Engineering TALE-linked deaminases to facilitate precision adenine base editing in mitochondrial DNA.

DddA-derived cytosine base editors (DdCBEs) and transcription activator-like effector (TALE)-linked deaminases (TALEDs) catalyze targeted base editing of mitochondrial DNA (mtDNA) in eukaryotic cells, a method useful for modeling of mitochondrial genetic disorders and developing novel therapeutic modalities. Here, we report that A-to-G-editing TALEDs but not C-to-T-editing DdCBEs induce tens of thousands of transcriptome-wide off-target edits in human cells. To avoid these unwanted RNA edits, we engineered the substrate-binding site in TadA8e, the deoxy-adenine deaminase in TALEDs, and created TALED variants with fine-tuned deaminase activity. Our engineered TALED variants not only reduced RNA off-target edits by >99% but also minimized off-target mtDNA mutations and bystander edits at a target site. Unlike wild-type versions, our TALED variants were not cytotoxic and did not cause developmental arrest of mouse embryos. As a result, we obtained mice with pathogenic mtDNA mutations, associated with Leigh syndrome, which showed reduced heart rates.

Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing.

Gene silencing is instrumental to interrogate gene function and holds promise for therapeutic applications. Here, we repurpose the endogenous retroviruses' silencing machinery of embryonic stem cells to stably silence three highly expressed genes in somatic cells by epigenetics. This was achieved by transiently expressing combinations of engineered transcriptional repressors that bind to and synergize at the target locus to instruct repressive histone marks and de novo DNA methylation, thus ensuring long-term memory of the repressive epigenetic state. Silencing was highly specific, as shown by genome-wide analyses, sharply confined to the targeted locus without spreading to nearby genes, resistant to activation induced by cytokine stimulation, and relieved only by targeted DNA demethylation. We demonstrate the portability of this technology by multiplex gene silencing, adopting different DNA binding platforms and interrogating thousands of genomic loci in different cell types, including primary T lymphocytes. Targeted epigenome editing might have broad application in research and medicine.

A six-amino-acid motif is a major determinant in functional evolution of HOX1 proteins.

Gene duplication and divergence is a major driver in the emergence of evolutionary novelties. How variations in amino acid sequences lead to loss of ancestral activity and functional diversification of proteins is poorly understood. We used cross-species functional analysis of Drosophila Labial and its mouse HOX1 orthologs (HOXA1, HOXB1, and HOXD1) as a paradigm to address this issue. Mouse HOX1 proteins display low (30%) sequence similarity with Drosophila Labial. However, substituting endogenous Labial with the mouse proteins revealed that HOXA1 has retained essential ancestral functions of Labial, while HOXB1 and HOXD1 have diverged. Genome-wide analysis demonstrated similar DNA-binding patterns of HOXA1 and Labial in mouse cells, while HOXB1 binds to distinct targets. Compared with HOXB1, HOXA1 shows an enrichment in co-occupancy with PBX proteins on target sites and exists in the same complex with PBX on chromatin. Functional analysis of HOXA1-HOXB1 chimeric proteins uncovered a novel six-amino-acid C-terminal motif (CTM) flanking the homeodomain that serves as a major determinant of ancestral activity. In vitro DNA-binding experiments and structural prediction show that CTM provides an important domain for interaction of HOXA1 proteins with PBX. Our findings show that small changes outside of highly conserved DNA-binding regions can lead to profound changes in protein function.

'Building a perfect body': control of vertebrate organogenesis by PBX-dependent regulatory networks.

Pbx genes encode transcription factors that belong to the TALE (three-amino-acid loop extension) superclass of homeodomain proteins. We have witnessed a surge in information about the roles of this gene family as leading actors in the transcriptional control of development. PBX proteins represent a clear example of how transcription factors can regulate developmental processes by combinatorial properties, acting within multimeric complexes to implement activation or repression of transcription depending on their interaction partners. Here, we revisit long-emphasized functions of PBX transcription factors as cofactors for HOX proteins, major architects of the body plan. We further discuss new knowledge on roles of PBX proteins in different developmental contexts as upstream regulators of Hox genes-as factors that interact with non-HOX proteins and can work independently of HOX-as well as potential pioneer factors. Committed to building a perfect body, PBX proteins govern regulatory networks that direct essential morphogenetic processes and organogenesis in vertebrate development. Perturbations of PBX-dependent networks can cause human congenital disease and cancer.

