TAL effector science
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TAL effector science
infos on novel DNA-binding proteins of bacteria and their biotech use
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Ectopic expression of the TAL effector AvrXa7 in Xanthomonas citri subsp. citri hinders citrus canker symptom formation by modulating transcriptional profile of citrus genes - Biochem Biophys Res C...

Ectopic expression of the TAL effector AvrXa7 in Xanthomonas citri subsp. citri hinders citrus canker symptom formation by modulating transcriptional profile of citrus genes - Biochem Biophys Res C... | TAL effector science | Scoop.it

Sun et al, 2018

Xanthomonas citri subsp. citri (Xcc) is the causal agent of citrus canker, a serious bacterial disease that affects citrus trees worldwide. The ectopic expression of TAL effector AvrXa7 in Xcc suppressed canker development. The Xcc strain expressing avrXa7 induced a yellow symptom around the inoculation site. Transcriptome analysis revealed 315 differentially expressed genes, which were categorized into several functional groups. The more interesting genes were those involved in the biosynthesis of terpene and ethylene. In particular, the linoleate 13 S-lipoxygenase gene CsLOX2-1 was found to possess the AvrXa7 binding sequence in the promoter region. The recognition of AvrXa7 to the CsLOX2-1 promoter was subsequently confirmed by yeast one-hybrid and electrophoretic mobility shift experiments. This demonstrated that the TALE effector AvrXa7 promotes CsLOX2-1 expression by directly binding to the promoter sequence. Our findings contribute a valuable clue to identifying the potential genes that can be used to prevent citrus canker.

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A cautionary TALE: how plant breeding may have favoured expanded TALE repertoires in Xanthomonas - Mol. Plant Pathol.

A cautionary TALE: how plant breeding may have favoured expanded TALE repertoires in Xanthomonas - Mol. Plant Pathol. | TAL effector science | Scoop.it

Schandry et al, 2018

Xanthomonas oryzae strains overcome recognition by traditional R genes, Xo1 and Xa1, by deploying structural variants of TALEs that seem to interfere with R gene function, possibly by competitively binding the R protein without eliciting an immune response (Zuluaga et al., 2017). It is conceivable that the loss of TALEs in some US strains is a response to the widespread presence of these R genes in cultivated rice in North America. Consequently, these strains avoid recognition and resistance at the expense of a significant loss of virulence.

We believe that these described counter‐defence mechanisms are a testament to the effect of the deployment of R genes on TALome evolution. Indeed, to date, these mechanisms have been found almost exclusively in X. oryzae strains with expanded TALomes (Fig. 1). We propose that the expansion of TALomes is thus a feature that has been selected for in response to plant resistance, as it allows the bacteria to develop counter‐defence strategies and to quickly adapt to new cultivars.

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Engineering altered protein–DNA recognition specificity - Nucl Acids Res.

Engineering altered protein–DNA recognition specificity - Nucl Acids Res. | TAL effector science | Scoop.it

Bogdanove et al, 2018

Protein engineering is used to generate novel protein folds and assemblages, to impart new properties and functions onto existing proteins, and to enhance our understanding of principles that govern protein structure. While such approaches can be employed to reprogram protein–protein interactions, modifying protein–DNA interactions is more difficult. This may be related to the structural features of protein–DNA interfaces, which display more charged groups, directional hydrogen bonds, ordered solvent molecules and counterions than comparable protein interfaces. Nevertheless, progress has been made in the redesign of protein–DNA specificity, much of it driven by the development of engineered enzymes for genome modification. Here, we summarize the creation of novel DNA specificities for zinc finger proteins, meganucleases, TAL effectors, recombinases and restriction endonucleases. The ease of re-engineering each system is related both to the modularity of the protein and the extent to which the proteins have evolved to be capable of readily modifying their recognition specificities in response to natural selection. The development of engineered DNA binding proteins that display an ideal combination of activity, specificity, deliverability, and outcomes is not a fully solved problem, however each of the current platforms offers unique advantages, offset by behaviors and properties requiring further study and development.

