collmer et al., 2009) to hundreds (oomycetes and fungi; Schmidt & Panstruga, 2011) of effector proteins. They often do not share any considerable sequence relatedness to known proteins and therefore can be considered as ‘pioneer proteins’, which renders their functional analysis a formidable task. Nevertheless, owing to the key role effector proteins play in plant–microbe interactions, their molecular analysis lately became very popular and is a flourishing research field. This development, which is evidenced by the substantial increase in literature devoted to ‘effectors’ during the last decade (Fig. 1), has also been appreciated by New Phytologist as documented by the organization of two symposia, in 2009 and 2012, with an emphasis on effectors in plant–microbe interactions (22nd New Phytologist Symposium and 30th New Phytologist Symposium; Lee et al., 2013). Besides proteinaceous effectors, secreted small molecules can also exhibit effector activity. Prominent examples from the phytopathogenic bacterium Pseudomonas syringae comprise syringolin (a proteasome inhibitor) and coronatine (a mimic of the phytohormone jasmonic acid), but also fungal secondary metabolites can have defense-suppressing activities (e.g. host-selective toxins; Tsuge et al., 2013).

 

In this Virtual Special Issue we compile a number of papers that were published recently in New Phytologist which all deal with various aspects of effector biology, ranging from bacterial to oomycete and fungal as well as nematode effectors. These papers cover effector functions related to suppression of plant immune responses as well as nutrient acquisition and the identification of plant effector targets.


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Methods in Molecular Biology: Two-Dimensional Data Binning for the Analysis of Genome Architecture in Filamentous Plant Pathogens and Other Eukaryotes (2014)

Methods in Molecular Biology: Two-Dimensional Data Binning for the Analysis of Genome Architecture in Filamentous Plant Pathogens and Other Eukaryotes (2014) | Pathogenomics and Bioinformatics | Scoop.it

Genome architecture often reflects an organism’s lifestyle and can therefore provide insights into gene function, regulation, and adaptation. In several lineages of plant pathogenic fungi and oomycetes, characteristic repeat-rich and gene-sparse regions harbor pathogenicity-related genes such as effectors. In these pathogens, analysis of genome architecture has assisted the mining for novel candidate effector genes and investigations into patterns of gene regulation and evolution at the whole genome level. Here we describe a two-dimensional data binning method in R with a heatmap-style graphical output to facilitate analysis and visualization of whole genome architecture. The method is flexible, combining whole genome architecture heatmaps with scatter plots of the genomic environment of selected gene sets. This enables analysis of specific values associated with genes such as gene expression and sequence polymorphisms, according to genome architecture. This method enables the investigation of whole genome architecture and reveals local properties of genomic neighborhoods in a clear and concise manner.


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rougeforfire's curator insight, April 7, 2014 4:42 AM

This seems pretty interesting.. That's a nice gift idea

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MPMI: Single amino acid mutations in the potato immune receptor R3a expand response to Phytophthora effectors (2014)

MPMI: Single amino acid mutations in the potato immune receptor R3a expand response to Phytophthora effectors (2014) | Pathogenomics and Bioinformatics | Scoop.it

Both plants and animals rely on nucleotide-binding domain and leucine-rich repeat-containing proteins (NB-LRRs or NLRs) to respond to invading pathogens and activate immune responses. How plant NB-LRR proteins respond to pathogens is poorly understood. We undertook a gain-of-function random mutagenesis screen of the potato NB-LRR immune receptor R3a to study how this protein responds to the effector protein AVR3a from the oomycete pathogen Phytophthora infestans. R3a response can be extended to the stealthy AVR3aEM isoform of the effector while retaining recognition of AVR3aKI. Each one of 8 single amino acid mutations is sufficient to expand the R3a response to AVR3aEM and other AVR3a variants. These mutations occur across the R3a protein, from the N-terminus to different regions of the LRR domain. Further characterization of these R3a mutants revealed that at least one of them was sensitized, exhibiting a stronger response than the wild-type R3a protein to AVR3aKI. Remarkably, the N336Y mutation, near the R3a nucleotide-binding pocket, conferred response to the effector protein PcAVR3a4 from the vegetable pathogen Phytophthora capsici. This work contributes to understanding how NB-LRR receptor specificity can be modulated. Together with knowledge of pathogen effector diversity, this strategy can be exploited to develop synthetic immune receptors.


