TAL effector science

Sakono et al. 2022

Transcriptional activator-like effector (TALE), a DNA-binding protein, is widely used in genome editing. However, the recognition of the target sequence by the TALE is adversely affected by the number of mismatches. Therefore, the association constant of DNA-TALE complex formation can be controlled by appropriately introducing a mismatch into the TALE recognition sequence. This study aimed to construct a TALE that can distinguish a single nucleotide difference. Our results show that a single mismatch present in repeats 2 or 3 of TALE did not interfere with the complex formation with DNA, whereas continuous mismatches present in repeats 2 and 3 significantly reduced association with the target DNA. Based on these findings, we constructed a detection system of the one nucleotide difference in gene with high accuracy and constructed a TALE-nuclease (TALEN) that selectively cleaves DNA with a single mismatch.

(via T. Lahaye, thx)

Xu et al. 2022

• This study, for the first time, uncover a naturally-emerging Xa23-breaking Xoo isolate.

• The ability of AvrXa23 to be trapped by the EBE of Xa23 gene can be altered by one or more RVDs of AvrXa23.

• Seven AvrXa23-like TALEs determine the “arms-race” in the AvrXa23 and Xa23 pair.

• This study provide new insights into the diversified strategies used by Xoo to evade host resistance.

• Planting single R gene (like Xa23) rice in a great region is risk to make Xoo minority to majority.

(via T. Lahaye, thx)

Sakono et al. 2022

Transcriptional activator-like effector (TALE), a DNA-binding protein, is widely used in genome editing. However, the recognition of the target sequence by the TALE is adversely affected by the number of mismatches. Therefore, the association constant of DNA-TALE complex formation can be controlled by appropriately introducing a mismatch into the TALE recognition sequence. This study aimed to construct a TALE that can distinguish a single nucleotide difference. Our results show that a single mismatch present in repeats 2 or 3 of TALE did not interfere with the complex formation with DNA, whereas continuous mismatches present in repeats 2 and 3 significantly reduced association with the target DNA. Based on these findings, we constructed a detection system of the one nucleotide difference in gene with high accuracy and constructed a TALE-nuclease (TALEN) that selectively cleaves DNA with a single mismatch.

(via T. Lahaye, thx)

Lee et al. 2022

Bacterial toxin DddA-derived cytosine base editors (DdCBEs)—composed of split DddAtox (a cytosine deaminase specific to double-stranded DNA), custom-designed TALE (transcription activator-like effector) DNA-binding proteins, and a uracil glycosylase inhibitor—enable mitochondrial DNA (mtDNA) editing in human cells, which may pave the way for therapeutic correction of pathogenic mtDNA mutations in patients. The utility of DdCBEs has been limited by off-target activity, which is probably caused by spontaneous assembly of the split DddAtox deaminase enzyme, independent of DNA-binding interactions. We engineered high-fidelity DddA-derived cytosine base editors (HiFi-DdCBEs) with minimal off-target activity by substituting alanine for amino acid residues at the interface between the split DddAtox halves. The resulting domains cannot form a functional deaminase without binding of their linked TALE proteins at adjacent sites on DNA. Whole mitochondrial genome sequencing shows that, unlike conventional DdCBEs, which induce hundreds of unwanted off-target C-to-T conversions in human mtDNA, HiFi-DdCBEs are highly efficient and precise, avoiding collateral off-target mutations, and as such, they will probably be desirable for therapeutic applications.

(via T. Lahaye, thx)

Cho et al. 2022
Mitochondrial DNA (mtDNA) editing paves the way for disease modeling of mitochondrial genetic disorders in cell lines and animals and also for the treatment of these diseases in the future. Bacterial cytidine deaminase DddA-derived cytosine base editors (DdCBEs) enabling mtDNA editing, however, are largely limited to C-to-T conversions in the 5′-TC context (e.g., TC-to-TT conversions), suitable for generating merely 1/8 of all possible transition (purine-to-purine and pyrimidine-to-pyrimidine) mutations. Here, we present transcription-activator-like effector (TALE)-linked deaminases (TALEDs), composed of custom-designed TALE DNA-binding arrays, a catalytically impaired, full-length DddA variant or split DddA originated from Burkholderia cenocepacia, and an engineered deoxyadenosine deaminase derived from the E. coli TadA protein, which induce targeted A-to-G editing in human mitochondria. Custom-designed TALEDs were highly efficient in human cells, catalyzing A-to-G conversions at a total of 17 target sites in various mitochondrial genes with editing frequencies of up to 49%.

