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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Mitochondrial DNA editing in mice with DddA-TALE fusion deaminases - Nature Communications

Mitochondrial DNA editing in mice with DddA-TALE fusion deaminases - Nature Communications | TAL effector science | Scoop.it

Lee et al, 2021
DddA-derived cytosine base editors (DdCBEs), composed of the split interbacterial toxin DddAtox, transcription activator-like effector (TALE), and uracil glycosylase inhibitor (UGI), enable targeted C-to-T base conversions in mitochondrial DNA (mtDNA). Here, we demonstrate highly efficient mtDNA editing in mouse embryos using custom-designed DdCBEs. We target the mitochondrial gene, MT-ND5 (ND5), which encodes a subunit of NADH dehydrogenase that catalyzes NADH dehydration and electron transfer to ubiquinone, to obtain several mtDNA mutations, including m.G12918A associated with human mitochondrial diseases and m.C12336T that incorporates a premature stop codon, creating mitochondrial disease models in mice and demonstrating a potential for the treatment of mitochondrial disorders.

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Simple and efficient custom transcription activator-like effector gene synthesis via twin primer assembly. - BioTechniques

Simple and efficient custom transcription activator-like effector gene synthesis via twin primer assembly. - BioTechniques | TAL effector science | Scoop.it

Wang et al., 2021

Transcription activator-like effector (TALE) nucleases (TALENs) efficiently recognize and cleave DNA in a sequence-dependent manner. However, current TALE custom synthesis methods are either complicated or expensive. Here we report a simple and low-cost method for TALE construct assembly. This method utilizes the denaturation/reannealing nature of double-stranded DNA to create a unique single-stranded DNA overhang for proper ordering of TALEmonomers in an engineered multimer.We successfully synthesized two TALEN pairs targeting the endogenous TET1
locus in human embryonic kidney cells and demonstrated their editing efficiency. Our method provides an alternative simple, low-cost method for effective TALEN assembly, which may improve the application of TALE-based technology.

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MitoTALENs for mtDNA editing - ScienceDirect

MitoTALENs for mtDNA editing - ScienceDirect | TAL effector science | Scoop.it

Bacman & Moraes 2020
The mixture of mutant and wild-type mitochondrial DNA (mtDNA) in cells from patients with mitochondrial diseases provides an opportunity to develop therapies by selectively eliminating the mutant fraction. Our laboratory has adapted TALENs, a gene editing platform, to specifically cleave mutant mtDNA. Ex vivo and in vivo experiments have provided proof-of-principle that the approach works in changing mtDNA heteroplasmy toward the wild-type mtDNA.

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EVOLUTION OF TALENS - US Patent Application 20200277587

Liu, Hubbard, Badran 2020, US Patent application

Engineered transcriptional activator-like effectors (TALEs) are versatile tools for genome manipulation with applications in research and clinical contexts. One current drawback of TALEs is that the 5′ nucleotide of the target is specific for thymine (T). TALE domains with alternative 5′ nucleotide specificities could expand the scope of DNA target sequences that can be bound by TALEs. Another drawback of TALEs is their tendency to bind and cleave off-target sequence, which hampers their clinical application and renders applications requiring high-fidelity binding unfeasible. This disclosure provides methods and strategies for the continuous evolution of proteins comprising DNA-binding domains, e.g., TALE domains. In some aspects, this disclosure provides methods and strategies for evolving such proteins under positive selection for a desired DNA-binding activity and/or under negative selection against one or more undesired (e.g., off-target) DNA-binding activities. Some aspects of this disclosure provide engineered TALE domains and TALEs comprising such engineered domains, e.g., TALE nucleases (TALENs), TALE transcriptional activators, TALE transcriptional repressors, and TALE epigenetic modification enzymes, with altered 5′ nucleotide specificities of target sequences. Engineered TALEs that target ATM with greater specificity are also provided.