Via Kamoun Lab @ TSL
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Stephen Bolus's curator insight, March 30, 2014 2:09 AM

I just really love the idea of synthetic immune receptors!

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New Phytologist: Cospeciation vs host-shift speciation: methods for testing, evidence from natural associations and relation to coevolution (2013)

New Phytologist: Cospeciation vs host-shift speciation: methods for testing, evidence from natural associations and relation to coevolution (2013) | Pathogenomics and Bioinformatics | Scoop.it

Hosts and their symbionts are involved in intimate physiological and ecological interactions. The impact of these interactions on the evolution of each partner depends on the time-scale considered. Short-term dynamics – ‘coevolution’ in the narrow sense – has been reviewed elsewhere. We focus here on the long-term evolutionary dynamics of cospeciation and speciation following host shifts. Whether hosts and their symbionts speciate in parallel, by cospeciation, or through host shifts, is a key issue in host–symbiont evolution. In this review, we first outline approaches to compare divergence between pairwise associated groups of species, their advantages and pitfalls. We then consider recent insights into the long-term evolution of host–parasite and host–mutualist associations by critically reviewing the literature. We show that convincing cases of cospeciation are rare (7%) and that cophylogenetic methods overestimate the occurrence of such events. Finally, we examine the relationships between short-term coevolutionary dynamics and long-term patterns of diversification in host–symbiont associations. We review theoretical and experimental studies showing that short-term dynamics can foster parasite specialization, but that these events can occur following host shifts and do not necessarily involve cospeciation. Overall, there is now substantial evidence to suggest that coevolutionary dynamics of hosts and parasites do not favor long-term cospeciation.


Via Kamoun Lab @ TSL
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TASSEL-GBS: A High Capacity Genotyping by Sequencing Analysis Pipeline

TASSEL-GBS: A High Capacity Genotyping by Sequencing Analysis Pipeline | Pathogenomics and Bioinformatics | Scoop.it

Genotyping by sequencing (GBS) is a next generation sequencing based method that takes advantage of reduced representation to enable high throughput genotyping of large numbers of individuals at a large number of SNP markers. The relatively straightforward, robust, and cost-effective GBS protocol is currently being applied in numerous species by a large number of researchers. Herein we describe a bioinformatics pipeline, TASSEL-GBS, designed for the efficient processing of raw GBS sequence data into SNP genotypes. The TASSEL-GBS pipeline successfully fulfills the following key design criteria: (1) Ability to run on the modest computing resources that are typically available to small breeding or ecological research programs, including desktop or laptop machines with only 8–16 GB of RAM, (2) Scalability from small to extremely large studies, where hundreds of thousands or even millions of SNPs can be scored in up to 100,000 individuals (e.g., for large breeding programs or genetic surveys), and (3) Applicability in an accelerated breeding context, requiring rapid turnover from tissue collection to genotypes. Although a reference genome is required, the pipeline can also be run with an unfinished “pseudo-reference” consisting of numerous contigs. We describe the TASSEL-GBSpipeline in detail and benchmark it based upon a large scale, species wide analysis in maize (Zea mays), where the average error rate was reduced to 0.0042 through application of population genetic-based SNP filters. Overall, the GBS assay and the TASSEL-GBS pipeline provide robust tools for studying genomic diversity.


Via Niklaus Grunwald
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Steve Marek's curator insight, March 13, 2014 9:57 AM

More impetus to get your bug sequenced.

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A promoter-level mammalian expression atlas : Nature : Nature Publishing Group

A promoter-level mammalian expression atlas : Nature : Nature Publishing Group | Pathogenomics and Bioinformatics | Scoop.it

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Comparative Pathogenomics Reveals Horizontally Acquired Novel Virulence Genes in Fungi Infecting Cereal Hosts

Comparative Pathogenomics Reveals Horizontally Acquired Novel Virulence Genes in Fungi Infecting Cereal Hosts | Pathogenomics and Bioinformatics | Scoop.it

Comparative analyses of pathogen genomes provide new insights into how pathogens have evolved common and divergent virulence strategies to invade related plant species. Fusarium crown and root rots are important diseases of wheat and barley world-wide. In Australia, these diseases are primarily caused by the fungal pathogen Fusarium pseudograminearum.


