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Scooped by
dromius
April 27, 2017 7:42 AM
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(via T. Lahaye, thx) Malzahn et al, 2017 Genome editing promises giant leaps forward in advancing biotechnology, agriculture, and basic research. The process relies on the use of sequence specific nucleases (SSNs) to make DNA double stranded breaks at user defined genomic loci, which are subsequently repaired by two main DNA repair pathways: non-homologous end joining (NHEJ) and homology directed repair (HDR). NHEJ can result in frameshift mutations that often create genetic knockouts. These knockout lines are useful for functional and reverse genetic studies but also have applications in agriculture. HDR has a variety of applications as it can be used for gene replacement, gene stacking, and for creating various fusion proteins. In recent years, transcription activator-like effector nucleases and clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR associated protein 9 or CRISPR from Prevotella and Francisella 1 have emerged as the preferred SSNs for research purposes. Here, we review their applications in plant research, discuss current limitations, and predict future research directions in plant genome editing.
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Scooped by
dromius
April 1, 2017 6:37 AM
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Zuluaga et al, 2017 Plants are constantly challenged by a wide range of pathogens and have therefore evolved an array of mechanisms to defend against them. In response to these defense systems, pathogens have evolved strategies to avoid recognition and suppress plant defenses (Brown & Tellier 2011). Three recent reports dealing with the resistance of rice to Xanthomonas oryzae have added a new twist to our understanding of this fascinating co-evolutionary arms race (Ji et al. 2016; Read et al. 2016 and Triplett et al. 2016). They show that pathogens also develop sophisticated effector mimics to trick recognition.
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Scooped by
dromius
March 24, 2017 6:36 AM
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Douglas 2017 Molecular engineers have become increasingly adept at repurposing life's building blocks to make custom self-assembled shapes. Because a single drop of solution contains billions of such shapes, DNA origami smiley faces ( 1 ), RNA stars ( 2 ), and designer protein polyhedra ( 3 ) may vastly outnumber most other human-made objects on Earth. These shapes lack immediate practical utility, but they transmit a powerful message: Researchers are beginning to understand how molecules self-assemble. On page 1283 of this issue, Praetorius and Dietz make another leap forward by demonstrating a novel class of nanostructures, namely DNA-protein hybrid shapes ( 4 ). This is an important advance because it provides a method to create human-designed shapes out of ingredients that are generally compatible with living systems.
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Scooped by
dromius
March 23, 2017 2:50 PM
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Kaya et al, 2017 Although transcription activator-like effector nucleases (TALENs) are one of the most prevalent SSNs and have a potential to provide higher target specificity by their dimeric property, TALENs are sensitive to methylated cytosines that are present not only in transposons but also in active genes in plants. In mammalian cells, the methylation sensitivity of TALENs could be overcome by using a base-recognition module (N∗) that has a higher affinity to methylated cytosine. In contrast to mammals, plants carry DNA methylation at all cytosine contexts (CG, CHG, and CHH, where H represents A, C, or T) with various degrees and effectiveness of N∗ module in genome editing in plants has not been explored. In this study, we designed sets of TALENs with or without N∗ modules and examined their efficiency in genome editing of methylated regions in rice. Although improvement in genome editing efficiency was observed with N∗-TALENs designed to a stably methylated target, another target carrying cytosines with various levels of methylation showed resistance to both normal and N∗-TALENs. The results suggest that variability of cytosine methylation in target regions is an additional factor affecting the genome editing efficiency of TALENs.
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Scooped by
dromius
March 21, 2017 4:44 AM
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Streubel et al, 2017 Plant-pathogenic Xanthomonas bacteria inject transcription activator-like effector proteins (TALEs) into host cells to specifically induce transcription of plant genes and enhance susceptibility. Although the DNA-binding mode is well-understood it is still ambiguous how TALEs initiate transcription and whether additional promoter elements are needed to support this. To systematically dissect prerequisites for transcriptional initiation the activity of one TALE was compared on different synthetic Bs4 promoter fragments. In addition, a large collection of artificial TALEs spanning the OsSWEET14 promoter was compared. We show that the presence of a TALE alone is not sufficient to initiate transcription suggesting the requirement of additional supporting promoter elements. At the OsSWEET14 promoter TALEs can initiate transcription from various positions, in a synergistic manner of multiple TALEs binding in parallel to the promoter, and even by binding in reverse orientation. TALEs are known to shift the transcriptional start site, but our data show that this shift depends on the individual position of a TALE within a promoter context. Our results implicate that TALEs function like classical enhancer-binding proteins and initiate transcription in both orientations which has consequences for in planta target gene prediction and design of artificial activators.