Schenstnyi et al. 2022
The Xanthomonas transcription activator-like effector (TALE) protein AvrBs3 transcriptionally activates the executor-type resistance (R) gene Bs3 from pepper (Capsicum annuum), thereby triggering a hypersensitive response (HR). AvrBs3 also triggers HR in tomato (Solanum lycopersicum) upon recognition by the nucleotide-binding leucine-rich repeat (NLR) R protein Bs4. Whether the executor-type R protein Bs3 and the NLR-type R protein Bs4 use common or distinct signalling components to trigger HR remains unclear.
CRISPR/Cas9-mutagenesis revealed, that the immune signalling node EDS1 is required for Bs4- but not for Bs3-dependent HR, suggesting that NLR- and executor-type R proteins trigger HR via distinct signalling pathways. CRISPR/Cas9-mutagenesis also revealed that tomato Bs4 suppresses the virulence function of both TALEs, the HR-inducing AvrBs3 protein and of AvrHah1, a TALE that does not trigger HR in tomato.
Analysis of AvrBs3- and AvrHah1-induced host transcripts and disease phenotypes in CRISPR/Cas9-induced bs4 mutant plants indicates that both TALEs target orthologous transcription factor genes to promote disease in tomato and pepper host plants.
Our studies display that tomato mutants lacking the TALE-sensing Bs4 protein provide a novel platform to either uncover TALE-induced disease phenotypes or genetically dissect components of executor-triggered HR.

Lei et al. 2022

DddA-derived cytosine base editors (DdCBEs)—which are fusions of split DddA halves and transcription activator-like effector (TALE) array proteins from bacteria—enable targeted C•G-to-T•A conversions in mitochondrial DNA1. However, their genome-wide specificity is poorly understood. Here we show that the mitochondrial base editor induces extensive off-target editing in the nuclear genome. Genome-wide, unbiased analysis of its editome reveals hundreds of off-target sites that are TALE array sequence (TAS)-dependent or TAS-independent. TAS-dependent off-target sites in the nuclear DNA are often specified by only one of the two TALE repeats, challenging the principle that DdCBEs are guided by paired TALE proteins positioned in close proximity. TAS-independent off-target sites on nuclear DNA are frequently shared among DdCBEs with distinct TALE arrays. Notably, they co-localize strongly with binding sites for the transcription factor CTCF and are enriched in topologically associating domain boundaries. We engineered DdCBE to alleviate such off-target effects. Collectively, our results have implications for the use of DdCBEs in basic research and therapeutic applications, and suggest the need to thoroughly define and evaluate the off-target effects of base-editing tools.

(via T. Schreiber, thx)

Tang et al. 2022

Recent studies have shown that reprogramming of gene expression in a genome can induce the production of proteins enabling yield increase. The transcription activator-like effectors (TALEs) from several species of bacterial Xanthomonas have been extensively studied, and a series of research tools, such as genome editing tool TALENs and gene expression activators, have been developed based on the specific protein–nucleic acid recognition and binding mechanisms of TALEs. In this proof-of-principle study, we designed and constructed a designer TALE (dTALE), designated as dTALE-NOG1, to specifically target the promoter of OsNOG1 gene in rice, and demonstrated that this dTALE can be used as a new type of plant growth regulator for better crop growth and harvest. In doing so, the dTALE-NOG1 was transferred into the non-pathogenic Xanthomonas oryzae pv. oryzae (Xoo) strain PH to generate a genetically engineered bacteria (GEB) strain called PH-dtNOG1. Functional verification showed that dTALE-NOG1 could significantly induce the expression of OsNOG1. By spraying cell suspension of PH-dtNOG1 on the rice plants during the tillering stage, the transcription level of OsNOG1 was highly enhanced, the grain number of rice plants was increased by more than 11.40%, and the grain yield per plant increased by more than 11.08%, demonstrating that the dTALE-NOG1 was highly effective in enhancing rice yield. This work provided a new strategy for manipulating agronomical traits by reprogrammin