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Identification of a cell-penetrating peptide applicable to a protein-based transcription activator-like effector expression system for cell engineering - Biomaterials

Takashina et al, 2018

Here, we identified a cell-penetrating peptide composed of 10 amino acids (RIFIHFRIGC) with nuclear trafficking activity and found that it was significantly more potent than a Tat-derived peptide or polyarginine peptide (R11). We named the peptide “nuclear trafficking peptide” (NTP) and applied it to a protein-based artificial transcription factor (NTP-ATF), which was composed of a transcription activator-like effector and transcription domain (VP64). An NTP-ATF designed to the proximal promoter region of the microRNA-302/367 cluster efficiently induced endogenous RNA expression at an extremely low concentration (0.25 nM), and repetitive treatment of mouse embryonic fibroblasts with NTP-ATF generated induced pluripotent stem-like cells, which gave chimeric mice. Together with the observation that recombinant NTP-ATF protein did not induce any apparent cytotoxicity, we propose that NTP-ATF is a promising system for cellular reprogramming applicable to regenerative medicine.

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Recognition of Epigenetic Nucleobases - Phil. Trans. R. Soc. B

Recognition of Epigenetic Nucleobases - Phil. Trans. R. Soc. B | TAL effector science | Scoop.it

Rathi et al, 2018

The epigenetic DNA nucleobases 5-methylcytosine (5mC) and N4-methylcytosine (4mC) coexist in bacterial genomes and have important functions in host defence and transcription regulation. To better understand the individual biological roles of both methylated nucleobases, analytical strategies for distinguishing unmodified cytosine (C) from 4mC and 5mC are required. Transcription-activator-like effectors (TALEs) are programmable DNA-binding repeat proteins, which can be re-engineered for the direct detection of epigenetic nucleobases in user-defined DNA sequences. We here report the natural, cytosine-binding TALE repeat to not strongly differentiate between 5mC and 4mC. To engineer repeats with selectivity in the context of C, 5mC and 4mC, we developed a homogeneous fluorescence assay and screened a library of size-reduced TALE repeats for binding to all three nucleobases. This provided insights into the requirements of size-reduced TALE repeats for 4mC binding and revealed a single mutant repeat as a selective binder of 4mC. Employment of a TALE with this repeat in affinity enrichment enabled the isolation of a user-defined DNA sequence containing a single 4mC but not C or 5mC from the background of a bacterial genome. Comparative enrichments with TALEs bearing this or the natural C-binding repeat provides an approach for the complete, programmable decoding of all cytosine nucleobases found in bacterial genomes.

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The pepper Bs4C proteins are localized to the ER membrane and confer disease resistance to bacterial blight in transgenic rice - Mol.Plant Pathol.

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Wang et al, 2018

Transcription activator‐like effector (TALE)‐dependent dominant disease resistance (R) genes in plants, also referred to as executor R genes, are induced upon infection by phytopathogenic bacteria of the genus Xanthomonas harbouring the corresponding TALE genes. Unlike the traditional R proteins, the executor R proteins do not determine the resistance specificity and may function broadly in different plant species. The executor R gene Bs4C‐R in the resistant genotype PI 235047 of the pepper species Capsicum pubescens (CpBs4C‐R) confers disease resistance to Xanthomonas campestris pv. vesicatoria (Xcv) harbouring the TALE genes avrBsP/avrBs4. In this study, the synthetic genes of CpBs4C‐R and two other Bs4C‐like genes, the susceptible allele in the genotype PI585270 of C. pubescens (CpBs4C‐S) and the CaBs4C‐R homolog gene in the cultivar “CM334” of Capsicum annum (CaBs4C), were characterized in tobacco (Nicotiana benthamiana) and rice (Oryza sativa). The Bs4C genes induced cell death in N. benthamiana. The functional Bs4C‐eCFP fusion proteins were localized to the ER membrane in the leaf epidermal cells of N. benthamiana. The Xa10 promoter‐Bs4C fusion genes in transgenic rice conferred strain‐specific disease resistance to Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight in rice, and were specifically induced by the Xa10‐incompatible Xoo strain PXO99A(pHM1avrXa10). The results indicated that the Bs4C proteins from the pepper species function broadly in rice and the Bs4C protein‐mediated cell death from the ER is conserved between dicotyledonous and monocotyledonous plants, which can be utilized to engineer novel and enhanced disease resistance in heterologous plants. This article is protected by copyright. All rights reserved.