Comparative genomic analyses showed that the F. pseudograminearum genome encodes proteins that are present in other fungal pathogens of cereals but absent in non-cereal pathogens. In some cases, these cereal pathogen specific genes were also found in bacteria associated with plants. 


Phylogenetic analysis of selected F. pseudograminearum genes supported the hypothesis of horizontal gene transfer into diverse cereal pathogens. Two horizontally acquired genes with no previously known role in fungal pathogenesis were studied functionally via gene knockout methods and shown to significantly affect virulence of F. pseudograminearum on the cereal hosts wheat and barley.


Our results indicate using comparative genomics to identify genes specific to pathogens of related hosts reveals novel virulence genes and illustrates the importance of horizontal gene transfer in the evolution of plant infecting fungal pathogens.

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Australian Bioinformatics Network

Australian Bioinformatics Network | Pathogenomics and Bioinformatics | Scoop.it
The Australian Bioinformatics Network aims to connect people, resources and opportunities to increase the benefits Australian bioinformatics can deliver.

While our focus is on Australia, we want to help science make an even more positive difference to the world.

We hope the Australian Bioinformatics Network will help foster science links internationally and we welcome participation from anyone interested in bioinformatics.
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Interesting collection of bioinformatics presentations curated by the AUstralian Bioinformatics Network

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Accurate prediction of secondary metabolite gene clusters in filamentous fungi

Accurate prediction of secondary metabolite gene clusters in filamentous fungi | Pathogenomics and Bioinformatics | Scoop.it

In this study, we design and apply a DNA expression array forAspergillus nidulans in combination with legacy data to form a comprehensive gene expression compendium. We apply a guilt-by-association–based analysis to predict the extent of the biosynthetic clusters for the 58 synthases active in our set of experimental conditions.

A comparison with legacy data shows the method to be accurate in 13 of 16 known clusters and nearly accurate for the remaining 3 clusters. Furthermore, we apply a data clustering approach, which identifies cross-chemistry between physically separate gene clusters (superclusters), and validate this both with legacy data and experimentally by prediction and verification of a supercluster consisting of the synthase AN1242 and the prenyltransferase AN11080, as well as identification of the product compound nidulanin A.

We have used A. nidulans for our method development and validation due to the wealth of available biochemical data, but the method can be applied to any fungus with a sequenced and assembled genome, thus supporting further secondary metabolite pathway elucidation in the fungal kingdom.

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XSim: Simulation of Descendants from Ancestors with Sequence Data

XSim: Simulation of Descendants from Ancestors with Sequence Data | Pathogenomics and Bioinformatics | Scoop.it

Real or imputed high-density SNP genotypes are routinely used for genomic prediction and genome-wide association studies. Many researchers are moving toward the use of actual or imputed next-generation sequence data in whole-genome analyses. Simulation studies are useful to mimic complex scenarios and test different analytical methods. We have developed the software tool XSim to efficiently simulate sequence data in descendants in arbitrary pedigrees. In this software, a strategy to drop-down origins and positions of chromosomal segments rather than every allele state is implemented to simulate sequence data and to accommodate complicated pedigree structures across multiple generations. Both C++ and Julia versions of XSim have been developed.


Via Niklaus Grunwald
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Compact graphical representation of phylogenetic data and metadata with GraPhlAn

Compact graphical representation of phylogenetic data and metadata with GraPhlAn | Pathogenomics and Bioinformatics | Scoop.it
The increased availability of genomic and metagenomic data poses challenges at multiple analysis levels, including visualization of very large-scale microbial and microbial community data paired with rich metadata. We developed GraPhlAn (Graphical Phylogenetic Analysis), a computational tool that produces high-quality, compact visualizations of microbial genomes and metagenomes. This includes phylogenies spanning up to thousands of taxa, annotated with metadata ranging from microbial community abundances to microbial physiology or host and environmental phenotypes. GraPhlAn has been developed as an open-source command-driven tool in order to be easily integrated into complex, publication-quality bioinformatics pipelines. It can be executed either locally or through an online Galaxy web application. We present several examples including taxonomic and phylogenetic visualization of microbial communities, metabolic functions, and biomarker discovery that illustrate GraPhlAn’s potential for modern microbial and community genomics.