(via T. Lahaye, thx)

Mok et al, 2022

The all-protein cytosine base editor DdCBE uses TALE proteins and a double-stranded DNA-specific cytidine deaminase (DddA) to mediate targeted C•G-to-T•A editing. To improve editing efficiency and overcome the strict TC sequence-context constraint of DddA, we used phage-assisted non-continuous and continuous evolution to evolve DddA variants with improved activity and expanded targeting scope. Compared to canonical DdCBEs, base editors with evolved DddA6 improved mitochondrial DNA (mtDNA) editing efficiencies at TC by 3.3-fold on average. DdCBEs containing  evolved DddA11 offered a broadened HC (H = A, C or T) sequence compatibility for both mitochondrial and nuclear base editing, increasing average editing efficiencies at AC and CC targets from less than 10% for canonical DdCBE to 15–30% and up to 50% in cell populations sorted to express both halves of DdCBE. We used these evolved DdCBEs to efficiently install disease-associated mtDNA mutations in human cells at non-TC target sites. DddA6 and DddA11 substantially increase the effectiveness and applicability of all-protein base editing.

(via T. Schreiber, thx)

Sivaram et al, 2022

The molecular mechanism of pomegranate susceptibility to bacterial blight, a serious threat to pomegranate production in India, is largely unknown.

In the current study, we have used PacBio and Illumina sequencing of Xanthomonas citri pv. punicae (Xcp) strain 119 genome to identify tal genes and RNA-Seq analysis to identify putative host targets in the susceptible pomegranate variety Bhagwa challenged with Xcp119. Xcp119 genome encodes seven transcription activator-like effectors (TALEs), three of which are harbored by a plasmid. RVD-based phylogenetic analysis of TALEs of Xanthomonas citri pathovars indicate the TALEs of Xcp as evolutionarily and functionally close to Xanthomonas citri pv. malvacearum and Xanthomonas citri pv. glycines. Comparative RNA-Seq of Xcp and mock-inoculated leaf tissues revealed Xcp-induced pomegranate transcription modulation. The prediction of TALE binding elements (EBEs) in the promoters of up-regulated genes identified a set of TALE-targeted candidate genes in pomegranate–Xcp interaction.

The predicted candidate susceptibility genes include two oxoglutarate-dependent dioxygenase gene, ethylene-responsive transcription factor and flavanone 3-hydroxylase-like gene, and the further characterization of these would enable blight resistance engineering in pomegranate.

Ji and Guo et al, 2022

Executor (E) genes comprise a new type of plant resistance (R) genes, identified from host–Xanthomonas interactions. The Xanthomonas-secreted transcription activation-like effectors (TALEs) usually function as major virulence factors, which activate the expression of the so-called “susceptibility” (S) genes for disease development. This activation is achieved via the binding of the TALEs to the effector-binding element (EBE) in the S gene promoter. However, host plants have evolved EBEs in the promoters of some otherwise silent R genes, whose expression directly causes a host cell death that is characterized by a hypersensitive response (HR). Such R genes are called E genes because they trap the pathogen TALEs in order to activate expression, and the resulting HR prevents pathogen growth and disease development. Currently, deploying E gene resistance is becoming a major component in disease resistance breeding, especially for rice bacterial blight resistance. Currently, the biochemical mechanisms, or the working pathways of the E proteins, are still fuzzy. There is no significant nucleotide sequence homology among E genes, although E proteins share some structural motifs that are probably associated with the signal transduction in the effector-triggered immunity. Here, we summarize the current knowledge regarding TALE-type avirulence proteins, E gene activation, the E protein structural traits, and the classification of E genes, in order to sharpen our understanding of the plant E genes.