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Scooped by
dromius
February 10, 2017 3:52 AM
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(via T. Schreiber, thx) Includes Table1: Comparison between TALE-TF and CRISPR0-dCas9 in the use for epigenetic editing Lo et al, 2017 Enzymes involved in epigenetic processes such as methyltransferases or demethylases are becoming highly utilized for their persistent DNA or histone modifying efficacy. Herein, we have developed an optogenetic toolbox fused to the catalytic domain (CD) of DNA-methyltransferase3A (DNMT3A-CD) or Ten-Eleven Dioxygenase-1 (TET1-CD) for loci-specific alteration of the methylation state at the promoter of Ascl1 (Mash1), a candidate proneuron gene. Optogenetical protein pairs, CRY2 linked to DNMT3A-CD or TET1-CD and CIB1 fused to a Transcription Activator-Like Element (TALE) locating an Ascl1 promoter region, were designed for site specific epigenetic editing. A differentially methylated region at the Ascl1 promoter, isolated from murine dorsal root ganglion (hypermethylated) and striated cells (hypomethylated), was targeted with these optogenetic-epigenetic constructs. Optimized blue-light illumination triggered the co-localization of TALE constructs with DNMT3A-CD or TET1-CD fusion proteins at the targeted site of the Ascl1 promoter. We found that this spatiotemporal association of the fusion proteins selectively alters the methylation state and also regulates gene activity. This proof of concept developed herein holds immense promise for the ability to regulate gene activity via epigenetic modulation with spatiotemporal precision.
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Scooped by
dromius
February 2, 2017 3:50 AM
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(via T. Lahaye, thx) Wang et al, 2017 Transcription activator-like effector (TALE) nucleases (TALENs) are increasingly used as a powerful tool for genome editing in a variety of organisms. We have previously cloned the TALE-coding gene avrXa23 from Xanthomonas oryzae pv. oryzae and developed an AvrXa23-based assembly system for designer TALEs or TALENs. Here, we exploit TALENs to induce mutagenesis of the rice ethylene response factor (ERF) transcription factor OsERF922 for testing the gene-editing efficiency of AvrXa23-based TALENs system. A pair of TALENs (T-KJ9/KJ10) was assembled and their nuclease activities were first confirmed in rice protoplast transient assay. The TALENs-expressing construct pT-KJ9/KJ10 was then used for rice transformation. We observed targeting somatic mutagenesis frequency of 15.0% in positive transgenic rice calli and obtained two mutant plants with nucleotide deletion or insertion at the designer target region. Our work demonstrates that the AvrXa23-based TALENs system can be used for site-specific genome editing in rice.