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Immune modulation in pigs through TALEN-based editing of the RELA locus

Ballantyne 2018

 

Livestock animals are an ancient, vital renewable natural resource. Many livestock
species have the ability to convert inedible crops and waste food into food fit for human
consumption, in the form of meat, eggs and dairy products. As the global demand for
high value animal protein is ever increasing, the livestock market continues to play a
major role in worldwide economics.
Animal disease has the potential to be a huge burden on the livestock industry, impacting both welfare and production. Major outbreaks of transboundary diseases, such as foot and mouth disease, rinderpest and classical swine disease, have resulted in devastating global economic losses. As a result, scientific research is engaged in lowering this impact by generating effective preventative measures and treatments.
One way to reduce livestock disease is to select animals that are genetically
resistant, traditionally carried out through selective animal breeding programs; however, this is a time- consuming process and requires that appropriate genetic variation exists within the population. Advances in genome engineering technologies offer us an alternative approach, with the capability to make genetic improvements in livestock within a single generation. It is hypothesised that resilience to a disease, known as African
swine fever (ASF), could be genetically engineered into the domestic pig.
ASF is a highly contagious disease of domestic pigs and is a re- emerging global threat to the swine industry. It is a lethal haemorrhagic disease caused by a virus, known as the African swine fever virus (ASFV). At present, there is no vaccine or treatment for ASF, and disease control relies on rapid diagnosis, quarantine and the mass slaughter
of animals. Unlike the domestic pig, swine indigenous to Sub-Saharan Africa, such as
the warthog, show no clinical signs of disease following infection with ASFV. A
comparative study was carried out to identify host genetic variation that could underlie
the difference in response to ASFV, with candidate genes selected based on their
potential involvement with the viral protein A238L, involved in immune evasion.
Functional polymorphisms where identified in the porcine RELA gene, encoding
RelA, a subunit of the NF-κB transcription factor family. This evolutionary conserved protein family plays a vital role in mediating inflammatory and immune responses.
The specific RELA polymorphisms identified alter potential phosphorylation sites
within the C-terminal transactivation domain of RelA which have been found to
modulate NF-κB transcriptional activity
in vitro. We set out to investigate whether
genome editing tools could be employed to engineer the RELA sequence of domestic
pigs. Initial attempts targeted the final exon of RELA, producing animals with a
truncatedRelA protein; modified animals lack the final 60 amino acids of the C-terminal transactivation domain. The aim of this thesis was to genotype and characterise the effects of this RELA modification at a molecular, cellular, morphological and whole organism
level. The ultimate goal of this project was to
investigate whether this RELA modification altered the domestic pig’s response to
ASFV in vitro and in vivo. Unlike rela-/
-mice which have an embryonic lethal phenotype, these RELA-edited pigs were born healthy and were fully viable when housed in a typical farm environment. Phenotypic analysis of lymphoid tissues from the RELA-edited pigs demonstrated no significant anatomical or histological changes compared to unmodified counterparts. Pigs homozygous for the RELA mutation had a significantly lower body weight compared to wild-type pigs. Molecular studies of samples from, these pigs have shown that the modified RelA has an altered activity; however, the RELA modified pigs do develop the characteristic disease phenotype when challenged with ASFV. Finally, genome editors have been developed to introduce a specific warthog allele into the domestic pig RELA locus, these editors are currently being
taken forward to produce a novel pig line.
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Zinc Fingers, TALEs, and CRISPR Systems: A Comparison of Tools for Epigenome Editing

Zinc Fingers, TALEs, and CRISPR Systems: A Comparison of Tools for Epigenome Editing | TAL effector science | Scoop.it

Waryah et al, 2018

The completion of genome, epigenome, and transcriptome mapping in multiple cell types has created a demand for precision biomolecular tools that allow researchers to functionally manipulat

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Interaction of Rice and Xanthomonas TAL Effectors - Rice Genomics, Genetics and Breeding

Interaction of Rice and Xanthomonas TAL Effectors - Rice Genomics, Genetics and Breeding | TAL effector science | Scoop.it

Char et al, 2018

Bacterial blight of rice, caused by the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) in rice, represents one of the most well-studied crop diseases and is also well-known as a model for studying host/microbe interaction. TALEs (transcription activator-like effectors), as a group of pathogenesis factors and once translocated into the host cells from pathogen, recognize and activate host genes to condition disease susceptibility and also trigger host resistance responses dependent on the nature of target genes in plants. TALEs and their target genes have become the foci of the molecular battles between Xoo and rice. The continuing battles have led to incredibly diverse virulence mechanisms in pathogen and counteracting defense mechanisms in rice. Extensive efforts have been made to understand the TALE biology, identify host target genes, and elucidate their interaction and resulting physiological relevance to rice blight and other crop diseases. This review aims to summarize how much we have learned about TALEs and their role in bacterial blight of rice, as well as associated susceptibility and resistance genes in the host. The review also intends to provide a prospect of engineering genetic resistance by applying precise genome editing of TALE-associated target genes in rice.