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PLOS Genetics: Genome Sequencing and Comparative Genomics of the Broad Host-Range Pathogen Rhizoctonia solani AG8 (2014)

PLOS Genetics: Genome Sequencing and Comparative Genomics of the Broad Host-Range Pathogen Rhizoctonia solani AG8 (2014) | Pathogenomics and Bioinformatics | Scoop.it

Rhizoctonia solani is a soil-borne basidiomycete fungus with a necrotrophic lifestyle which is classified into fourteen reproductively incompatible anastomosis groups (AGs). One of these, AG8, is a devastating pathogen causing bare patch of cereals, brassicas and legumes. R. solani is a multinucleate heterokaryon containing significant heterozygosity within a single cell. This complexity posed significant challenges for the assembly of its genome. We present a high quality genome assembly of R. solani AG8 and a manually curated set of 13,964 genes supported by RNA-seq. The AG8 genome assembly used novel methods to produce a haploid representation of its heterokaryotic state. The whole-genomes of AG8, the rice pathogen AG1-IA and the potato pathogen AG3 were observed to be syntenic and co-linear. Genes and functions putatively relevant to pathogenicity were highlighted by comparing AG8 to known pathogenicity genes, orthology databases spanning 197 phytopathogenic taxa and AG1-IA. We also observed SNP-level “hypermutation” of CpG dinucleotides to TpG between AG8 nuclei, with similarities to repeat-induced point mutation (RIP). Interestingly, gene-coding regions were widely affected along with repetitive DNA, which has not been previously observed for RIP in mononuclear fungi of the Pezizomycotina. The rate of heterozygous SNP mutations within this single isolate of AG8 was observed to be higher than SNP mutation rates observed across populations of most fungal species compared. Comparative analyses were combined to predict biological processes relevant to AG8 and 308 proteins with effector-like characteristics, forming a valuable resource for further study of this pathosystem. Predicted effector-like proteins had elevated levels of non-synonymous point mutations relative to synonymous mutations (dN/dS), suggesting that they may be under diversifying selection pressures. In addition, the distant relationship to sequenced necrotrophs of the Ascomycota suggests the R. solani genome sequence may prove to be a useful resource in future comparative analysis of plant pathogens.


Via Kamoun Lab @ TSL
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PNAS | Gene silencing and gene expression in phytopathogenic fungi using a plant virus vector

PNAS | Gene silencing and gene expression in phytopathogenic fungi using a plant virus vector | Pathogenomics and Bioinformatics | Scoop.it

RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including phytopathogenic fungi. In such fungi, RNAi has been induced by expressing hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. All these approaches have some drawbacks ranging from instability of hairpin constructs in fungal cells to difficulties in preparing and handling transgenic plants to silence homologous sequences in fungi grown on these plants.

Here we show that RNAi can be expressed in the phytopathogenic fungus Colletotrichum acutatum (strain C71) by virus-induced gene silencing (VIGS) without a plant intermediate, but by using the direct infection of a recombinant virus vector based on the plant virus, tobacco mosaic virus (TMV). We provide evidence that a wild-type isolate of TMV is able to enter C71 cells grown in liquid medium, replicate, and persist therein. With a similar approach, a recombinant TMV vector carrying a gene for the ectopic expression of the green fluorescent protein (GFP) induced the stable silencing of the GFP in the C. acutatumtransformant line 10 expressing GFP derived from C71.

The TMV-based vector also enabled C. acutatum to transiently express exogenous GFP up to six subcultures and for at least 2 mo after infection, without the need to develop transformation technology. With these characteristics, we anticipate this approach will find wider application as a tool in functional genomics of filamentous fungi.

 

TMV graphic from Russell Kightley Media

 


Via Ed Rybicki
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Ed Rybicki's curator insight, March 5, 2014 8:02 AM

This is a nice paper for two main reasons: one, they were able to get VIGS - virus-induced gene silencing - working in a non-model fungus; two, they did it with TMV.