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One-Day TALEN Assembly Protocol and a Dual-Tagging System for Genome Editing - ACS Omega

One-Day TALEN Assembly Protocol and a Dual-Tagging System for Genome Editing - ACS Omega | TAL effector science | Scoop.it

(via T. Schreiber, thx)

Zhang et al, 2020

This study developed a new rapid transcription activator-like effector nuclease (TALEN) preparation protocol by thoroughly redesigning the widely used Golden Gate TALEN and TAL Effector Kit 2.0. The new protocol can be used to prepare any custom 18-bp binding TALENs in just one day (about 12 h), more rapidly than CRISPR. This protocol used a set of linear monomers, a final TALE-FokI backbone plasmid, and a pipeline to assemble the ready-to-use TALEN expression plasmid, which were all newly developed for this study. The set of linear monomers can be easily produced and reproduced by high-fidelity polymerase chain reaction (PCR) amplification in a 96-well plate using a pair of universal primers. Most important of all, our rapid TALEN construction pipeline can easily obtain many positive colonies with high efficiency (over 80%). By preparing five pairs of TALENs targeting five NF-κB genes (RELA, RELB, CREL,NFKB1, and NFKB2) and editing these genes in different cell lines (293T, HepG2, and PANC1), this study demonstrated that the new protocol has high efficiency, reproducibility, reliability, and applicability. Moreover, this study showed that the fabricated TALEN has much higher editing efficiency than CRISPR. Finally, this study developed a dual-tagging system for simultaneously tagging target proteins and successfully edited cells with a streptavidin-binding peptide (SBP) or AviTag via homology-directed repair, which could have wide applications in protein (antigen) preparation, immunoprecipitation, and a transcription factor chromatin immunoprecipitation assay.

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New Gene Editing Tool (with old TALEN domains ;)) Corrects Mutations in Mitochondrial DNA

New Gene Editing Tool (with old TALEN domains ;)) Corrects Mutations in Mitochondrial DNA | TAL effector science | Scoop.it
An enzyme pulled from toxic bacteria can enter the organelle and perform single-nucleotide DNA swaps.
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Targeted genome editing in tetraploid potato through transient TALEN expression by Agrobacterium infection - Plant Biotech.

(via T. Schreiber, thx)

Yasumoto et al, 2020

Genome editing using site-specific nucleases, such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat–CRISPR-associated protein 9 (CRISPR-Cas9), is a powerful technology for crop breeding. For plant genome editing, the genome-editing reagents are usually expressed in plant cells from stably integrated transgenes within the genome. This requires crossing processes to remove foreign nucleotides from the genome to generate null segregants. However, in highly heterozygous plants such as potato, the progeny lines have different agronomic traits from the parent cultivar and do not necessarily become elite lines. Agrobacteria can transfer exogenous genes on T-DNA into plant cells. This has been used both to transform plants stably and to express the genes transiently in plant cells. Here, we infected potato, with Agrobacterium tumefaciens harboring TALEN-expression vector targeting sterol side chain reductase 2 (SSR2) gene and regenerated shoots without selection. We obtained regenerated lines with disrupted-SSR2 gene and without transgene of the TALEN gene, revealing that their disruption should be caused by transient gene expression. The strategy using transient gene expression by Agrobacterium that we call Agrobacterial mutagenesis, developed here should accelerate the use of genome-editing technology to modify heterozygous plant genomes.

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Modification of Paraburkholderia TALE-Like Protein to Activate Transcription of Target Genes in Plant - Biosci. Biotechnol. Res. Asia

Atef et al, 2020

The technology of transcription activator-like effector (TALE) is based on designing and synthesizing DNA binding protein as a molecular tool for specific targeting of any desired gene. TALE-like proteins were originally identified in bacteria including two plant pathogenic bacteria and one fungal symbiotic bacteria (namely Paraburkholderia rhizoxinica). Plant transcription activator protein can be customly designed based on the information of the original Paraburkholderia TALE-like protein (BAT). In the present study, we designed a new protein in order to reorder the repeat domain to specifically target the minimal BS3 promoter. This modified BAT sequence was fused to nuclear localization signal (NLS) at the 5` end and Herpes simplex virus VP16 activation domain at the 3` end, then designed protein sequence was reverse translated, de novo synthesized and cloned in a plant expression system. Nicotiana benthamiana plants were co-transformed via agro-infiltration method with the expression cassette with the modified BAT sequence to target the effector binding element (EBE) of the minimal BS3 promoter that drives GUS gene of the mBS3::GUS cassette. Two days post inoculation, plant leaves were GUS-stained and strong signal was detected as indicator for GUS gene activation by the modified BAT sequence. This data suggests that the synthesized BAT protein could be used as a custom transcription activator of any desired genes in plant.