Xanthomonas oryzae pv. oryzicola effector Tal10a directly activates rice OsHXK5 expression to facilitate pathogenesis.

Bacterial leaf streak (BLS), caused by Xanthomonas oryzae pv. oryzicola (Xoc), is a major bacterial disease in rice. Transcription activator-like effectors (TALEs) from Xanthomonas can induce host susceptibility (S) genes and facilitate infection. However, knowledge of the function of Xoc TALEs in promoting bacterial virulence is limited. In this study, we demonstrated the importance of Tal10a for the full virulence of Xoc. Through computational prediction and gene expression analysis, we identified the hexokinase gene OsHXK5 as a host target of Tal10a. Tal10a directly binds to the gene promoter region and activates the expression of OsHXK5. CRISPR/Cas9-mediated gene editing in the effector binding element (EBE) of OsHXK5 significantly increases rice resistance to Xoc, while OsHXK5 overexpression enhances the susceptibility of rice plants and impairs rice defense responses. Moreover, simultaneous editing of the promoters of OsSULTR3;6 and OsHXK5 confers robust resistance to Xoc in rice. Taken together, our findings highlight the role of Tal10a in targeting OsHXK5 to promote infection and suggest that OsHXK5 represents a potential target for engineering rice resistance to Xoc.

Ancestral TALE homeobox protein transcription factor regulates actin dynamics and cellular activities of protozoan parasite Entamoeba invadens.

Entamoeba histolytica causes invasive amoebiasis, an important neglected tropical disease with a significant global health impact. The pathogenicity and survival of E. histolytica and its reptilian equivalent, Entamoeba invadens, relies on its ability to exhibit efficient motility, evade host immune responses, and exploit host resources, all of which are governed by the actin cytoskeleton remodeling. Our study demonstrates the early origin and the regulatory role of TALE homeobox protein EiHbox1 in actin-related cellular processes. Several genes involved in different biological pathways, including actin dynamics are differentially expressed in EiHbox1 silenced cells. EiHbox1 silenced parasites showed disrupted F-actin organization and loss of cellular polarity. EiHbox1's presence in the anterior region of migrating cells further suggests its involvement in maintaining cellular polarity. Loss of polarized morphology of EiHbox1 silenced parasites leads to altered motility from fast, directionally persistent, and highly chemotactic to slow, random, and less chemotactic, which subsequently leads to defective aggregation during encystation. EiHbox1 knockdown also resulted in a significant reduction in phagocytic capacity and poor capping response. These findings highlight the importance of EiHbox1 of E. invadens in governing cellular processes crucial for their survival, pathogenicity, and evasion of the host immune system.

DNA base editing in nuclear and organellar genomes.

Genome editing continues to revolutionize biological research. Due to its simplicity and flexibility, CRISPR/Cas-based editing has become the preferred technology in most systems. Cas nucleases tolerate fusion to large protein domains, thus allowing combination of their DNA recognition properties with new enzymatic activities. Fusion to nucleoside deaminase or reverse transcriptase domains has produced base editors and prime editors that, instead of generating double-strand breaks in the target sequence, induce site-specific alterations of single (or a few adjacent) nucleotides. The availability of protein-only genome editing reagents based on transcription activator-like effectors has enabled the extension of base editing to the genomes of chloroplasts and mitochondria. In this review, we summarize currently available base editing methods for nuclear and organellar genomes. We highlight recent advances with improving precision, specificity, and efficiency and discuss current limitations and future challenges. We also provide a brief overview of applications in agricultural biotechnology and gene therapy.

Three-amino acid loop extension homeodomain proteins regulate stress responses and encystation in Entamoeba.

It is interesting to identify factors involved in the regulation of the encystation of Entamoeba histolytica that differentiate trophozoites into cysts. Evolutionarily conserved three amino acid loop extension (TALE) homeodomain proteins act as transcription factors and execute a variety of functions that are essential for life. A TALE homeodomain (EhHbox) protein-encoding gene has been identified in E. histolytica (Eh) that is highly upregulated during heat shock, glucose, and serum starvation. Its ortholog, EiHbox1, a putative homeobox protein in E. invadens (Ei), is also highly upregulated during the early hours of encystation, glucose starvation, and heat shock. They belong to the PBX family of TALE homeobox proteins and have conserved residues in the homeodomain that are essential for DNA binding. Both are localized in the nucleus during encystation and under different stress conditions. The electrophoretic mobility shift assay confirmed that the recombinant GST-EhHbox binds to the reported TGACAG and TGATTGAT motifs. Down-regulation of EiHbox1 by gene silencing reduced Chitin synthase, Jacob, and increased Jessie gene expression, resulting in defective cysts and decreased encystation efficiency and viability. Overall, our results suggest that the TALE homeobox family has been conserved during evolution and acts as a transcription factor to control the differentiation of Entamoeba by regulating the key encystation-induced genes.