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Reprogramming cell fate with artificial transcription factors - FEBS Letters

Reprogramming cell fate with artificial transcription factors - FEBS Letters | TAL effector science | Scoop.it

Heiderscheidt et al, 2018

Transcription factors (TFs) reprogram cell states by exerting control over gene regulatory networks and the epigenetic landscape of a cell. Artificial transcription factors (ATFs) are designer regulatory proteins comprised of modular units that can be customized to overcome challenges faced by natural TFs in establishing and maintaining desired cell states. Decades of research on DNA-binding proteins and synthetic molecules has provided a molecular toolkit for ATF design and the construction of genome-scale libraries of ATFs capable of phenotypic manipulation and reprogramming of cell states. Here, we compare the unique strengths and limitations of different ATF platforms, highlight the advantages of cooperative assembly, and present the potential of ATF libraries in revealing gene regulatory networks that govern cell fate choices.

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Multiple Chemical Inducible Tal Effectors for Genome Editing and Transcription Activation

Multiple Chemical Inducible Tal Effectors for Genome Editing and Transcription Activation | TAL effector science | Scoop.it

(via T. Lahaye, thx)

Zhao et al, 2018

Inducible modulation is often required for precise investigations and manipulations of dynamic biological processes. Transcription activator-like effectors (TALEs) provide a powerful tool for targeted gene editing and transcriptional programming. We designed a series of chemical inducible systems by coupling TALEs with a mutated human estrogen receptor (ERT2), which renders them 4-hydroxyl-tamoxifen (4-OHT) inducible for access of the genome. Chemical inducible genome editing was achieved via fusing two tandem ERT2 domains to customized transcription activator-like effector nuclease (TALEN), which we termed “Hybrid Inducible Technology” (HIT-TALEN). Those for transcription activation were vigorously optimized using multiple construct designs. Most efficient drug induction for endogenous gene activation was accomplished with minimal background activity using an optimized inducible TALE based SunTag system (HIT-TALE-SunTag). The HIT-SunTag system is rapid, tunable, selective to 4-OHT over an endogenous ligand, and reversible in drug induced transcriptional activation. Versatile systems developed in this study can be easily applied for editing and transcriptional programming of potentially any genomic loci in a tight and effective chemical inducible fashion.

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Robust transcriptional activation in plants using multiplexed CRISPR-Act2.0 and mTALE-Act systems - Mol. Plant

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Lowder et al, 2017

Previously, we have shown transcriptional activation of endogenous genes in plants using dCas9-VP64. Here, we develop a second generation of vector systems for enhanced transcriptional activation in plants. We tested multiple strategies for CRISPR-Cas9 based transcriptional activation. Simultaneous recruitment of VP64 by dCas9 and a modified guide RNA scaffold gRNA2.0 (designated CRISPR-Act2.0) yielded stronger transcriptional activation than our first generation dCas9-VP64 activators. In addition, we have built a multiplex transcription activator-like effector activation (mTALE-Act) system for simultaneous activation of up to four genes in plants. Our results suggest that mTALE-Act is even more effective than CRISPR-Act2.0. Moreover, we explored tissue specific gene activation using positive feedback loops. Interestingly, our study revealed that certain endogenous genes are more amenable than others to transcriptional activation, and tightly regulated genes may cause target gene silencing when perturbed by activation probes. Hence, these new tools may be used to investigate gene regulatory networks and their control mechanisms. Assembly of multiplex CRISPR-Act2.0 and mTALE-Act systems are both based on streamlined and PCR-independent Golden Gate and Gateway cloning strategies. The systems will enable transcriptional activation applications in both dicots and monocots, and the vectors in this new toolbox are publicly available to the research community through Addgene.

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A multiplexable TALE-based binary expression system for in vivo cellular interaction studies - Nature Comm.

A multiplexable TALE-based binary expression system for in vivo cellular interaction studies - Nature Comm. | TAL effector science | Scoop.it

(via T. Schreiber, thx)

Toegel et al, 2017

Binary expression systems have revolutionised genetic research by enabling delivery of loss-of-function and gain-of-function transgenes with precise spatial-temporal resolution in vivo. However, at present, each existing platform relies on a defined exogenous transcription activator capable of binding a unique recognition sequence. Consequently, none of these technologies alone can be used to simultaneously target different tissues or cell types in the same organism. Here, we report a modular system based on programmable transcription activator-like effector (TALE) proteins, which enables parallel expression of multiple transgenes in spatially distinct tissues in vivo. Using endogenous enhancers coupled to TALE drivers, we demonstrate multiplexed orthogonal activation of several transgenes carrying cognate variable activating sequences (VAS) in distinct neighbouring cell types of the Drosophila central nervous system. Since the number of combinatorial TALE–VAS pairs is virtually unlimited, this platform provides an experimental framework for highly complex genetic manipulation studies in vivo.