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Scooped by
dromius
January 20, 2017 12:53 PM
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Barak et al, 2016 Multiple species of Xanthomonas cause bacterial spot of tomato (BST) and pepper. We sequenced five Xanthomonas euvesicatoria strains isolated from three continents (Africa, Asia, and South America) to provide a set of representative genomes with temporal and geographic diversity. LMG strains 667, 905, 909, and 933 were pathogenic on tomato and pepper, except LMG 918 which was pathogenic on pepper but elicited a hypersensitive reaction (HR) on tomato. Furthermore, LMG 667, 909, and 918 elicited a HR on Early Cal Wonder 30R containing Bs3. We examined pectolytic activity and starch hydrolysis, two tests which are useful in differentiating X. euvesicatoria from X. perforans, both causal agents of BST. LMG strains 905, 909, 918, and 933 were nonpectolytic while only LMG 918 was amylolytic. These results suggest that these strains are all atypical to both X. euvesicatoria and X. perforans. Sequence analysis of all the publicly available X. euvesicatoria and X. perforans strains comparing seven housekeeping genes identified seven haplotypes with few polymorphisms. Whole genome comparison by average nucleotide identity (ANI) resulted in values of >99% among the LMG strains 667, 905, 909, 918, and 933 and X. euvesicatoria strains and >99.6% among the LMG strains and a subset of X. perforans strains. These results suggest that X. euvesicatoria and X. perforans should be considered a single species. ANI values between strains of X. euvesicatoria, X. perforans, X. allii, X. alfalfa subsp. citrumelonis, X. dieffenbachiae, and a recently described pathogen of rose were >97.8% suggesting these pathogens should be a single species and recognized as X. euvesicatoria as well. Analysis of the newly sequenced X. euvesicatoria strains revealed interesting findings among the type 3 (T3) effectors, relatively ancient stepwise erosion of some T3 effectors, additional X. euvesicatoria-specific T3 effectors among the causal agents of BST, orthologs of avrBs3 and avrBs4, and T3 effectors shared among xanthomonads pathogenic on various hosts. The results from this study support the finding that T3 effector repertoire and host range are fundamental for the study of host – microbe interaction but of little relevance to bacterial speciation.
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Scooped by
dromius
January 19, 2017 7:58 AM
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Kumar Guha et al, 2016 Targeted genome editing has become a powerful genetic tool for studying gene function or for modifying genomes by correcting defective genes or introducing genes. A variety of reagents have been developed in recent years that can generate targeted double-stranded DNA cuts which can be repaired by the error-prone, non-homologous end joining repair system or via the homologous recombination-based double-strand break repair pathway provided a suitable template is available. These genome editing reagents require components for recognizing a specific DNA target site and for DNA-cleavage that generates the double-stranded break. In order to reduce potential toxic effects of genome editing reagents, it might be desirable to control the in vitro or in vivo activity of these reagents by incorporating regulatory switches that can reduce off-target activities and/or allow for these reagents to be turned on or off. This review will outline the various genome editing tools that are currently available and describe the strategies that have so far been employed for regulating these editing reagents. In addition, this review will examine potential regulatory switches/strategies that can be employed in the future in order to provide temporal control for these reagents.
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Scooped by
dromius
January 12, 2017 11:00 AM
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(via T. Schreiber, thx) Hu et al, 2017 Tracking the dynamics of genomic loci is important for understanding the mechanisms of fundamental intracellular processes. However, fluorescent labeling and imaging of such loci in live cells have been challenging. One of the major reasons is the low signal-to-background ratio (SBR) of images mainly caused by the background fluorescence from diffuse full-length fluorescent proteins (FPs) in the living nucleus, hampering the application of live cell genomic labeling methods. Here, combining bimolecular fluorescence complementation (BiFC) and transcription activator-like effector (TALE) technologies, we developed a novel method for labeling genomic loci (BiFC-TALE), which largely reduces the background fluorescence level. Using BiFC-TALE, we demonstrated a significantly improved SBR by imaging telomeres and centromeres in living cells in comparison with the methods using full-length FP.
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Scooped by
dromius
January 3, 2017 3:41 AM
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Subburaj et al, 2016 Improving crops through plant breeding, an important approach for sustainable agriculture, has been utilized to increase the yield and quality of foods and other biomaterials for human use. Crops, including cereals, vegetables, ornamental flowers, fruits, and trees, have long been cultivated to produce high-quality products for human consumption. Conventional breeding technologies, such as natural cross-hybridization, mutation induction through physical or chemical mutagenesis, and modern transgenic tools are often used to enhance crop production. However, these breeding methods are sometimes laborious and complicated, especially when attempting to improve desired traits without inducing pleiotropic effects. Recently, targeted genome editing (TGE) technology using engineered nucleases, including meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR) nucleases, has been used to improve the traits of economically important plants. TGE has emerged as a novel plant-breeding tool that represents an alternative approach to classical breeding, but with higher mutagenic efficiency. Here, we briefly describe the basic principles of TGE and the types of engineered nucleases utilized, along with their advantages and disadvantages. We also discuss their potential use to improve the traits of horticultural crops through genome engineering.