TALE gene family: identification, evolutionary and expression analysis under various exogenous hormones and waterlogging stress in Cucumis sativus L.

BACKGROUND: Three Amino acid Loop Extension (TALE) belongs to the homeobox group of genes that are important constituents of plant systems. The TALE gene family is instrumental not only in growth and development but also plays an essential role in regulating plant response to environmental adversaries. RESULTS: In the present study, we isolated 21 CsTALE genes from the cucumber (Cucumis sativus L.) genome database. Bioinformatics tools were put in place to understand the structural and functional components of the CsTALE gene family. The evolutionary analysis dissected them into seven subclades (KNOX-I, KNOX-II, and BELL-I to BELL-V). The cis-acting elements in the promoter region of CsTALE genes disclosed that they are key regulators of hormonal and stress-related processes. Additionally, the STRING database advocated the concerting role of CsTALE proteins with other key transcription factors potent in plant developmental biology. The CsmiR319 and CsmiR167a-3p targeting the CsTALE15 and CsTALE16, respectively, further assert the importance of the CsTALE gene family posttranscriptional-related processes. Tissue-specific gene expression unfolded the fundamental involvement of CsTALE genes as they were expressed throughout the developmental stages. Under waterlogging stress, the CsTALE17 expressed significantly higher values in WL, WL-NAA, and WL-ETH but not in WL-MeJA-treated samples. CONCLUSIONS: The present study reveals the evolution and functions of the CsTALE gene family in cucumber. Our work will provide a platform that will help future researchers address the issue of waterlogging stress in the Yangtze River Delta.

Evolution in the Cycles of Life.

The life cycles of eukaryotes alternate between haploid and diploid phases, which are initiated by meiosis and gamete fusion, respectively. In both ascomycete and basidiomycete fungi and chlorophyte algae, the haploid-to-diploid transition is regulated by a pair of paralogous homeodomain protein encoding genes. That a common genetic program controls the haploid-to-diploid transition in phylogenetically disparate eukaryotic lineages suggests this may be the ancestral function for homeodomain proteins. Multicellularity has evolved independently in many eukaryotic lineages in either one or both phases of the life cycle. Organisms, such as land plants, exhibiting a life cycle whereby multicellular bodies develop in both the haploid and diploid phases are often referred to as possessing an alternation of generations. We review recent progress on understanding the genetic basis for the land plant alternation of generations and highlight the roles that homeodomain-encoding genes may have played in the evolution of complex multicellularity in this lineage.

Shaping leaves through TALE homeodomain transcription factors.

The first TALE homeodomain transcription factor gene to be described in plants was maize knotted1 (kn1). Dominant mutations in kn1 disrupt leaf development, with abnormal knots of tissue forming in the leaf blade. kn1 was found to be expressed in the shoot meristem but not in a peripheral region that gives rise to leaves. Furthermore, KN1 and closely related proteins were excluded from initiating and developing leaves. These findings were a prelude to a large body of work wherein TALE homeodomain proteins have been identified as vital regulators of meristem homeostasis and organ development in plants. KN1 homologues are widely represented across land plant taxa. Thus, studying the regulation and mechanistic action of this gene class has allowed investigations into the evolution of diverse plant morphologies. This review will focus on the function of TALE homeodomain transcription factors in leaf development in eudicots. Here, we discuss how TALE homeodomain proteins contribute to a spectrum of leaf forms, from the simple leaves of Arabidopsis thaliana to the compound leaves of Cardamine hirsuta and species beyond the Brassicaceae.

TALE-based organellar genome editing and gene expression in plants.