TMV! A plant virus in good standing, not previously shown to infect fungi productively, even if it has been studied in yeast as far as replication requirements go.

This is very interesting, not the least because it opens up the possibility that TMV NATURALLY infects some soil / leaf surface fungi.

Which could open up some investigation of just how the virus gets around, because it has always been touted as being only "mechanically" transmissible - even though we and others have shown it CAN be transmitted by aphids (reasonably inefficiently).

Mind you, Barbara von Wechmar and others in our lab showed in the 1980s that wheat stem and leaf rust fungi could transmit Brome mosaic virus; they just did not have the techniques to look at whether or not it replicated too.

As far as my last post here is concerned, I think there is going to be a LOT of stuff coming out in the next few years on how "plant" and "insect" and "fungal" viruses are in fact considerably more promiscuous in choice of host(s) than we have hitherto been aware.

Now, just to prove what Barbara always said, that Tobacco necrosis virus is also a bacteriophage....

Thanks to Gary Foster (@Prof_GD_Foster) for pointing this out! 

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Bananageddon: Millions face hunger as deadly fungus Panama disease decimates global banana crop

Bananageddon: Millions face hunger as deadly fungus Panama disease decimates global banana crop | Pathogenomics and Bioinformatics | Scoop.it
Scientists have warned that the world’s banana crop, worth £26 billion and a crucial part of the diet of more than 400 million people, is facing “disaster” from virulent diseases immune to pesticides or other forms of control.

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PRADA: Pipeline for RNA sequencing Data Analysis

Technological advances in high throughput sequencing necessitate improved computational tools for processing and analyzing large-scale datasets in a systematic, automated manner. For that purpose, we have developed PRADA (Pipeline for RNA-Sequencing Data Analysis), a flexible, modular, highly scalable software platform that provides many different types of information available by multifaceted analysis starting from raw paired-end RNA-seq data: gene expression levels, quality metrics, detection of unsupervised and supervised fusion transcripts, detection of intragenic fusion variants, homology scores and fusion frame classification. PRADA uses a dual-mapping strategy that increases sensitivity and refines the analytical endpoints. PRADA has been used extensively and successfully in the glioblastoma and renal clear cell projects of The Cancer Genome Atlas program.
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Note: Who puts a comp sci paper behind a paywall these days, honestly!
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Prevalence of transcription factors in ascomycete and basidiomycete fungi

Prevalence of transcription factors in ascomycete and basidiomycete fungi | Pathogenomics and Bioinformatics | Scoop.it

In this study [the authors] have analysed the number of members for 37 regulator classes in 77 ascomycete and 31 basidiomycete fungal genomes and revealed significant differences between ascomycetes and basidiomycetes. In addition, [the authors] determined the presence of 64 regulators characterised in ascomycetes across these 108 genomes. This demonstrated that overall the highest presence of orthologs is in the filamentous ascomycetes. A significant number of regulators lacked orthologs in the ascomycete yeasts and the basidiomycetes. Conversely, of seven basidiomycete regulators included in the study, only one had orthologs in ascomycetes.


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TeselaGen Is Building A Platform For Rapid Prototyping in Synthetic Biology | TechCrunch

TeselaGen Is Building A Platform For Rapid Prototyping in Synthetic Biology | TechCrunch | Pathogenomics and Bioinformatics | Scoop.it
As the costs of DNA sequencing and synthesis drop precipitously, a host of computer science-meets-biotech startups are cropping up in Silicon Valley...

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From dead leaf, to new life: TAL effectors as tools for synthetic biology - Plant Journal

From dead leaf, to new life: TAL effectors as tools for synthetic biology - Plant Journal | Pathogenomics and Bioinformatics | Scoop.it

de Lange et al, 2014

Whether rice, yeast, or fly there is barely a model organism not yet reached by transcription activator like effectors (TALEs) and their derivative fusion proteins. Insights into fundamental biology are now arriving on the back of work in the last years to develop these proteins as tools for molecular biology. This began with the publication of the simple cipher determining base-specific DNA recognition by TALEs in 2009 and now encompasses a huge variety of established fusion proteins mediating targeted modifications to transcriptome, genome, and recently, epigenome. Straightforward design and exquisite specificity, allowing unique sites to be targeted even within complex eukaryote genomes, are key to the popularity of this system. Synthetic biology is one field that is just beginning to make use of these properties with a number of recent publications demonstrating TALE-mediated regulation of synthetic genetic circuits. Intense interest has surrounded the CRISPR/Cas9 system within the last twelve months and it is already proving its mettle as a tool for targeted gene modifications and transcriptional regulation. However, questions over off-target activity and means for independent regulation of multiple Cas9-guide RNA pairs will have to be resolved before this method enters into the synthetic biology toolbox. TALEs are already showing promise as regulators of synthetic biological systems, a role that will likely be developed further in the coming years.