You et al. 2022

Bacteria inject effector proteins into host cells to manipulate cellular processes that promote disease. Since bacteria deliver minuscule amounts of effectors only into targeted host cells, it is technically challenging to capture effector-dependent cellular changes from bulk-infected host tissues. Here, we report a new technique called effector-inducible isolation of nuclei tagged in specific cell types (eINTACT), which facilitates affinity-based purification of nuclei from Arabidopsis plant cells that have received Xanthomonas bacterial effectors. Analysis of purified nuclei reveals that the Xanthomonas effector XopD manipulates the expression of Arabidopsis abscisic acid signalling-related genes and activates OSCA1.1, a gene encoding a calcium-permeable channel required for stomatal closure in response to osmotic stress. The loss of OSCA1.1 causes leaf wilting and reduced bacterial growth in infected leaves, suggesting that OSCA1.1 promotes host susceptibility. eINTACT allows us to uncover that XopD exploits host OSCA1.1/abscisic acid osmosignalling-mediated stomatal closure to create a humid habitat that favours bacterial growth and opens up a new avenue for accurately elucidating functions of effectors from numerous gram-negative plant bacteria in native infection contexts. eINTACT uncovers Xanthomonas bacterial exploitation of plant osmosignalling by its effector XopD to enhance virulence. This provides the basis for accurately elucidating functions of bacterial type-III effectors in natively infected plants.

Xu et al. 2022

Transcription activator-like effectors (TALEs) are proteins produced by plant pathogenic Xanthomonas spp. TALEs exhibit a conserved structure and have the ability to directly bind to the promoter region of host target genes where they activate transcription. TALEs in Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight (BB) in rice, play important roles in triggering resistance (ETI) and susceptibility (ETS) for rice immunity. This review briefly describes rice resistance breeding in China, TALE properties and their roles, BB resistance (R) and susceptibility (S) genes in rice, the arms-race between TALEs and TALE-targets, and strategies for breeding disease-resistant crops. A systematic overview of the complex roles of TALEs are presented along with ongoing efforts to breed crops with durable and broad-spectrum resistance to the pathogenic bacterium.

(via T. Lahaye, thx)

Mok et al. 2022

Chloroplast DNA (cpDNA) encodes up to 315 (typically, 120–130) genes1, including those for essential components in photosystems I and II and the large subunit of RuBisCo, which catalyses CO2 fixation in plants. Targeted mutagenesis in cpDNA will be broadly useful for studying the functions of these genes in molecular detail and for developing crops and other plants with desired traits. Unfortunately, CRISPR–Cas9 and CRISPR-derived base editors, which enable targeted genetic modifications in nuclear DNA, are not suitable for organellar DNA editing2, owing to the difficulty of delivering guide RNA into organelles. CRISPR-free, protein-only base editors (including DddA-derived cytosine base editors3–8 and zinc finger deaminases9), originally developed for mitochondrial DNA editing in mammalian cells, can be used for C-to-T, rather than A-to-G, editing in cpDNA10–12. Here we show that heritable homoplasmic A-to-G edits can be induced in cpDNA, leading to phenotypic changes, using transcription activator-like effector-linked deaminases13. Plant-optimized transcription activator-like effector-linked deaminases enable site-specific A-to-G base editing in the chloroplast genome, leading to heritable homoplasmic base conversions and phenotypic changes.

(via T. Lahaye, thx)

Lee et al. 2022

We present two methods for enhancing the efficiency of mitochondrial DNA (mtDNA) editing in mice with DddA-derived cytosine base editors (DdCBEs). First, we fused DdCBEs to a nuclear export signal (DdCBE-NES) to avoid off-target C-to-T conversions in the nuclear genome and improve editing efficiency in mtDNA. Second, mtDNA-targeted TALENs (mitoTALENs) are co-injected into mouse embryos to cleave unedited mtDNA. We generated a mouse model with the m.G12918A mutation in the MT-ND5 gene, associated with mitochondrial genetic disorders in humans. The mutant mice show hunched appearances, damaged mitochondria in kidney and brown adipose tissues, and hippocampal atrophy, resulting in premature death.

(via T. Schreiber, thx)

Roeschlin et al, 2019

Transcription activator-like effectors (TALEs) are important effectors of Xanthomonas spp. that manipulate the transcriptome of the host plant, conferring susceptibility or resistance to bacterial infection. Xanthomonas citri ssp. citri variant AT (X. citri AT) triggers a host-specific hypersensitive response (HR) that suppresses citrus canker development. However, the bacterial effector that elicits this process is unknown. In this study, we show that a 7.5-repeat TALE is responsible for triggering the HR. PthA4AT was identified within the pthA repertoire of X. citri AT followed by assay of the effects on different hosts. The mode of action of PthA4AT was characterized using protein-binding microarrays and testing the effects of deletion of the nuclear localization signals and activation domain on plant responses. PthA4AT is able to bind DNA and activate transcription in an effector binding element-dependent manner. Moreover, HR requires PthA4AT nuclear localization, suggesting the activation of executor resistance (R) genes in host and non-host plants. This is the first case where a TALE of unusually short length performs a biological function by means of its repeat domain, indicating that the action of these effectors to reprogramme the host transcriptome following nuclear localization is not limited to ‘classical’ TALEs.