KEY MESSAGE: TALE-based editors provide an alternative way to engineer the organellar genomes in plants. We update and discuss the most recent developments of TALE-based organellar genome editing in plants. Gene editing tools have been widely used to modify the nuclear genomes of plants for various basic research and biotechnological applications. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 editing platform is the most commonly used technique because of its ease of use, fast speed, and low cost; however, it encounters difficulty when being delivered to plant organelles for gene editing. In contrast, protein-based editing technologies, such as transcription activator-like effector (TALE)-based tools, could be easily delivered, expressed, and targeted to organelles in plants via Agrobacteria-mediated nuclear transformation. Therefore, TALE-based editors provide an alternative way to engineer the organellar genomes in plants since the conventional chloroplast transformation method encounters technical challenges and is limited to certain species, and the direct transformation of mitochondria in higher plants is not yet possible. In this review, we update and discuss the most recent developments of TALE-based organellar genome editing in plants.

Systems for Targeted Silencing of Gene Expression and Their Application in Plants and Animals.

At present, there are a variety of different approaches to the targeted regulation of gene expression. However, most approaches are devoted to the activation of gene transcription, and the methods for gene silencing are much fewer in number. In this review, we describe the main systems used for the targeted suppression of gene expression (including RNA interference (RNAi), chimeric transcription factors, chimeric zinc finger proteins, transcription activator-like effectors (TALEs)-based repressors, optogenetic tools, and CRISPR/Cas-based repressors) and their application in eukaryotes-plants and animals. We consider the advantages and disadvantages of each approach, compare their effectiveness, and discuss the peculiarities of their usage in plant and animal organisms. This review will be useful for researchers in the field of gene transcription suppression and will allow them to choose the optimal method for suppressing the expression of the gene of interest depending on the research object.

Recent Advances in Tomato Gene Editing.

The use of gene-editing tools, such as zinc finger nucleases, TALEN, and CRISPR/Cas, allows for the modification of physiological, morphological, and other characteristics in a wide range of crops to mitigate the negative effects of stress caused by anthropogenic climate change or biotic stresses. Importantly, these tools have the potential to improve crop resilience and increase yields in response to challenging environmental conditions. This review provides an overview of gene-editing techniques used in plants, focusing on the cultivated tomatoes. Several dozen genes that have been successfully edited with the CRISPR/Cas system were selected for inclusion to illustrate the possibilities of this technology in improving fruit yield and quality, tolerance to pathogens, or responses to drought and soil salinity, among other factors. Examples are also given of how the domestication of wild species can be accelerated using CRISPR/Cas to generate new crops that are better adapted to the new climatic situation or suited to use in indoor agriculture.

Supporting each other towards independence: A narrative analysis of first-year nursing students' collaborative process.

Collaboration for nursing is a core competence and therefore educational interventions are essentials for collaborative skills. To identify such interventions, we carried out a study to understand nursing students' collaborative process. A narrative inquiry method was used to explore the collaborative process of first-year undergraduate nursing students. The analysis was conducted on field notes from 70 h of observation of 87 nursing students' collaboration during skills lab activities. It also included transcriptions of four focus group discussions with 11 students. The results are presented as a sequential process of (1) navigating in unfamiliar territory, (2) navigating together to cope, and (3) navigating together towards independency and the future nursing profession. We identified a transition from teacher-led assistance and guidance to student interdependency and reciprocal learning, ending with student-led assistance supporting independency. In line with Vygotsky's theory of zone of proximal development, different scaffolding interventions are needed depending on where the students are in the collaborative process.

Does Dietitian Involvement During Pregnancy Improve Birth Outcomes? A Systematic Review.

Maternal diet during pregnancy can have a significant impact on maternal and offspring health. As nutrition counselling is an important component of prenatal care, registered dietitians (RDs) are uniquely trained professionals who can provide personalized nutrition counselling customized to an individual's sociocultural needs. The objective of this systematic review was to determine if RD involvement during pregnancy is associated with a lower prevalence of adverse birth outcomes in the United States and Canada. The review was conducted through a search of four databases: PubMed, CINAHL, Embase, and Web of Science. A total of 14 studies were identified. Women had a lower prevalence of low birth weight and preterm infants when RDs were involved during prenatal care. While RD involvement during pregnancy was not associated with macrosomia, more research is needed to assess its relationship with small for gestational age, large for gestational age, and infant mortality. Future research should also investigate the specific dietary advice provided by RDs and the extent and timing of their involvement throughout pregnancy to better understand the mechanisms surrounding nutrition counselling, in utero development, and health outcomes.