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Xanthomonas diversity, virulence and plant–pathogen interactions - Nature Rev. Microbiol.

Xanthomonas diversity, virulence and plant–pathogen interactions - Nature Rev. Microbiol. | TAL effector science | Scoop.it

(via. T. Schreiber, thx)

Timilsina et al, 2020

Xanthomonas spp. encompass a wide range of plant pathogens that use numerous virulence factors for pathogenicity and fitness in plant hosts. In this Review, we examine recent insights into host–pathogen co-evolution, diversity in Xanthomonas populations and host specificity of Xanthomonas spp. that have substantially improved our fundamental understanding of pathogen biology. We emphasize the virulence factors in xanthomonads, such as type III secreted effectors including transcription activator-like effectors, type II secretion systems, diversity resulting in host specificity, evolution of emerging strains, activation of susceptibility genes and strategies of host evasion. We summarize the genomic diversity in several Xanthomonas spp. and implications for disease outbreaks, management strategies and breeding for disease resistance. In this Review, Jones and colleagues describe the extremely diverse Xanthomonas spp. and how these plant pathogens use their extensive repertoire of effectors for virulence and immune evasion. Understanding these prototypical plant pathogens paves the way to combat disease.

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Identification of a virulence tal gene in the cotton pathogen, Xanthomonas citri pv. malvacearum strain Xss-V 2 –18 - BMC Microbiology

Identification of a virulence tal gene in the cotton pathogen, Xanthomonas citri pv. malvacearum strain Xss-V 2 –18 - BMC Microbiology | TAL effector science | Scoop.it

(via T. Schreiber, thx)

Haq et al, 2020

Bacterial blight of cotton (BBC), which is caused by the bacterium Xanthomonas citri pv. malvacearum (Xcm), is a destructive disease in cotton. Transcription activator-like effectors (TALEs), encoded by tal-genes, play critical roles in the pathogenesis of xanthomonads. Characterized strains of cotton pathogenic Xcm harbor 8–12 different tal genes and only one of them is functionally decoded. Further identification of novel tal genes in Xcm strains with virulence contributions are prerequisite to decipher the Xcm-cotton interactions.

In this study, we identified six tal genes in Xss-V2–18, a highly-virulent strain of Xcm from China, and assessed their role in BBC. RFLP-based Southern hybridization assays indicated that Xss-V2–18 harbors the six tal genes on a plasmid. The plasmid-encoded tal genes were isolated by cloning BamHI fragments and screening clones by colony hybridization. The tal genes were sequenced by inserting a Tn5 transposon in the DNA encoding the central repeat region (CRR) of each tal gene. Xcm TALome evolutionary relationship based on TALEs CRR revealed relatedness of Xss-V2–18 to MSCT1 and MS14003 from the United States. However, Tal2 of Xss-V2–18 differs at two repeat variable diresidues (RVDs) from Tal6 and Tal26 in MSCT1 and MS14003, respectively, inferred functional dissimilarity. The suicide vector pKMS1 was then used to construct tal deletion mutants in Xcm Xss-V2–18. The mutants were evaluated for pathogenicity in cotton based on symptomology and growth in planta. Four mutants showed attenuated virulence and all contained mutations in tal2. One tal2 mutant designated M2 was further investigated in complementation assays. When tal2 was introduced into Xcm M2 and expressed in trans, the mutant was complemented for both symptoms and growth in planta, thus indicating that tal2 functions as a virulence factor in Xcm Xss-V2–18.

Overall, the results demonstrated that Tal2 is a major pathogenicity factor in Xcm strain Xss-V2–18 that contributes significantly in BBC. This study provides a foundation for future efforts aimed at identifying susceptibility genes in cotton that are targeted by Tal2.