(via T. Schreiber, thx)

Young Geun Mok et al. 2022

Inter-bacterial toxin DddA-derived cytosine base editors (DdCBEs) enable targeted C-to-T conversions in nuclear and organellar DNA. DddAtox, the deaminase catalytic domain derived from Burkholderia cenocepacia, is split into two inactive halves to avoid its cytotoxicity in eukaryotic cells, when fused to transcription activator-like effector (TALE) DNA-binding proteins to make DdCBEs. As a result, DdCBEs function as pairs, which hampers gene delivery via viral vectors with a small cargo size. Here, we present non-toxic, full-length DddAtox variants to make monomeric DdCBEs (mDdCBEs), enabling mitochondrial DNA editing with high efficiencies of up to 50%, when transiently expressed in human cells. We demonstrate that mDdCBEs expressed via AAV in cultured human cells can achieve nearly homoplasmic C-to-T editing in mitochondrial DNA. Interestingly, mDdCBEs often produce mutation patterns different from those obtained with conventional dimeric DdCBEs. Furthermore, mDdCBEs allow base editing at sites for which only one TALE protein can be designed. We also show that transfection of mDdCBE-encoding mRNA, rather than plasmid, can reduce off-target editing in human mitochondrial DNA.

Danila et al. 2022
In biological discovery and engineering research, there is a need to spatially and/or temporally regulate transgene expression. However, the limited availability of promoter sequences that are uniquely active in specific tissue-types and/or at specific times often precludes co-expression of multiple transgenes in precisely controlled developmental contexts. Here, we developed a system for use in rice that comprises synthetic designer transcription activator-like effectors (dTALEs) and cognate synthetic TALE-activated promoters (STAPs). The system allows multiple transgenes to be expressed from different STAPs, with the spatial and temporal context determined by a single promoter that drives expression of the dTALE. We show that two different systems—dTALE1-STAP1 and dTALE2-STAP2—can activate STAP-driven reporter gene expression in stable transgenic rice lines, with transgene transcript levels dependent on both dTALE and STAP sequence identities. The relative strength of individual STAP sequences is consistent between dTALE1 and dTALE2 systems but differs between cell-types, requiring empirical evaluation in each case. dTALE expression leads to off-target activation of endogenous genes but the number of genes affected is substantially less than the number impacted by the somaclonal variation that occurs during the regeneration of transformed plants. With the potential to design fully orthogonal dTALEs for any genome of interest, the dTALE-STAP system thus provides a powerful approach to fine-tune the expression of multiple transgenes, and to simultaneously introduce different synthetic circuits into distinct developmental contexts.

Mishra et al. 2022
Network science identifies key players in diverse biological systems including host-pathogen interactions. We demonstrated a scale-free network property for a comprehensive rice protein-protein interactome (RicePPInets) that exhibits nodes with increased centrality indices. While weighted k-shell decomposition was shown efficacious to predict pathogen effector targets in Arabidopsis, we improved its computational code for a broader implementation on large-scale networks including RicePPInets. We determined that nodes residing within the internal layers of RicePPInets are poised to be the most influential, central, and effective information spreaders. To identify central players and modules through network topology analyses, we integrated RicePPInets and co-expression networks representing susceptible and resistant responses to strains of the bacterial pathogens Xanthomonas oryzae pv. oryzae and X. oryzae pv. oryzicola (Xoc) and generated a RIce-Xanthomonas INteractome (RIXIN). This revealed that previously identified candidate targets of pathogen transcription activator-like (TAL) effectors are enriched in nodes with enhanced connectivity, bottlenecks, and information spreaders that are located in the inner layers of the network, and these nodes are involved in several important biological processes. Overall, our integrative multi-omics network-based platform provides a potentially useful approach to prioritizing candidate pathogen effector targets for functional validation, suggesting that this computational framework can be broadly translatable to other complex pathosystems.