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Scooped by
dromius
December 13, 2016 2:33 AM
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Tsuji et al, 2016 A 5mC-selective TALE-repeat was created by screening a TALE repeat library containing randomized amino acids at repeat variable diresidues and their neighboring residues. The new repeat showed high 5mC discrimination ability. An artificial TALE containing the new repeat activated an endogenous gene in a genomic methylation status-dependent manner.
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Scooped by
dromius
December 12, 2016 3:33 AM
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Geiger-Schuller et al, 2016 Transcription activator-like effector proteins (TALEs) contain large numbers of repeats that bind double-stranded DNA, wrapping around DNA to form a continuous superhelix. Since unbound TALEs retain superhelical structure, it seems likely that DNA binding requires a significant structural distortion or partial unfolding. In this study, we use nearest-neighbor “Ising” analysis of consensus TALE (cTALE) repeat unfolding to quantify intrinsic folding free energies, coupling energies between repeats, and the free energy distribution of partly unfolded states, and to determine how those energies depend on the sequence that determines DNA-specificity (called the “RVD”). We find a moderate level of cooperativity for both the HD and NS RVD sequences (stabilizing interfaces combined with unstable repeats), as has been seen in other linear repeat proteins. Surprisingly, RVD sequence identity influences both the overall stability and the balance of intrinsic repeat stability and interfacial coupling energy. Using parameters from the Ising analysis, we have analyzed the distribution of partly folded states as a function of cTALE length and RVD sequence. We find partly unfolded states where one or more repeats are unfolded to be energetically accessible. Mixing repeats with different RVD sequences increases the population of partially folded states. Local folding free energies plateau for central repeats, suggesting that TALEs access partially folded states where a single internal repeat is unfolded while adjacent repeats remain folded. This breakage should allow TALEs to access superhelically-broken states, and may facilitate DNA binding.
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Scooped by
dromius
April 6, 2017 3:06 AM
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(via P. Carella, thx) Kopischke et al, 2017 The liverwort Marchantia polymorpha occupies a crucial position in land plant evolution and provides the opportunity to investigate adaptations to a terrestrial plant life style. Marchantia reverse genetic analyses have thus far been conducted by employing a homologous recombination approach, which yields an efficiency of around 3%. Availability of the characterized and suitable endogenous MpEF1α promoter prompted us to establish the TALEN gene targeting technique for Marchantia. Here, two different TALEN techniques, using custom and self-assembled TALEN constructs, were applied and compared. The MpNOP1 gene was selected as a candidate gene, as the respective knockout mutant has been shown to lack air chamber formation, representing an easily traceable phenotype. We demonstrate that both TALEN approaches are successful in Marchantia yielding high gene targeting efficiencies of over 20%. Investigation of selected G1 up to G4 generations proved the stability of the knockout mutants. In 392 analyzed T1 plants, no additional phenotypes were observed and only one chimeric knockout plant was detected after an extended cultivation period. Interestingly, two out of the 24 sequenced mutants harbored indels causing in-frame mutations and revealed novel Mpnop1-related phenotypes. This demonstrates the potential to detect crucial amino acids and motives of targeted proteins, which is of special interest for essential genes where full knockouts are lethal. The FastTALE™ TALEN assembly kit enables the rapid assembly and ligation of the TALEN arms within half a day. For transformations, custom and assembled constructs were subcloned into Marchantia binary vectors possessing the MpEF1α promoter. Considering time, costs and practicability, the assembly TALEN approach represents a rapid and highly efficient gene targeting system to generate Marchantia knockout mutants, which can be further adapted for future advanced genome-editing applications.