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TALE - Mediated Inhibition of Replication of Begomoviruses - Int. J. Agr. Biol.

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Khan et al, 2018

During the last decade, unprecedented progress in the field of genome modification has been witnessed with various applications in basic and applied biology. Genome editing with specific DNA binding proteins has shown higher specificity and fidelity. Artificially engineered proteins such as zinc fingers (ZFs), transcription activator-like effectors (TALEs) and clustered regularly interspaced short palindromic repeats (CRISPR) RNA-guided nucleases (e.g., Cas9) have been used to edit genomes of several plants species such as wheat, rice, soybean, potato, tomato, tobacco, Arabidopsis etc. Engineered proteins with nuclease domain, ZFNs, TALENs, CRISPR/Cas9, can be used to induce double-strand breaks (DSB) in the target genomes. In eukaryotic systems, double strand breaks are repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR) based repair mechanisms resulting in knockdown or malfunction of the targeted gene. Begomoviruses are becoming a serious threat to a number of crops in Pakistan. The present study was initiated with the objective to demonstrate suppression of replication of cotton leaf curl virus (CLCuV) using TALE technology. The most conserved DNA sequence of begomoviruses, nonanucleotide, was targeted to achieve a broad-spectrum resistance against CLCuV prevalent in Pakistan. Activity of TALEs for virus suppression was successfully demonstrated in Nicotiana benthamiana by challenging with infectious clones of cotton leaf curl Kokhran virus (CLCuKV). Virus accumulation was determined by qPCR. The plants showed varying degrees of resistance to CLCuKV in three ways; attenuated virus infection, delayed symptoms and lower virus titer. Our results successfully demonstrated the potential of TALE technology for CLCuV suppression and offer a broader genome targeting platform for suppression of other viruses.

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Conformational heterogeneity allows access to DNA in longer Transcription Activator-Like Effector (TALE) arrays. - BioRxiv

Geiger-Schuller et al, 2018

Transcription activator-like effectors (TALEs) bind DNA through an array of tandem 34-residue repeats. Here, we examine the kinetics of DNA binding for a set of TALE arrays with identical repeats of varying length (and repeat number) using single molecule microscopy. Using a new deterministic modeling approach to test kinetic models consistent with data, we find evidence for conformational heterogeneity in both the free- and DNA-bound TALE arrays. We connect these results with previous work demonstrating populations of partly folded TALE states. TALEs forming less than one superhelical turn around DNA access partly folded open states that inhibit DNA binding, whereas TALEs forming more than one turn access partly folded open states that facilitate DNA binding. Overall, we find that increasing repeat number results in significantly slower interconversion between and among DNA-free and DNA-bound states. These findings highlight the role conformational dynamics can play in facilitating the assembly of large complexes.

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Xanthomonas oryzae pv. oryzae TALE proteins recruit OsTFIIAγ1 to compensate for the absence of OsTFIIAγ5 in bacterial blight in rice - Mol Plant Pathol.

Ma et al, 2018

Xanthomonas oryzae pv. oryzae (Xoo), causal agent of bacterial blight (BB) of rice, uses transcription activator‐like effectors (TALEs) to interact with the basal transcription factor gama subunit OsTFIIAγ5 (Xa5) and activates transcription of host genes. However, how OsTFIIAγ1, the other OsTFIIAγ protein, functions in the presence of TALEs remains unclear. In this study, we show that OsTFIIAγ1 plays a compensatory role in the absence of Xa5. The expression of OsTFIIAγ1, which is activated by TALE PthXo7, increased the expression of host genes targeted by avirulent and virulent TALEs. Defective OsTFIIAγ1 rice lines showed reduced expression of the TALE‐targeted susceptibility (S) genes, OsSWEET11 and OsSWEET14, which resulted in increased BB resistance. Selected TALEs (PthXo1, AvrXa7, and AvrXa27) were evaluated for interactions with OsTFIIAγ1, Xa5 and xa5 (naturally‐occurring mutant form of Xa5) using biomolecular fluorescence complementation (BiFC) and microscale thermophoresis (MST). BiFC and MST demonstrated that the three TALEs bind Xa5 and OsTFIIAγ1 with a stronger affinity than xa5. These results provide insight into the complex roles of OsTFIIAγ1 and OsTFIIAγ5 in TALE‐mediated host gene transcription. This article is protected by copyright. All rights reserved.

