TAL Effector

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Adam J Bogdanove - One of the best experts on this subject based on the ideXlab platform.

  • functional analysis of african xanthomonas oryzae pv oryzae TALomes reveals a new susceptibility gene in bacterial leaf blight of rice
    PLOS Pathogens, 2018
    Co-Authors: Tuan Tu Tran, Yanhua Yu, Valerie Verdier, Alvaro L Perezquintero, Issa Wonni, Sara C D Carpenter, Li Wang, Jan E Leach, Sebastien Cunnac, Adam J Bogdanove
    Abstract:

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

  • Virulence function of African X. oryzae pv. oryzae TAL Effectors delivered by X. oryzae X11-5A.
    2018
    Co-Authors: Tuan T. Tran, Valerie Verdier, Issa Wonni, Sara C D Carpenter, Li Wang, Jan E Leach, Sebastien Cunnac, Alvaro L. Pérez-quintero, Adam J Bogdanove
    Abstract:

    Mean lesion length was measured 15 days post leaf-clip inoculation of the rice variety Azucena with X. oryzae X11-5A derivatives carrying the empty vector (EV), pTALCBAI3 used as positive control, or each of the nine individual TAL Effector genes from MAI1 and four of those from BAI3 that display RVD polymorphisms relative to their MAI1 counterparts. An asterisk denotes a significant difference from X11-5A(EV) (P < 0.0001). Values represent the averages of at least 10 inoculated leaves. This experiment was repeated four times with similar results.

  • The effect of increasing numbers of repeats on TAL Effector DNA binding specificity.
    Nucleic acids research, 2017
    Co-Authors: Fabio C. Rinaldi, Barry L Stoddard, Lindsey Doyle, Adam J Bogdanove
    Abstract:

    Transcription activator-like Effectors (TALEs) recognize their DNA targets via tandem repeats, each specifying a single nucleotide base in a one-to-one sequential arrangement. Due to this modularity and their ability to bind long DNA sequences with high specificity, TALEs have been used in many applications. Contributions of individual repeat-nucleotide associations to affinity and specificity have been characterized. Here, using in vitro binding assays, we examined the relationship between the number of repeats in a TALE and its affinity, for both target and non-target DNA. Each additional repeat provides extra binding energy for the target DNA, with the gain decaying exponentially such that binding energy saturates. Affinity for non-target DNA also increases non-linearly with the number of repeats, but with a slower decay of gain. The difference between the effect of length on affinity for target versus non-target DNA manifests in specificity increasing then diminishing with increasing TALE length, peaking between 15 and 19 repeats. Modeling across different hypothetical saturation levels and rates of gain decay, reflecting different repeat compositions, yielded a similar range of specificity optima. This range encompasses the mean and median length of native TALEs, suggesting that these proteins as a group have evolved for maximum specificity.

  • TAL Effectors Drive Transcription Bidirectionally in Plants
    Molecular plant, 2016
    Co-Authors: Li Wang, Fabio C. Rinaldi, Boris Szurek, Erin L Doyle, Tuan Tu Tran, Alvaro L. Pérez-quintero, Pallavi Singh, Zoe E. Dubrow, Adam J Bogdanove
    Abstract:

    TAL Effectors delivered by phytopathogenic Xanthomonas species are DNA-sequence-specific transcriptional activators of host susceptibility genes and sometimes resistance genes. The modularity of DNA recognition by TAL Effectors makes them important also as tools for gene targeting and genome editing. Effector binding elements (EBEs) recognized by native TAL Effectors in plants have been identified only on the forward strand of target promoters. Here, we demonstrate that TAL Effectors can drive plant transcription from EBEs on either strand and in both directions. Furthermore, we show that a native TAL Effector from Xanthomonas oryzae pv. oryzicola drives expression of a target with an EBE on each strand of its promoter. By inserting that promoter and derivatives between two reporter genes oriented head to head, we show that the TAL Effector drives expression from either EBE in the respective orientations, and that activity at the reverse-strand EBE also potentiates forward transcription driven by activity at the forward-strand EBE. Our results reveal new modes of action for TAL Effectors, suggesting the possibility of yet unrecognized targets important in plant disease, expanding the search space for off-targets of custom TAL Effectors, and highlighting the potential of TAL Effectors for probing fundamenTAL aspects of plant transcription.