Forner et al. 2022

The development of technologies for the genetic manipulation of mitochondrial genomes remains a major challenge. Here we report a method for the targeted introduction of mutations into plant mitochondrial DNA (mtDNA) that we refer to as transcription activator-like effector nuclease (TALEN) gene-drive mutagenesis (GDM), or TALEN-GDM. The method combines TALEN-induced site-specific cleavage of the mtDNA with selection for mutations that confer resistance to the TALEN cut. Applying TALEN-GDM to the tobacco mitochondrial nad9 gene, we isolated a large set of mutants carrying single amino acid substitutions in the Nad9 protein. The mutants could be purified to homochondriomy and stably inherited their edited mtDNA in the expected maternal fashion. TALEN-GDM induces both transitions and transversions, and can access most nucleotide positions within the TALEN binding site. Our work provides an efficient method for targeted mitochondrial genome editing that produces genetically stable, homochondriomic and fertile plants with specific point mutations in their mtDNA. A method for targeted mutagenesis of mitochondrial genomes is presented. It combines site-specific DNA cleavage with selection for mutations that confer cleavage resistance, and produces genetically stable plants with edited mitochondrial genomes.

Becker et al, 2022

Transcription activator-like effectors (TALEs) are bacterial proteins with a programmable DNA-binding domain, which turned them into exceptional tools for biotechnology. TALEs contain a central array of consecutive 34 amino acid long repeats to bind DNA in a simple one-repeat-to-one-nucleotide manner. However, a few naturally occurring aberrant repeat variants break this strict binding mechanism, allowing for the recognition of an additional sequence with a −1 nucleotide frameshift. The limits and implications of this extended TALE binding mode are largely unexplored. Here, we analyse the complete diversity of natural and artificially engineered aberrant repeats for their impact on the DNA binding of TALEs. Surprisingly, TALEs with several aberrant repeats can loop out multiple repeats simultaneously without losing DNA-binding capacity. We also characterized members of the only natural TALE class harbouring two aberrant repeats and confirmed that their target is the major virulence factor OsSWEET13 from rice. In an aberrant TALE repeat, the position and nature of the amino acid sequence strongly influence its function. We explored the tolerance of TALE repeats towards alterations further and demonstrate that inserts as large as GFP can be tolerated without disrupting DNA binding. This illustrates the extraordinary DNA-binding capacity of TALEs and opens new uses in biotechnology.

Dinh et al, 2021 - Preprint
Host resistance is considered the most effective means to control plant diseases; however, individually deployed resistance genes are often rapidly overcome by pathogen adaptation. Combining multiple effective resistance genes is the optimal approach to durable resistance, but the lack of functional markers for resistance genes has hampered implementation. Leaf rust, caused by Puccinia hordei, is an economically significant disease of barley, but only a few major Resistance genes to P. hordei (Rph) have been cloned. In this study, gene Rph3 was isolated by positional cloning and confirmed by mutational analysis and transgenic complementation. The Rph3 gene, which originated from wild barley and was first introgressed into cultivated Egyptian germplasm, encodes a unique transmembrane resistance protein that differs from all known plant disease resistance proteins at the amino acid sequence level. Genetic profiles of diverse accessions indicated limited genetic diversity in Rph3 in domesticated germplasm, and higher diversity in wild barley from the Eastern Mediterranean region. Expression profiling using P. hordei isolates with contrasting pathogenicity for the Rph3 host locus showed that the Rph3 gene was expressed only in interactions with Rph3-avirulent isolates, a phenomenon also observed for transcription activator-like effector-dependent genes known as executors conferring resistance to Xanthomonas spp. Like the known transmembrane executors such as Bs3 and Xa7 heterologous expression of Rph3 in N. benthamiana induced a cell death response. Given that Rph3 shares several features with executor genes, it seems likely that P. hordei contains effectors similar to the transcription activator-like effectors that target host executor genes. The isolation of Rph3 highlights convergent evolutionary processes in diverse plant-pathogen interaction systems, where similar defence mechanisms evolved independently in monocots and dicots and provide evidence for executor genes in the Triticeae tribe.