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Complete, Programmable Decoding of Oxidized 5-Methylcytosine Nucleobases in DNA by Chemoselective Blockage of Universal TALE-Binders - JACS

Complete, Programmable Decoding of Oxidized 5-Methylcytosine Nucleobases in DNA by Chemoselective Blockage of Universal TALE-Binders - JACS | TAL effector science | Scoop.it

Giess et al, 2018

5-methylcytosine (5mC) and its oxidized derivatives are regulatory elements of mammalian genomes involved in development and disease. These nucleobases do not selectively modulate Watson-Crick pairing, preventing their programmable targeting and analysis by traditional hybridiza-tion probes. Transcription-activator-like effectors (TALEs) can be engineered for use as programmable probes with epi-genetic nucleobase selectivity. However, only partial selectivi-ties for oxidized 5mC have been achieved so far, preventing unambiguous target binding. We here overcome this limitation by destroying and re-inducing nucleobase selectivity in TA-LEs via protein engineering and chemoselective nucleobase blocking. We engineer cavities in TALE repeats and identify a cavity that accommodates all eight human DNA nucleobases. We then introduce substituents with varying size, flexibility and branching degree at each oxidized 5mC. Depending on the nucleobase, substituents with distinct properties effectively block TALE-binding and induce full nucleobase selectivity in the universal repeat. Successful transfer to affinity enrichment in a human genome background indicates that this approach now enables the fully selective detection of each oxidized 5mC in complex DNA samples by programmable probes.

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Two ancestral genes shaped the Xanthomonas campestris TAL effector gene repertoire - New Phytol.

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Denance et al, 2018

  • Xanthomonas transcription activator‐like effectors (TALEs) are injected inside plant cells to promote host susceptibility by enhancing transcription of host susceptibility genes. TALE‐encoding (tal) genes were thought to be absent from Brassicaceae‐infecting Xanthomonas campestris (Xc) genomes based on four reference genomic sequences.
  • We discovered tal genes in 26 of 49 Xc strains isolated worldwide and used a combination of single molecule real time (SMRT) and tal amplicon sequencing to yield a near‐complete description of the TALEs found in Xc (Xc TALome).
  • The 53 sequenced tal genes encode 21 distinct DNA binding domains that sort into seven major DNA binding specificities. In silico analysis of the Brassica rapa promoterome identified a repertoire of predicted TALE targets, five of which were experimentally validated using quantitative reverse transcription polymerase chain reaction. The Xc TALome shows multiple signs of DNA rearrangements that probably drove its evolution from two ancestral tal genes. We discovered that Tal12a and Tal15a of Xcc strain Xca5 contribute together in the development of disease symptoms on susceptible B. oleracea var. botrytis cv Clovis.
  • This large and polymorphic repertoire of TALEs opens novel perspectives for elucidating TALE‐mediated susceptibility of Brassicaceae to black rot disease and for understanding the molecular processes underlying TALE evolution.
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Functional characterization of the citrus canker susceptibility gene CsLOB1 - Mol. Plant Pathol.

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Duan et al, 2018

Xanthomonas citri ssp. citri (Xcc) is an important plant‐pathogenic bacterium that causes citrus canker disease worldwide. PthA, a transcriptional activator‐like (TAL) effector, directs the expression of the canker susceptibility gene CsLOB1. Here, we report our recent progress in the functional characterization of CsLOB1. Subcellular localization analysis of CsLOB1 protein in citrus protoplast revealed that CsLOB1 is primarily localized in the nucleus. We showed that CsLOB1 expression driven by dexamethasone (DEX) in CsLOB1‐GR transgenic plants is associated with pustule formation following treatment with DEX. Pustule formation was not observed in DEX‐treated wild‐type plants and in non‐treated CsLOB1‐GR transgenic plants. Water soaking is typically associated with symptoms of citrus canker. Weaker water soaking was observed with pustule formation in CsLOB1‐GR transgenic plants following DEX treatment. When CsLOB1‐GR‐transgenic Duncan grapefruit leaves were inoculated with Xcc306ΔpthA4 and treated with DEX, typical canker symptoms, including hypertrophy, hyperplasia and water soaking symptoms, were observed on DEX‐treated transgenic plant leaves, but not on mock‐treated plants. Twelve citrus genes that are induced by PthA4 are also stimulated by the DEX‐induced expression of CsLOB1. As CsLOB1 acts as a transcriptional factor, we identified putative targets of CsLOB1 via bioinformatic and electrophoretic mobility shift assays. Cs2g20600, which encodes a zinc finger C3HC4‐type RING finger protein, has been identified to be a direct target of CsLOB1. This study advances our understanding of the function of CsLOB1 and the molecular mechanism of how Xcc causes canker symptoms via CsLOB1.