  • a resistance locus in the american heirloom rice variety carolina gold select is triggered by TAL Effectors with diverse predicted targets and is effective against african strains of xanthomonas oryzae pv oryzicola
    Plant Journal, 2016
    Co-Authors: Lindsay R. Triplett, Valerie Verdier, Adam J Bogdanove, Stephen P Cohen, Christopher Heffelfinger, Clarice Schmidt, Alejandra I Huerta, Cheick Tekete
    Abstract:

    The rice pathogens Xanthomonas oryzae pathovar (pv.) oryzae and pv. oryzicola produce numerous transcription activator-like (TAL) Effectors that increase bacterial virulence by activating expression of host susceptibility genes. Rice resistance mechanisms against TAL Effectors include polymorphisms that prevent Effector binding to susceptibility gene promoters, or that allow Effector activation of resistance genes. This study identifies, in the heirloom variety Carolina Gold Select, a third mechanism of rice resistance involving TAL Effectors. This resistance manifests through strong suppression of disease development in response to diverse TAL Effectors from both X. oryzae pathovars. The resistance can be triggered by an Effector with only 3.5 central repeats, is independent of the composition of the repeat variable di-residues that determine TAL Effector binding specificity, and is independent of the transcriptional activation domain. We determined that the resistance is conferred by a single dominant locus, designated Xo1, that maps to a 1.09 Mbp fragment on chromosome 4. The Xo1 interval also confers complete resistance to the strains in the African clade of X. oryzae pv. oryzicola, representing the first dominant resistance locus against bacterial leaf streak in rice. The strong phenotypic similarity between the TAL Effector-triggered resistance conferred by Xo1 and that conferred by the tomato resistance gene Bs4 suggests that monocots and dicots share an ancient or convergently evolved mechanism to recognize analogous TAL Effector epitopes.

Frank F White - One of the best experts on this subject based on the ideXlab platform.

  • an efficient method to clone TAL Effector genes from xanthomonas oryzae using gibson assembly
    Molecular Plant Pathology, 2019
    Co-Authors: Jose C Huguettapia, Frank F White, Bing Yang, Hansong Dong
    Abstract:

    Transcription Activator-Like Effectors (TALes) represent the largest family of type III Effectors among pathogenic bacteria and play a critical role in the process of infection. Strains of Xanthomonas oryzae pv. oryzae (Xoo) and some strains of other Xanthomonas pathogens contain large numbers of TALe genes. Previous techniques to clone individual or a complement of TALe genes through conventional strategies are inefficient and time-consuming due to multiple genes (up to 29 copies) in a given genome, and technically challenging due to the repetitive sequences (up to 33 nearly identical 102-nucleotide repeats) of individual TALe genes. Thus, only a limited number of TALe genes have been molecularly cloned and characterized, and the functions of most TALe genes remain unknown. Here, we present an easy and efficient cloning technique to clone TALe genes selectively through in vitro homologous recombination and single-strand annealing, and demonstrate the feasibility of this approach with four different Xoo strains. Based on the Gibson assembly strategy, two complementary vectors with scaffolds that can preferentially capture all TALe genes from a pool of genomic fragments were designed. Both vector systems enabled cloning of a full complement of TALe genes from each of four Xoo strains and functional analysis of individual TALes in rice in approximately 1 month compared to 3 months by previously used methods. The results demonstrate a robust tool to advance TALe biology and a potential for broad usage of this approach to clone multiple copies of highly competitive DNA elements in any genome of interest.

  • an efficient method to clone TAL Effector genes from xanthomonas oryzae using gibson assembly
    bioRxiv, 2019
    Co-Authors: Jose C Huguettapia, Frank F White, Hansong Dong, Bing Yang
    Abstract:

    TALes (Transcription Activator-Like Effectors) represent the largest family of type III Effectors among pathogenic bacteria and play a critical role in the process of infection. Strains of Xanthomonas oryzae pv. oryzae (Xoo) and some strains of other Xanthomonas pathogens contain large numbers of TALe genes. Previous techniques to clone individual or a complement of TALe genes through conventional strategies are inefficient and time-consuming due to multiple genes (up to 29 copies) in a given genome and technically challenging due to the repetitive sequences (up to 33 nearly identical 102-nucleotide repeats) of individual TALe genes. Thus, only a limited number of TALe genes have been molecularly cloned and characterized, and the functions of most TALe genes remain unknown. Here, we present an easy and efficient cloning technique to clone TALe genes selectively through in vitro homologous recombination and single strand annealing and demonstrate the feasibility of this approach with four different Xoo strains. Based on the Gibson assembly strategy, two complementary vectors with scaffolds that can preferentially capture all TALe genes from a pool of genomic fragments were designed. Both vector systems enabled cloning of a full complement of TALe genes from each of four Xoo strains and functional analysis of individual TALes in rice in approximately one month compared to three months by previously used methods. The results demonstrate a robust tool to advance TALe biology and a potential for broad usage of this approach to clone multiple copies of highly competitive DNA elements in any genome of interest.