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A TAL effector-like protein of symbiotic Mycetohabitans (Burkholderia rhizoxinica) increases stress tolerance and alters the transcriptome of the fungal host Rhizopus microsporus - bioRxiv

A TAL effector-like protein of symbiotic Mycetohabitans (Burkholderia rhizoxinica) increases stress tolerance and alters the transcriptome of the fungal host Rhizopus microsporus - bioRxiv | TAL effector science | Scoop.it

(Via T. Lahaye, thx)

Carter et al, 2020, Preprint

Symbioses of bacteria with fungi have only recently been described and are poorly understood. In the symbiosis of Mycetohabitans (formerly Burkholderia ) rhizoxinica with the fungus Rhizopus microsporus, bacterial type III (T3) secretion is known to be essential. Proteins resembling T3-secreted transcription activator-like (TAL) effectors of plant pathogenic bacteria are encoded in the three sequenced Mycetohabitans spp. genomes. TAL effectors nuclear localize in plants, where they bind and activate genes important in disease. The Burkholderia TAL-like (Btl) proteins bind DNA but lack the N- and C-terminal regions in which TAL effectors harbor their T3 and nuclear localization signals, and activation domain. We characterized a Btl protein, Btl19-13, and found that, despite the structural differences, it can be T3-secreted and can nuclear localize. A btl19-13 gene knockout did not prevent the bacterium from infecting the fungus, but the fungus became less tolerant to cell membrane stress. Btl19-13 did not alter transcription in a plant-based reporter assay, but 15 R. microsporus genes were differentially expressed in comparisons both of the fungus infected with the wildtype bacterium vs the mutant and with the mutant vs. a complemented strain. Southern blotting revealed btl genes in 14 diverse Mycetohabitans isolates. However, banding patterns and available sequences suggest variation, and the btl19-13 phenotype could not be rescued by a btl gene from a different strain. Our findings support the conclusion that Btl proteins are effectors that act on host DNA and play important but varied or possibly host-genotype-specific roles in the M. rhizoxinica-R. microsporus symbiosis.

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Extensive genomic rearrangements along with distinct mobilome and TALome is associated with extreme pathotypes of a rice pathogen - Genome Biol Evolution

Extensive genomic rearrangements along with distinct mobilome and TALome is associated with extreme pathotypes of a rice pathogen - Genome Biol Evolution | TAL effector science | Scoop.it

(via. T Schreiber, thx)

Kaur et al, 2020

Xanthomonas oryzae pv. oryzae (Xoo) is a serious pathogen of rice which displays tremendous inter-strain variation. The emergence of highly virulent strains of Xoo is a major threat to rice cultivation. Evolutionary insights into genome dynamics of highly virulent strains as compared to the less virulent ones are crucial for understanding the molecular basis of exceptional success of Xoo as a highly evolved plant pathogen. In the present study, we report a complete genome sequence of Xoo strains with extreme virulent pathotypes (XVPs) characterized based on their reaction towards ten resistance (Xa) genes. One strain, IXO1088 can overcome resistance mediated by all the ten resistance genes while the other strain IXO704 cannot overcome any of them. Interestingly, our investigation revealed that XVPs display dramatic variation in the genome structure with numerous rearrangements/inversions. Moreover, XPVs also possess distinct transposon content and prophage elements that may provide genomic flux required for the acquisition of novel gene cassettes and structural changes in the genome. Interestingly, analysis of transcription activator-like effector (TALE) proteins, which are major virulence determinants of Xanthomonas pathogen show marked variation in the TALE content and DNA binding domain of tal genes. Overall, the present study indicates the possible role of mobilomes and repetitive elements in major structural and sequence alterations, which may be leading to the emergence of novel and extreme pathotypes. The knowledge and resource of XVPs will be invaluable in the further systematic understanding of evolution and management of variant pathotypes of Xoo.