Is lower fetal heart rate variability a susceptibility marker to the impact of negative coparenting on infant regulatory capacity?

Lower fetal heart rate variability (FHRV) may be a prenatal endophenotypic susceptibility marker and increase the impact of both positive and negative coparenting on infant regulatory capacity. This study analyzed the moderator role of FHRV in the association between positive and negative coparenting and infant regulatory capacity at 3 months. The sample comprised 86 first-born infants and their mothers and fathers recruited at a public Health Service in Northern Portugal. FHRV was recorded during routine cardiotocography examination at the third trimester of gestation. Mothers and fathers reported on coparenting and infant regulatory capacity at 2 weeks and 3 months postpartum. FHRV moderated the association between mother's and father's negative coparenting at 2 weeks postpartum and infant regulatory capacity at three months. Infants with low FHRV presented higher regulatory capacity when mothers or fathers reported less negative coparenting, while lower regulatory capacity when mothers or fathers reported more negative coparenting, than infants with high FHRV. Findings suggested lower FHRV as a prenatal endophenotypic susceptibility marker that increases the impact of negative coparenting on infant regulatory capacity.

La más baja variabilidad del pulso cardíaco fetal (FHRV) pudiera ser un marcador determinante de la susceptibilidad endofenotípica prenatal y aumentar el impacto de la crianza compartida tanto positiva como negativa sobre la capacidad regulatoria del infante. Este estudio analizó el papel moderador de FHRV en la asociación entre la positiva y negativa crianza compartida y la capacidad regulatoria del infante a los tres meses. El grupo muestra estaba compuesto de 86 infantes primerizos y sus mamás y papás. Se grabó la FHRV durante una examinación de cardiotocografía de rutina en el tercer trimestre de la gestación. Las mamás y los papás reportaron acerca de la crianza compartida y la capacidad regulatoria del infante a las dos semanas y a los tres meses después del parto. La FHRV moderó la asociación entre la crianza compartida negativa de la mamá y del papá a las dos semanas después del parto y la capacidad regulatoria del infante a los tres meses. Los infantes con baja FHRV presentaron una capacidad regulatoria más alta cuando las mamás o los papás reportaron una crianza compartida menos negativa, mientras que la capacidad regulatoria más baja se dio cuando las mamás o los papás reportaron una crianza compartida más negativa, que los infantes con una FHRV alta. Los resultados señalan la más baja FHRV como un marcador determinante de la susceptibilidad endofenotípica prenatal que aumenta el impacto de la crianza compartida negativa sobre la capacidad regulatoria del infante. Los infantes con baja FHRV pueden ser aquellos que mejor desarrollan mecanismos de autorregulación en la presencia de una crianza compartida menos negativa, mientras que están bajo alto riesgo de problemas regulatorios en la presencia de una crianza compartida más negativa.

Une variabilité de la fréquence cardiaque fœtale (VFCF) plus basse pourrait être un marqueur de sensibilité endophénotypique prénatale et augmenter l'impact du coparentage positif et négatif sur la capacité régulatoire du nourrisson. Cette étude a analysé le rôle modérateur de la VFCF dans le lien entre le coparentage positif et négatif et la capacité régulatoire du nourrisson à trois mois. Cet échantillon a inlu 86 nourrisson premiers nés et leurs mères et pères. La VFCF a été enregistrée penfdant un examen de cardiotocographie de routine au troisième trimestre de la grossesse. Les mères et les pères ont fait état de leur coparentage et de la capacité régulatoire du nourrisson à deux semaines et à trois mois postpartum. La VFCF a modéré le lien entre le coparentage négatif de la mère et du père à deux mois postpartum et la capacité régulatoire du nourrisson à trois mois. Les nourrissons avec une VFCF basse ont présenté une capacité régulatoire plus élevée lorsque les mères ou les pères ont signalé moins de coparentage négatif, alors qu'ils ont présenté une capacité régulatoire plus basse lorsque les mères ou les pères ont signalé un coparentage plus négatif, que les nourrissons avec une VFCF élevée. Les résultats ont suggéré une VFCF plus basse comme un marqueur de sensibilité endophénotypique prénatale qui augmente l'impact de coparentage négatif sur la capacité régulatoire du nourrisson. Les enfants avec une VFCF basse peuvent être ceux qui développent mieux leurs mécanismes auto-régulatoires en présence de moins de coparentage négatif, tout en étant à haut risque de problèmes régulatoires en présence de plus de coparentage négatif.