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Scooped by
dromius
March 28, 2017 4:13 AM
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Vikal & Bhatia, 2017 Rice is an important food crop for half the world’s population and has been in cultivation for over 10,000 years. During the last few decades, rice has evolved intricate relationships with associated pathogens and pests, bacterial blight (BB) being one of the most important among them. Utilization of resistant varieties with agricultural management practices is a more effective way to control BB. Of the 42 different resistance (R) genes identified to confer BB resistance, 9 have been isolated and cloned, whereas a few of the avirulence genes and a large number of candidate pathogenicity genes have been isolated from Xanthomonas oryzae pv. oryzae. The complete genome sequences of two different rice subspecies japonica and indica and three different races of BB pathogen are available. Therefore, the interaction between rice-Xoo could be deciphered and pave a way to study the molecular aspects of bacterial pathogenesis and host counter measures like innate immunity and R gene–mediated immunity. Although several of the type III effectors of Xoo have been characterized and the host targets of a few of them identified, a relatively large number of candidate effectors remain to be studied and their functional analysis may provide key for developing broad spectrum and durable resistance to BB.
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Scooped by
dromius
March 24, 2017 6:34 AM
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(via T. Lahaye and R. Koebnik, thx) A wide variety of DNA nanostructures have been assembled by folding long DNA single strands with short DNA staples. However, such structures typically need annealing at elevated temperatures in order to form. To accommodate the formation of such structures in living cells, Praetorius and Dietz developed an approach in which custom protein staples based on transcription activator–like effector proteins fold double-stranded DNA templates (see the Perspective by Douglas). The structures folded into user-defined geometric shapes on the scale of tens to hundreds of nanometers. These nanostructures could self-assemble at room temperature in physiological buffers.
Science , this issue p. [eaam5488][1]; see also p. [1261][2]
[1]: /lookup/doi/10.1126/science.aam5488
[2]: /lookup/doi/10.1126/science.aam9541
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Scooped by
dromius
March 21, 2017 5:54 AM
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(via T. Lahaye, thx) Wang et al, 2017 Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae, is one of the most destructive bacterial diseases throughout the major rice growing regions in the world. The rice disease resistance (R) genes Xa10 confers race-specific disease resistance to X. oryzae pv. oryzae strains that deliver the corresponding transcription activator-like (TAL) effectors AvrXa10. Upon bacterial infection, AvrXa10 binds specifically to the effector binding element (EBE) in the promoter of the R gene and activates its expression. Xa10 encodes an executor R protein that triggers hypersensitive response and activates disease resistance. Rice cultivar Nipponbare carries two Xa10-like genes in its genome, of which one is the susceptible allele of the Xa23 gene, a Xa10-like TAL effector-dependent executor R gene isolated recently from rice cultivar CBB23. However, the function of the two Xa10-like genes in disease resistance to X. oryzae pv. oryzae strains has not been investigated. Here we designated the two Xa10-like genes as Xa10-Ni and Xa23-Ni and characterized their function for disease resistance to rice bacterial blight. Both Xa10-Ni and Xa23-Ni provided disease resistance to X. oryzae pv. oryzae strains that deliver the matching artificially designed TAL effectors (dTALEs). Transgenic rice plants containing Xa10-Ni and Xa23-Ni under the Xa10 promoter provided specific disease resistance to X. oryzae pv. oryzae strains that deliver AvrXa10. Xa10-Ni and Xa23-Ni knock-out mutants abolished dTALE-dependent disease resistance to X. oryzae pv. oryzae. Heterologous expression of Xa10-Ni and Xa23-Ni in Nicotiana benthamiana triggered cell death. The 19-amino acid residues at the N-terminal regions of XA10 or XA10-Ni are dispensable for their function in inducing cell death in N. benthamiana and the C-terminal regions of XA10, XA10-Ni and XA23-Ni are interchangeable among each other without affecting their function. Like XA10, both XA10-Ni and XA23-Ni locate to the endoplasmic reticulum (ER) membrane, show self-interaction and induce ER Ca2+ depletion in leaf cells of N. benthamiana. The results indicate that Xa10-Ni and Xa23-Ni in Nipponbare encode functional executor R proteins, which induce cell death in both monocotyledonous and dicotyledonous plants and have potential of being engineered to provide broad-spectrum disease resistance to plant pathogenic Xanthomonas species.
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Scooped by
dromius
February 25, 2017 5:47 AM
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Serif et al, 2017
• TALEN is an effective tool for genome-editing in the diatom P. tricornutum.