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TALEN-based editing of TFIIAy5 changes rice response to Xanthomonas oryzae pv. Oryzae

TALEN-based editing of TFIIAy5 changes rice response to Xanthomonas oryzae pv. Oryzae | TAL effector science | Scoop.it

(via T. Schreiber. thx)

Han et al, 2020

The xa5 gene encodes a basal transcription factor (TFIIAγ) protein with wide spectrum resistance to bacterial blight caused by Xanthomonas oryzae pv. Oryzae (Xoo) in rice. It was only found in a few rice ecotypes, and the recessive characteristics limited its application in breeding. Here, we employed a TALEN-based technique to edit its dominant allelic TFIIAγ5 and obtained many mutant TFIIAγ5 genes. Most of them reduced rice susceptibility to varying degrees when the plants were challenged with the Xoo. In particular, the knocked-out TFIIAγ5 can reduce the rice susceptibility significantly, although it cannot reach the xa5-mediated resistance level, indicating TFIIAγ5 is a major component involved in disease susceptibility. In addition, the mutant encoding the protein with deletion of the 32nd amino acid or amino acid insertion between 32nd and 33rd site confers rice with the similar resistance to that of the knocked-out TFIIAγ5. Thus, the amino acids around 32nd site are also the important action sites of TFIIAγ5 besides the 39th amino acid previously reported. Moreover, the integration of xa5 into TFIIAγ5-knockout plants conferred them with a similar resistance as IRBB5, the rice variety containing the homozygous xa5 gene. Thus, TFIIAγ5 was not simply regarded as a resistant or a susceptible locus, as the substitution of amino acids might shift its functions.

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TALEN outperforms Cas9 in editing heterochromatin target sites - Nature Communications

TALEN outperforms Cas9 in editing heterochromatin target sites - Nature Communications | TAL effector science | Scoop.it

(via T. Schreiber, thx)

Jain et al, 2021
Genome editing critically relies on selective recognition of target sites. However, despite recent progress, the underlying search mechanism of genome-editing proteins is not fully understood in the context of cellular chromatin environments. Here, we use single-molecule imaging in live cells to directly study the behavior of CRISPR/Cas9 and TALEN. Our single-molecule imaging of genome-editing proteins reveals that Cas9 is less efficient in heterochromatin than TALEN because Cas9 becomes encumbered by local searches on non-specific sites in these regions. We find up to a fivefold increase in editing efficiency for TALEN compared to Cas9 in heterochromatin regions. Overall, our results show that Cas9 and TALEN use a combination of 3-D and local searches to identify target sites, and the nanoscopic granularity of local search determines the editing outcomes of the genome-editing proteins. Taken together, our results suggest that TALEN is a more efficient gene-editing tool than Cas9 for applications in heterochromatin. While Cas9 outperforms TALENs in euchromatin, it is less efficient in heterochromatic regions. Here the authors, using single-molecule imaging, show that Cas9 uses a less efficient search strategy compared to TALENs in these regions.

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Assembly of TALEN and mTALE-Act for Plant Genome Engineering

Assembly of TALEN and mTALE-Act for Plant Genome Engineering | TAL effector science | Scoop.it

Mahlzahn & Qi 2020

Transcription activator-like effector (TALE) is a DNA-binding domain that can be paired with a nuclease to create DNA double-strand breaks, or with an effector protein to alter gene transcription. The ability to precisely alter plant genomes and transcriptomes has provided many insights into gene function and has recently been utilized for crop improvement. Easy design and construction of TALE make the tool more accessible to a variety of researchers. Here, we describe two TALE-based systems: transcription activator-like effector nucleases (TALEN), for creating targeted mutations in a gene of interest, and multiplex TALE activation (mTALE-Act), for activating one or a few genes of interest at the transcription level. Assembly of these tools is based on Golden Gate cloning and Gateway recombination, which are cost-effective and streamlined cloning methods.