Physiologically-based pharmacokinetic models to predict drug exposure during pregnancy.

As pregnant women are constantly exposed to drugs during pregnancy, either to treat long-term conditions or acute illnesses, drug safety is a major concern for the fetus and the mother. Clinical trials are rarely made in this population due to strict regulation and ethical reasons. However, drug pharmacokinetic (PK) parameters vary during pregnancy with an increase in distribution volume, renal clearance and more. In addition, the fetal distribution should be evaluated with the importance of placental diffusion, both active and passive. Therefore, there is a recent interest in the use of physiologically-based pharmacokinetic (PBPK) modeling to characterize these changes and complete the sparse data available on drug PK during pregnancy. Indeed, PBPK models integrate drug physicochemical and physiological parameters corresponding to each compartment of the body to estimate drug concentrations. This review establishes an overview on the current use of PBPK models in drug dosage determination for the pregnant woman, fetal exposure and drug interactions in the fetal compartment.

BEL transcription factors in prominent Solanaceae crops: the missing pieces of the jigsaw in plant development.

MAIN CONCLUSION: Understanding BEL transcription factors roles in potato and tomato varies considerably with little overlap. The review suggests reciprocal use of gained results to proceed with the knowledge in both crops The proper development of organs that plants use for reproduction, like fruits or tubers, is crucial for the survival and competitiveness of the species and thus subject to strict regulations. Interestingly, the controls of potato (Solanum tuberosum) tuber and tomato (S. lycopersicum) fruit development use common mechanisms, including the action of the BEL transcription factors (TFs). Although more than ten BEL genes have been identified in either genome, only a few of them have been characterized. The review summarizes knowledge of BEL TFs' roles in these closely related Solanaceae species, focusing on those that are essential for tuberization in potato, namely StBEL5, StBEL11 and StBEL29, and for fruit development in tomato - SlBEL11, SlBL2 and SIBL4. Comprehension of the roles of individual BEL TFs, however, is not yet sufficient. Different levels of understanding of important characteristics are described, such as BEL transcript accumulation patterns, their mobility, BEL protein interaction with KNOX partners, subcellular localisation, and their target genes during initiation and development of the organs in question. A comparison of the knowledge on BEL TFs and their mechanisms of action in potato and tomato may provide inspiration for faster progress in the study of both models through the exchange of information and ideas. Both crops are extremely important for human nutrition. In addition, their production is likely to be threatened by the upcoming climate change, so there is a particular need for breeding using a deep knowledge of control mechanisms.

A promoter trap in transgenic citrus mediates recognition of a broad spectrum of Xanthomonas citri pv. citri TALEs, including in planta-evolved derivatives.

Citrus bacterial canker (CBC), caused by Xanthomonas citri subsp. citri (Xcc), causes dramatic losses to the citrus industry worldwide. Transcription activator-like effectors (TALEs), which bind to effector binding elements (EBEs) in host promoters and activate transcription of downstream host genes, contribute significantly to Xcc virulence. The discovery of the biochemical context for the binding of TALEs to matching EBE motifs, an interaction commonly referred to as the TALE code, enabled the in silico prediction of EBEs for each TALE protein. Using the TALE code, we engineered a synthetic resistance (R) gene, called the Xcc-TALE-trap, in which 14 tandemly arranged EBEs, each capable of autonomously recognizing a particular Xcc TALE, drive the expression of Xanthomonas avrGf2, which encodes a bacterial effector that induces plant cell death. Analysis of a corresponding transgenic Duncan grapefruit showed that transcription of the cell death-inducing executor gene, avrGf2, was strictly TALE-dependent and could be activated by several different Xcc TALE proteins. Evaluation of Xcc strains from different continents showed that the Xcc-TALE-trap mediates resistance to this global panel of Xcc isolates. We also studied in planta-evolved TALEs (eTALEs) with novel DNA-binding domains and found that these eTALEs also activate the Xcc-TALE-trap, suggesting that the Xcc-TALE-trap is likely to confer durable resistance to Xcc. Finally, we show that the Xcc-TALE-trap confers resistance not only in laboratory infection assays but also in more agriculturally relevant field studies. In conclusion, transgenic plants containing the Xcc-TALE-trap offer a promising sustainable approach to control CBC.