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TALEN-Based Knockout System

TALEN-Based Knockout System | TAL effector science | Scoop.it

Yoshida & Treen, 2018

Targeted mutagenesis of genes-of-interest is a powerful method of addressing the functions of genes. Genome editing techniques, such as transcriptional activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems, have enabled this approach in various organisms because of their ease of use. In the ascidian, Ciona intestinalis, recent studies show that TALEN-based knockout can be applied to establishing both mutant lines and tissue-specific knockout for addressing gene functions. Here, we introduce recent updates to the TALEN toolkit that facilitate detailed functional analysis of genes in ascidians.

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Genome Editing B.C. (Before CRISPR): Lasting Lessons from the “Old Testament” - CRISPR Journal

Urnov 2018

Genome editing with engineered nucleases, a powerful tool for understanding biological function and revealing causality, was built in a joint effort by academia and industry in 1994–2010. Use of CRISPR-Cas9 is the most recent (2013–), and facile, implementation of the resulting editing toolbox. Principles and methods of genome editing from the pre-CRISPR era remain relevant and continue to be useful.

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Functional analysis of African Xanthomonas oryzae pv. oryzae TALomes reveals a new susceptibility gene in bacterial leaf blight of rice - Biorxiv

Functional analysis of African Xanthomonas oryzae pv. oryzae TALomes reveals a new susceptibility gene in bacterial leaf blight of rice - Biorxiv | TAL effector science | Scoop.it

(via T. Schreiber, thx)

Tran et al, 2018

Most Xanthomonas species translocate Transcription Activator-Like (TAL) effectors into plant cells where they function like plant transcription factors via a programmable DNA-binding domain. Characterized strains of rice pathogenic X. oryzae pv. oryzae harbor 9-16 different tal effector genes, but the function of only a few of them has been decoded. Using sequencing of entire genomes, we first performed comparative analyses of the complete repertoires of TAL effectors, herein referred to as TALomes, in three Xoo strains forming an African genetic lineage different from Asian Xoo. A phylogenetic analysis of the three TALomes combined with in silico predictions of TAL effector targets showed that African Xoo TALomes are highly conserved, genetically distant from Asian ones, and closely related to TAL effectors from the bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc). Nine clusters of TAL effectors could be identified among the three TALomes, including three showing higher levels of variation in their repeat variable diresidues (RVDs). Detailed analyses of these groups revealed recombination events as a possible source of variation among TAL effector genes. Next, to address contribution to virulence, nine TAL effector genes from the Malian Xoo strain MAI1 and four allelic variants from the Burkinabe Xoo strain BAI3, thus representing most of the TAL effector diversity in African Xoo strains, were expressed in the TAL effector-deficient X. oryzae strain X11-5A for gain-of-function assays. Inoculation of the susceptible rice variety Azucena lead to the discovery of three TAL effectors promoting virulence, including two TAL effectors previously reported to target the susceptibility (S) gene OsSWEET14 and a novel major virulence contributor, TalB. RNA profiling experiments in rice and in silico prediction of EBEs were carried out to identify candidate targets of TalB, revealing OsTFX1, a bZIP transcription factor previously identified as a bacterial blight S gene, and OsERF#123, which encodes a subgroup IXc AP2/ERF transcription factor. Use of designer TAL effectors demonstrated that induction of either gene resulted in greater susceptibility to strain X11-5A. The induction of OsERF#123 by BAI3Δ1, a talB knockout derivative of BAI3, carrying these designer TAL effectors increased virulence of BAI3Δ1, validating OsERF#123 as a new, bacterial blight S gene.

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Bacterial gene control by DNA looping using engineered dimeric transcription activator like effector (TALE) proteins - Nucl. Acids Res.

Bacterial gene control by DNA looping using engineered dimeric transcription activator like effector (TALE) proteins - Nucl. Acids Res. | TAL effector science | Scoop.it

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Becker et al, 2018

Genetic switches must alternate between states whose probabilities are dependent on regulatory signals. Classical examples of transcriptional control in bacteria depend on repressive DNA loops anchored by proteins whose structures are sensitive to small molecule inducers or co-repressors. We are interested in exploiting these natural principles to engineer artificial switches for transcriptional control of bacterial genes. Here, we implement designed homodimeric DNA looping proteins (‘Transcription Activator-Like Effector Dimers’; TALEDs) for this purpose in living bacteria. Using well-studied FKBP dimerization domains, we build switches that mimic regulatory characteristics of classical Escherichia coli lactose, galactose and tryptophan operon promoters, including induction or co-repression by small molecules. Engineered DNA looping using TALEDs is thus a new approach to tuning gene expression in bacteria. Similar principles may also be applicable for gene control in eukaryotes.