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The immunity of Meiwa kumquat against Xanthomonas citri is associated with a known susceptibility gene induced by a transcription activator-like effector - PLOS Pathogens

The immunity of Meiwa kumquat against Xanthomonas citri is associated with a known susceptibility gene induced by a transcription activator-like effector - PLOS Pathogens | TAL effector science | Scoop.it

(via T. Schreiber, thx)

Teper et al, 2020

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is one of the most devastating diseases in citrus. Meiwa kumquat (Fortunella crassifolia) has shown a durable resistance against Xcc. Here, we aimed to characterize the mechanisms responsible for such a durable resistance by characterizing the transcriptional and physiological responses of Meiwa kumquat to Xcc. Inoculation of Meiwa kumquat with Xcc promoted immune responses such as upregulation of PR genes, accumulation of salicylic acid, hypersensitive response (HR)-like cell death and early leaf abscission. Hypertrophy and hyperplasia symptoms, which are known to be caused by Xcc-induction of the canker susceptibility gene LOB1 through the transcription activator-like effector (TALE) PthA4, always appear prior to the development of cell death. Mutation of pthA4 in Xcc abolished the induction of LOB1, canker symptoms, cell death, and leaf abscission and reduced the expression of PR genes in inoculated kumquat leaves without reducing Xcc titers in planta. Transcriptome analysis demonstrated that PthA4 promotes plant biotic and abiotic stress responses and the biosynthesis of abscisic acid. Transcriptional induction of LOB1 homologs in Meiwa kumquat by Xcc pthA4 mutant strains carrying a repertoire of designer TALEs promoted the elicitation of HR-like phenotype and leaf abscission, suggesting that kumquat response to Xcc is associated with upregulation of LOB1. Our study suggests a novel mechanism of plant resistance to Xanthomonas via elicitation of immune responses by upregulation of a host susceptibility gene.

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Design and Application of DNA Modification-Specific Transcription-Activator-Like Effectors

Design and Application of DNA Modification-Specific Transcription-Activator-Like Effectors | TAL effector science | Scoop.it

Buchmueller et al, 2020
Transcription-activator like effectors (TALEs) are DNA-binding proteins used for genome targeting. TALEs contain a central domain of concatenated repeats, of which each selectively recognizes one nucleobase at the DNA major groove. Based on this simple and predictable interaction with little context dependence, TALEs offer programmable targeting of user-defined DNA sequences. Since many epigenetic DNA modifications protrude into the DNA major groove, natural and engineered TALE repeats can provide “epigenetic” selectivity, making TALEs a flexible platform to design probes for the analysis of epigenetic DNA modifications. Here, we describe guidelines for the design of TALE proteins with selectivity for epigenetic cytosine 5-modifications, the validation of their interaction with modified DNA nucleobases, and their employment in affinity enrichment assays. These techniques enable quantification of epigenetic nucleobases in user-defined genomic DNA sequences with nucleotide and strand resolution.

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The genetic arms race between plant and Xanthomonas : lessons learned from TALE biology - Science China Life Sci

The genetic arms race between plant and Xanthomonas : lessons learned from TALE biology - Science China Life Sci | TAL effector science | Scoop.it

Xue et al, 20020 The pathogenic bacterial genus Xanthomonas infects a wide variety of host plants and causes devastating diseases in many crops. Transcription activator-like effectors (TALEs) are important virulence factors secreted by Xanthomonas with the ability to directly bind to the promoters of target genes in plant hosts and activate their expression, which often facilitates the proliferation of pathogens. Understanding how plants cope with TALEs will provide mechanistic insights into crop breeding for Xanthomonas defense. Over the past 30 years, numerous studies have revealed the modes of action of TALEs in plant cells and plant defense strategies to overcome TALE attack. Based on these findings, new technologies were adopted for disease management to optimize crop production. In this article, we will review the most recent advances in the evolutionary arms race between plant resistance and TALEs from Xanthomonas, with a specific focus on TALE applications in the development of novel breeding strategies for durable and broad-spectrum resistance.

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A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing - Nature

A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing - Nature | TAL effector science | Scoop.it

(via T. Schreiber, thx)