• Guidelines for target site design and the screening process have been established.
• 21% of the obtained transformants were found to be bi-allelic knockout lines.
• Transformants were found to be genetically homogenous.
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Scooped by
dromius
February 4, 2017 4:10 AM
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Ren et al, 2017 Visualization of specific genomic loci in live cells is a prerequisite for the investigation of dynamic changes in chromatin architecture during diverse biological processes, such as cellular aging. However, current precision genomic imaging methods are hampered by the lack of fluorescent probes with high specificity and signal-to-noise contrast. We find that conventional transcription activator-like effectors (TALEs) tend to form protein aggregates, thereby compromising their performance in imaging applications. Through screening, we found that fusing thioredoxin with TALEs prevented aggregate formation, unlocking the full power of TALE-based genomic imaging. Using thioredoxin-fused TALEs (TTALEs), we achieved high-quality imaging at various genomic loci and observed aging-associated (epi) genomic alterations at telomeres and centromeres in human and mouse premature aging models. Importantly, we identified attrition of ribosomal DNA repeats as a molecular marker for human aging. Our study establishes a simple and robust imaging method for precisely monitoring chromatin dynamics in vitro and in vivo.
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Scooped by
dromius
January 27, 2017 2:56 AM
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(via T. Lahaye, thx) Zhao et al, 2017 Transcription-Activator Like (TAL) effectors, delivered by Xanthomonas pathogens bind specifically to UP-regulated by TAL effectors (UPT) box of the host gene promoter to arouse disease or trigger defense response. This type of protein-DNA interaction model has been applied in site-directed genome editing. However, the off-target effects of TAL have severely hindered the development of this promising technology. To better exploit the specific interaction and to deeper understand the TAL-induced host transcription rewiring, the binding between the central repeat region (CRR) of the TAL effector AvrXa27 and its UPT box variants was studied by kinetics analysis and TAL-blocked helicase unwinding assay. The results revealed that while AvrXa27 exhibited the highest affinity to the wild type UPT box, it could also bind to mutated UPT box variants, implying the possibility of non-specific interaction. Furthermore, some of these non-specific combinations restricted the helicase-elicited double-stranded DNA (dsDNA) separation to a greater extent. Our findings provide new insight into the mechanism of TAL transcriptional activation and are beneficial to TAL-mediated genome modification.
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Scooped by
dromius
January 20, 2017 12:50 PM
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Sun et al, 2016 Genome editing technologies enable precise modifications of DNA sequences in vivo and offer a great promise for harnessing plant genes in crop improvement. The precise manipulation of plant genomes relies on the induction of DNA double-strand breaks (DSBs) by sequence-specific nucleases (SSNs) to initiate DNA repair reactions that are based on either non-homologous end joining (NHEJ) or homology-directed repair (HDR). While complete knock-outs and loss-of-function mutations generated by NHEJ are very valuable in defining gene functions, their applications in crop improvement are somewhat limited because many agriculturally important traits are conferred by random point mutations or indels at specific loci in either the genes’ encoding or promoter regions. Therefore, genome modification through SSNs-mediated HDR for gene targeting (GT) that enables either gene replacement or knock-in will provide an unprecedented ability to facilitate plant breeding by allowing introduction of precise point mutations and new gene functions, or integration of foreign genes at specific and desired ‘safe’ harbor in a predefined manner. The emergence of three programmable SSNs such as zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems has revolutionized genome modification in plants in a more controlled manner. However, while targeted mutagenesis is becoming routine in plants, the potential of GT technology has not been well realized for traits improvement in crops, mainly due to the fact that NHEJ predominates DNA repair process in somatic cells and competes with the HDR pathway, and thus HDR-mediated GT is a relative rare event in plants. Here, we review recent research findings mainly focusing on development and applications of precise GT in plants using three SSNs systems described above, and the potential mechanisms underlying HDR events in plant cells. We then address the challenges and propose future perspectives in order to facilitate the implementation of precise genome modification through SSNs-mediated GT for crop improvement in a global context.