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Pacbio sequencing of copper-tolerant Xanthomonas citri reveals presence of a chimeric plasmid structure and provides insights into reassortment and shuffling of transcription activator-like effecto...

Gochez et al, 2018

Xanthomonas citri, a causal agent of citrus canker, has been a well-studied model system due to recent availability of whole genome sequences of multiple strains from different geographical regions. Major limitations in our understanding of the evolution of pathogenicity factors in X. citri strains sequenced by short-read sequencing methods have been tracking plasmid reshuffling among strains due to inability to accurately assign reads to plasmids, and analyzing repeat regions among strains. X. citri harbors major pathogenicity determinants, including variable DNA-binding repeat region containing Transcription Activator-like Effectors (TALEs) on plasmids. The long-read sequencing method, PacBio, has allowed the ability to obtain complete and accurate sequences of TALEs in xanthomonads. We recently sequenced Xanthomonas citri str. Xc-03-1638-1-1, a copper tolerant A group strain isolated from grapefruit in 2003 from Argentina using PacBio RS II chemistry. We analyzed plasmid profiles, copy number and location of TALEs in complete genome sequences of X. citri strains. We utilized the power of long reads obtained by PacBio sequencing to enable assembly of a complete genome sequence of strain Xc-03-1638-1-1, including sequences of two plasmids, 249 kb (plasmid harboring copper resistance genes) and 99 kb (pathogenicity plasmid containing TALEs). The pathogenicity plasmid in this strain is a hybrid plasmid containing four TALEs. Due to the intriguing nature of this pathogenicity plasmid with Tn3-like transposon association, repetitive elements and multiple putative sites for origins of replication, we might expect alternative structures of this plasmid in nature, illustrating the strong adaptive potential of X. citri strains. Analysis of the pathogenicity plasmid among completely sequenced X. citri strains, coupled with Southern hybridization of the pathogenicity plasmids, revealed clues to rearrangements of plasmids and resulting reshuffling of TALEs among strains. We demonstrate in this study the importance of long-read sequencing for obtaining intact sequences of TALEs and plasmids, as well as for identifying rearrangement events including plasmid reshuffling. Rearrangement events, such as the hybrid plasmid in this case, could be a frequent phenomenon in the evolution of X. citri strains, although so far it is undetected due to the inability to obtain complete plasmid sequences with short-read sequencing methods.

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Frontiers | A Conserved Basal Transcription Factor Is Required for the Function of Diverse TAL Effectors in Multiple Plant Hosts | Plant Science

Frontiers | A Conserved Basal Transcription Factor Is Required for the Function of Diverse TAL Effectors in Multiple Plant Hosts | Plant Science | TAL effector science | Scoop.it

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Huang et al, 2017

Many Xanthomonas bacteria use transcription activator-like effector (TALE) proteins to activate plant disease susceptibility (S) genes, and this activation contributes to disease. We recently reported that rice basal transcription factor IIA gamma subunit, OsTFIIAγ5, is hijacked by TALE-carrying Xanthomonas oryzae infecting the plants. However, whether TFIIAγs are also involved in TALE-carrying Xanthomonas-caused diseases in other plants is unknown. Here, molecular and genetic approaches were used to investigate the role of TFIIAγs in other plants. We found that TFIIAγs are also used by TALE-carrying Xanthomonas to cause disease in other plants. The TALEs of Xanthomonas citri pv. citri (Xcc) causing canker in citrus and Xanthomonas campestris pv. vesicatoria (Xcv) causing bacterial spot in pepper and tomato interacted with corresponding host TFIIAγs as in rice. Transcriptionally suppressing TFIIAγ led to resistance to Xcc in citrus and Xcv in pepper and tomato. The 39th residue of OsTFIIAγ5 and citrus CsTFIIAγ is vital for TALE-dependent induction of plant S genes. As mutated OsTFIIAγ5V39E, CsTFIIAγV39E, pepper CaTFIIAγV39E, and tomato SlTFIIAγV39E also did not interact with TALEs to prevent disease. These results suggest that TALE-carrying bacteria share a common mechanism for infecting plants. Using TFIIAγV39E-type mutation could be a general strategy for improving resistance to TALE-carrying pathogens in crops.

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