Mok et al, 2020

Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Such proteins include a group of predicted interbacterial toxins of the deaminase superfamily, members of which have found application in gene-editing techniques1,2. Because previously described cytidine deaminases operate on single-stranded nucleic acids3, their use in base editing requires the unwinding of double-stranded DNA (dsDNA)—for example by a CRISPR–Cas9 system. Base editing within mitochondrial DNA (mtDNA), however, has thus far been hindered by challenges associated with the delivery of guide RNA into the mitochondria4. As a consequence, manipulation of mtDNA to date has been limited to the targeted destruction of the mitochondrial genome by designer nucleases9,10.Here we describe an interbacterial toxin, which we name DddA, that catalyses the deamination of cytidines within dsDNA. We engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, transcription activator-like effector array proteins, and a uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyse C•G-to-T•A conversions in human mtDNA with high target specificity and product purity. We used DdCBEs to model a disease-associated mtDNA mutation in human cells, resulting in changes in respiration rates and oxidative phosphorylation. CRISPR-free DdCBEs enable the precise manipulation of mtDNA, rather than the elimination of mtDNA copies that results from its cleavage by targeted nucleases, with broad implications for the study and potential treatment of mitochondrial disorders.

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A TAL effector-like protein of an endofungal bacterium increases the stress tolerance and alters the transcriptome of the host - PNAS

A TAL effector-like protein of an endofungal bacterium increases the stress tolerance and alters the transcriptome of the host - PNAS | TAL effector science | Scoop.it

(via T Schreiber, thx)

Carter et al., 2020

Endosymbiotic bacteria are found in diverse fungi, but little is known about how they communicate with their hosts. Some plant pathogenic bacteria use type III-translocated TAL effectors to control host transcription, and TAL-like proteins are encoded in genomes of the fungal endosymbiotic bacterium Mycetohabitans rhizoxinica. In this paper, we present evidence that these proteins are, like TAL effectors, type III-secreted, nuclear-localizing effectors that perturb host transcription and show that one enhances tolerance of the fungal host to cell membrane stress. Our characterization of an effector in a bacterial–fungal symbiosis opens a new door to molecular understanding of these interkingdom partnerships. Our findings also provide insight into the functional diversity and evolution of the TAL effector protein family.

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In Silico Designing of Xa13 Locus Specific TALENs for Introducing Bacterial Blight Resistance in Rice - Ind. J. Pure App. Biosci.

(via T. Lahaye, thx)

Suman & Vikram Nain, 2020

Bacterial blight disease in rice is caused by Xanthomonas oryzae pv. oryzae (XOO) through binding of type III secretion system effectors to susceptibility genes in host. PthXo1 effectors secreted by XOO binds to promoter region of sugar transporter gene Os8N3 thereby activating dominant allele of Xa13 susceptibility gene in rice. Introgression of recessive xa13 allele by molecular breeding programs have successfully imparted resistance against PthXo1 effector mediated disease in few rice cultivars. However, molecular breeding mediated introgression of disease resistance in hundreds of susceptible cultivars is a daunting task. Recent advancements in the field of genome editing technology by use of engineered nucleases ZFN, TALEN and CRISPR-Cas9 system allow fast and précised modifications in the genome. Therefore, the present study focus on designing of Xa13 locus specific TALENs for introducing bacterial blight resistance in Indica rice through TALEN mediated genome editing. Total three hundred thirty-three TALENs were designed against promoter region of Xa13. Out of these, One-hundred thirty-nine pairs of TALENs that follow Streubel’s guidelines and having targets with >40% GC content were retained. Further, Screening of these selected TALENs on basis of distance of their putative cleavage site from PthXo1 effectors binding site in rice genome resulted into eleven TALEN pairs. These eleven TALEN pairs were further screened for their number of putative targets in host genome and on the TAL score. Finally only three pairs possess a unique target site and score below a cut-off of four. Best scoring TALEN pair was selected for designing of TALEN coding gene sequences codon-optimized for high level expression in rice. These TALEN coding genes can be used for introducing deletions in Xa13 promoter and impart resistance against bacterial blight disease in rice.