  • Long read and single molecule DNA sequencing simplifies genome assembly and TAL Effector gene analysis of Xanthomonas translucens
    BMC Genomics, 2016
    Co-Authors: Zhao Peng, Ying Hu, Neha Potnis, Alina Akhunova, Jeffrey Jones, Frank F White
    Abstract:

    Background The species Xanthomonas translucens encompasses a complex of bacterial strains that cause diseases and yield loss on grass species including important cereal crops. Three pathovars, X. translucens pv . undulosa , X. translucens pv . translucens and X. translucens pv .cerealis , have been described as pathogens of wheat, barley, and oats. However, no complete genome sequence for a strain of this complex is currently available. Results A complete genome sequence of X. translucens pv . undulosa strain XT4699 was obtained by using PacBio long read, single molecule, real time (SMRT) DNA sequences and Illumina sequences. Draft genome sequences of nineteen additional X. translucens strains, which were collected from wheat or barley in different regions and at different times, were generated by Illumina sequencing. Phylogenetic relationships among different Xanthomonas strains indicates that X. translucens are members of a distinct clade from so-called group 2 xanthomonads and three pathovars of this species, undulosa , translucens and cerealis , represent distinct subclades in the group 1 clade. Knockout mutation of type III secretion system of XT4699 eliminated the ability to cause water-soaking symptoms on wheat and barley and resulted in a reduction in populations on wheat in comparison to the wild type strain. Sequence comparison of X. translucens strains revealed the genetic variation on type III Effector repertories among different pathovars or within one pathovar. The full genome sequence of XT4699 reveals the presence of eight members of the Transcription-Activator Like (TAL) Effector genes, which are phylogenetically distant from previous known TAL Effector genes of group 2 xanthomonads. Microarray and qRT-PCR analyses revealed TAL Effector-specific wheat gene expression modulation. Conclusions PacBio long read sequencing facilitates the assembly of Xanthomonas genomes and the multiple TAL Effector genes, which are difficult to assemble from short read platforms. The complete genome sequence of X. translucens pv . undulosa strain XT4699 and draft genome sequences of nineteen additional X. translucens strains provides a resource for further genetic analyses of pathogenic diversity and host range of the X. translucens species complex. TAL Effectors of XT4699 strain play roles in modulating wheat host gene expressions.

  • Long read and single molecule DNA sequencing simplifies genome assembly and TAL Effector gene analysis of Xanthomonas translucens
    BMC genomics, 2016
    Co-Authors: Zhao Peng, Frank F White, Neha Potnis, Alina Akhunova, Jingzhong Xie, Jeffrey B. Jones, Zhaohui Liu, Sanzhen Liu
    Abstract:

    The species Xanthomonas translucens encompasses a complex of bacterial strains that cause diseases and yield loss on grass species including important cereal crops. Three pathovars, X. translucens pv. undulosa, X. translucens pv. translucens and X. translucens pv.cerealis, have been described as pathogens of wheat, barley, and oats. However, no complete genome sequence for a strain of this complex is currently available. A complete genome sequence of X. translucens pv. undulosa strain XT4699 was obtained by using PacBio long read, single molecule, real time (SMRT) DNA sequences and Illumina sequences. Draft genome sequences of nineteen additional X. translucens strains, which were collected from wheat or barley in different regions and at different times, were generated by Illumina sequencing. Phylogenetic relationships among different Xanthomonas strains indicates that X. translucens are members of a distinct clade from so-called group 2 xanthomonads and three pathovars of this species, undulosa, translucens and cerealis, represent distinct subclades in the group 1 clade. Knockout mutation of type III secretion system of XT4699 eliminated the ability to cause water-soaking symptoms on wheat and barley and resulted in a reduction in populations on wheat in comparison to the wild type strain. Sequence comparison of X. translucens strains revealed the genetic variation on type III Effector repertories among different pathovars or within one pathovar. The full genome sequence of XT4699 reveals the presence of eight members of the Transcription-Activator Like (TAL) Effector genes, which are phylogenetically distant from previous known TAL Effector genes of group 2 xanthomonads. Microarray and qRT-PCR analyses revealed TAL Effector-specific wheat gene expression modulation. PacBio long read sequencing facilitates the assembly of Xanthomonas genomes and the multiple TAL Effector genes, which are difficult to assemble from short read platforms. The complete genome sequence of X. translucens pv. undulosa strain XT4699 and draft genome sequences of nineteen additional X. translucens strains provides a resource for further genetic analyses of pathogenic diversity and host range of the X. translucens species complex. TAL Effectors of XT4699 strain play roles in modulating wheat host gene expressions.