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Scooped by
dromius
January 18, 2017 4:28 PM
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Schwartz et al. 2017 check out the watersoaking video: http://movie-usa.glencoesoftware.com/video/10.1073/pnas.1620407114/video-1 AvrHah1 [avirulence (avr) gene homologous to avrBs3 and hax2, no. 1] is a transcription activator-like (TAL) effector (TALE) in Xanthomonas gardneri that induces water-soaked disease lesions on fruits and leaves during bacterial spot of tomato. We observe that water from outside the leaf is drawn into the apoplast in X. gardneri-infected, but not X. gardneriΔavrHah1 (XgΔavrHah1)-infected, plants, conferring a dark, water-soaked appearance. The pull of water can facilitate entry of additional bacterial cells into the apoplast. Comparing the transcriptomes of tomato infected with X. gardneri vs. XgΔavrHah1 revealed the differential up-regulation of two basic helix–loop–helix (bHLH) transcription factors with predicted effector binding elements (EBEs) for AvrHah1. We mined our RNA-sequencing data for differentially up-regulated genes that could be direct targets of the bHLH transcription factors and therefore indirect targets of AvrHah1. We show that two pectin modification genes, a pectate lyase and pectinesterase, are targets of both bHLH transcription factors. Designer TALEs (dTALEs) for the bHLH transcription factors and the pectate lyase, but not for the pectinesterase, complement water soaking when delivered by XgΔavrHah1. By perturbing transcriptional networks and/or modifying the plant cell wall, AvrHah1 may promote water uptake to enhance tissue damage and eventual bacterial egression from the apoplast to the leaf surface. Understanding how disease symptoms develop may be a useful tool for improving the tolerance of crops from damaging disease lesions.
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Scooped by
dromius
January 4, 2017 5:36 AM
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(via T. Schreiber, thx) Crocker & Stern 2017 Regions of genomic DNA called enhancers encode binding sites for transcription factor proteins. Binding of activators and repressors increase and reduce transcription, respectively, but it is not understood how combinations of activators and repressors generate precise patterns of transcription during development. Here, we explore this problem using a fully synthetic transcriptional platform in Drosophila consisting of engineered transcription factor gradients and artificial enhancers. We found that binding sites for a transcription factor that makes DNA accessible are required together with binding sites for transcriptional activators to produce a functional enhancer. Only in this context can changes in the number of activator binding sites mediate quantitative control of transcription. Using an engineered transcriptional repressor gradient, we demonstrate that overlapping repressor and activator binding sites provide more robust repression and sharper expression boundaries than non-overlapping sites. This may explain why this common motif is observed in many developmental enhancers.
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Scooped by
dromius
December 31, 2016 9:07 AM
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(via T. Lahaye, thx) Lebar & Jerala, 2016 Transcriptional activator-like effector (TALE)- and CRISPR/Cas9-based designable recognition domains represent a technological breakthrough not only for genome editing but also for building designed genetic circuits. Both platforms are able to target rarely occurring DNA segments, even within complex genomes. TALE and dCas9 domains, genetically fused to transcriptional regulatory domains, can be used for the construction of engineered logic circuits. Here we benchmarked the performance of the two platforms, targeting the same DNA sequences, to compare their advantages for the construction of designed circuits in mammalian cells. Optimal targeting strands for repression and activation of dCas9-based designed transcription factors were identified; both platforms exhibited good orthogonality and were used to construct functionally complete NOR gates. Although the CRISPR/dCas9 system is clearly easier to construct, TALE-based activators were significantly stronger, and the TALE-based platform performed better, especially for the construction of layered circuits.
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Scooped by
dromius
December 13, 2016 2:31 AM
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Gurushidze et al, 2016 In this chapter, a detailed protocol for the induction of targeted mutations in barley using customized transcription activator-like effector nucleases (TALENs) is provided. As explant for the introduction of TALEN expression constructs, embryogenic pollen cultures are used which consist primarily of haploid cells able to be converted via pollen embryogenesis and genome duplication into fertile doubled haploid plants. Thanks to the haploid nature of these target cells, a mutation induced by TALE nucleases in just one gfp allele can be readily detected in vitro and thus genetically fixed primary mutant plants obtained in one generation.
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