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TALEN mediated gene editing in a mouse model of Fanconi anemia - Scientific Reports

TALEN mediated gene editing in a mouse model of Fanconi anemia - Scientific Reports | TAL effector science | Scoop.it

(Via T. Lahaye, thx)

Pino-Barrio et al, 2020

The promising ability to genetically modify hematopoietic stem and progenitor cells by precise gene editing remains challenging due to their sensitivity to in vitro manipulations and poor efficiencies of homologous recombination. This study represents the first evidence of implementing a gene editing strategy in a murine safe harbor locus site that phenotypically corrects primary cells from a mouse model of Fanconi anemia A. By means of the co-delivery of transcription activator-like effector nucleases and a donor therapeutic FANCA template to the Mbs85 locus, we achieved efficient gene targeting (23%) in mFA-A fibroblasts. This resulted in the phenotypic correction of these cells, as revealed by the reduced sensitivity of these cells to mitomycin C. Moreover, robust evidence of targeted integration was observed in murine wild type and FA-A hematopoietic progenitor cells, reaching mean targeted integration values of 21% and 16% respectively, that were associated with the phenotypic correction of these cells. Overall, our results demonstrate the feasibility of implementing a therapeutic targeted integration strategy into the mMbs85 locus, ortholog to the well-validated hAAVS1, constituting the first study of gene editing in mHSC with TALEN, that sets the basis for the use of a new safe harbor locus in mice.

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Secreted TAL effectors protect symbiotic bacteria from entrapment within fungal hyphae - bioRxiv

Secreted TAL effectors protect symbiotic bacteria from entrapment within fungal hyphae - bioRxiv | TAL effector science | Scoop.it

Richter et al, 2020

(Preprint)

The association of the agriculturally significant phytopathogenic fungus Rhizopus microsporus with the bacterial endosymbiont Burkholderia rhizoxinica is a remarkable example of bacteria controlling host physiology and reproduction. Here, we show that a group of transcription activator-like effectors (TALEs) called Burkholderia TALE-like proteins (BATs) from B. rhizoxinica are essential for the establishment of the symbiosis. Mutants lacking BAT proteins are unable to induce host sporulation. Utilising novel microfluidic devices in combination with fluorescence microscopy we observed the accumulation of BAT-deficient mutants in specific fungal side-hyphae with accompanying increased fungal re-infection. High-resolution live imaging revealed septa biogenesis at the base of infected hyphae leading to compartmental trapping of BAT-deficient endobacteria. Trapped endosymbionts showed reduced intracellular survival, suggesting a protective response from the fungal host against bacteria lacking specific effectors. These findings underscore the involvement of BAT proteins in maintaining a balance between mutualism and antagonism in bacterial-fungal interactions and provide deeper insights into the dynamic interactions between bacteria and eukaryotes.

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Precise Editing Enables Crop Broad-Spectrum Resistance - Mol. Plant

Precise Editing Enables Crop Broad-Spectrum Resistance - Mol. Plant | TAL effector science | Scoop.it

(via T. Lahaye, thx)

Tian et al, 2020

Pyramidization of resistance genes, such as nucleotide-binding leucine-rich repeat proteins, from diverse cultivars, landraces, and domesticated or wild germplasm accessions offers the most direct practice for this purpose. Nevertheless, the facts of a narrow resistance spectrum and limited number or even shortage of such resistance genes to many specific pathogens leave an immediate question to be resolved in such a situation. In light of the evidence that all compatible pathogens trigger a conserved and wide range of host genes, namely susceptibility genes for their proliferation, management of those genes are of equal importance and promising for improvement in crop disease resistance

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New Bicistronic TALENs Greatly Improve Genome Editing - Curr. Protocols Stem Cell Biol.

(via T. Lahaye, thx)

Martín Fernández et al, 2020

Genome editing has become one of the most powerful tools in present‐day stem cell and regenerative medicine research, but despite its rapid acceptance and widespread use, some elements of the technology still need improvement. In this unit, we present data regarding the use of a new, more efficient type of transcription activator‐like effector nuclease (TALEN) for gene editing. Our group has generated bicistronic genes in which classical TALEN coding sequences are linked by 2A elements to different reporter molecules, such as fluorochromes (TALEN‐F) or membrane receptors (TALEN‐M). This structure results in two proteins transcribed from the same transcript, of which the second (the reporter) can be used as the target for selection by fluorescence‐assisted cell sorting (FACS) or magnetic‐activated cell sorting (MACS). The application of these new TALEN genes allows a rapid enrichment of cells in which both members of the TALEN pair are active, thus eliminating the need for lengthy selection in culture and laborious characterization of a large number of clones.

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