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dromius's curator insight, March 7, 2014 6:28 AM

Cool review. I really like the figures!

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A comparative hidden Markov model analysis pipeline identifies proteins characteristic of cereal-infecting fungi

A comparative hidden Markov model analysis pipeline identifies proteins characteristic of cereal-infecting fungi | Pathogenomics and Bioinformatics | Scoop.it

We take advantage of the availability of sequenced fungal genomes and present an unbiased method for finding putative pathogen proteins and secreted effectors in a query genome via comparative hidden Markov model analyses followed by unsupervised protein clustering.

 

Our method returns experimentally validated fungal effectors in Stagonospora nodorum and Fusarium oxysporum as well as the N-terminal Y/F/WxC-motif from the barley powdery mildew pathogen.

 

Application to the cereal pathogen Fusarium graminearum reveals a secreted phosphorylcholine phosphatase that is characteristic of hemibiotrophic and necrotrophic cereal pathogens and shares an ancient selection process with bacterial plant pathogens.

 

Three F. graminearum protein clusters are found with an enriched secretion signal. One of these putative effector clusters contains proteins that share a [SG]-P-C-[KR]-P sequence motif in the N-terminal and show features not commonly associated with fungal effectors. This motif is conserved in secreted pathogenic Fusarium proteins and a prime candidate for functional testing.

Our pipeline has successfully uncovered conservation patterns, putative effectors and motifs of fungal pathogens that would have been overlooked by existing approaches that identify effectors as small, secreted, cysteine-rich peptides. It can be applied to any pathogenic proteome data, such as microbial pathogen data of plants and other organisms.
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Journal of Visualized Experiments: Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana (2014)

Journal of Visualized Experiments: Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana (2014) | Pathogenomics and Bioinformatics | Scoop.it

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plantNicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other's results.


Via Kamoun Lab @ TSL
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benthamiana's curator insight, November 9, 2014 11:24 AM

benthamiana in the Journal of Visualized Experiments

Kamoun Lab @ TSL's comment, November 9, 2014 11:28 AM
Wait! That's a potato leaf!
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Major Transcriptome Reprogramming Underlies Floral Mimicry Induced by the Rust Fungus Puccinia monoica in Boechera stricta

Major Transcriptome Reprogramming Underlies Floral Mimicry Induced by the Rust Fungus Puccinia monoica in Boechera stricta | Pathogenomics and Bioinformatics | Scoop.it

Puccinia monoica is a spectacular plant parasitic rust fungus that triggers the formation of flower-like structures (pseudoflowers) in its Brassicaceae host plant Boechera stricta.


Pseudoflowers mimic in shape, color, nectar and scent co-occurring and unrelated flowers such as buttercups. They act to attract insects thereby aiding spore dispersal and sexual reproduction of the rust fungus. 


We performed gene expression profiling to reveal 256 plant biological processes that are significantly altered in pseudoflowers. Among these biological processes, plant genes involved in cell fate specification, regulation of transcription, reproduction, floral organ development, anthocyanin (major floral pigments) and terpenoid biosynthesis (major floral volatile compounds) were down-regulated in pseudoflowers. In contrast, plant genes involved in shoot, cotyledon and leaf development, carbohydrate transport, wax biosynthesis, cutin transport and L-phenylalanine metabolism (pathway that results in phenylethanol and phenylacetaldehyde volatile production) were up-regulated.


These findings point to an extensive reprogramming of host genes by the rust pathogen to induce floral mimicry. We also highlight 31 differentially regulated plant genes that are enriched in the biological processes mentioned above, and are potentially involved in the formation of pseudoflowers.

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