  • TAL Effectors and the executor R genes.
    Frontiers in plant science, 2015
    Co-Authors: Junli Zhang, Zhongchao Yin, Frank F White
    Abstract:

    Transcription activation-like (TAL) Effectors are bacterial type III secretion proteins that function as transcription factors in plants during Xanthomonas/plant interactions, conditioning either host susceptibility and/or host resistance. Three types of TAL Effector associated resistance (R) genes have been characterized - recessive, dominant non-transcriptional and dominant TAL Effector-dependent transcriptional based resistance. Here, we discuss the last type of R genes, whose functions are dependent on direct TAL Effector binding to discrete Effector binding elements in the promoters. Only five of the so-called executor R genes have been cloned, and commonalities are not clear. We have placed the protein products in two groups for conceptual purposes. Group 1 consists solely of the protein from pepper, BS3, which is predicted to have caTALytic function on the basis of homology to a large conserved protein family. Group 2 consists of BS4C-R, XA27, XA10, and XA23, all of which are relatively short proteins from pepper or rice with multiple potential transmembrane domains. Group 2 members have low sequence similarity to proteins of unknown function in closely related species. Firm predictions await further experimentation on these interesting new members to the R gene repertoire, which have potential broad application in new strategies for disease resistance.

Boris Szurek - One of the best experts on this subject based on the ideXlab platform.

  • Functional and Genome Sequence-Driven Characterization of TAL Effector Gene Repertoires Reveals Novel Variants With Altered Specificities in Closely Related Malian Xanthomonas oryzae pv. oryzae Strains.
    Frontiers in microbiology, 2018
    Co-Authors: Hinda Doucoure, Ralf Koebnik, Boris Szurek, Alvaro L. Pérez-quintero, Ganna Reshetnyak, C. Tekete, Florence Auguy, Emilie Thomas, Ousmane Koita, Valerie Verdier
    Abstract:

    Rice Bacterial Leaf Blight (BLB) is caused by Xanthomonas oryzae pv. oryzae (Xoo) which injects Transcription Activator-Like Effectors (TALEs) into the host cell to modulate the expression of target disease susceptibility genes. Xoo major-virulence TALEs universally target susceptibility genes of the SWEET sugar transporter family. TALE-unresponsive alleles of OsSWEET genes have been identified in the rice germplasm or created by genome editing and confer resistance to BLB. In recent years BLB has become one of the major biotic constraints to rice cultivation in Mali. To inform the deployment of alternative sources of resistance in this country, rice lines carrying alleles of OsSWEET14 unresponsive to either TALF (formerly TAL5) or TALC, two important TALEs previously identified in West African Xoo, were challenged with a panel of strains recently isolated in Mali and were found to remain susceptible to these isolates. The characterization of TALE repertoires revealed that TALF and TALC specific molecular markers were simultaneously present in all surveyed Malian strains, suggesting that the corresponding TALEs are broadly deployed by Malian Xoo to redundantly target the OsSWEET14 gene promoter. Consistent with this, the capacity of most Malian Xoo to induce OsSWEET14 was unaffected by either TALC- or TALF-unresponsive alleles of this gene. Long-read sequencing and assembly of eight Malian Xoo genomes confirmed the widespread occurrence of active TALF and TALC variants and provided a detailed insight into the diversity of TALE repertoires. All sequenced strains shared nine evolutionary related TAL Effector genes. Notably, a new TALF variant that is unable to induce OsSWEET14 was identified. Furthermore, two distinct TALB variants were shown to have lost the ability to simultaneously induce two susceptibility genes as previously reported for the founding members of this group from strains MAI1 and BAI3. Yet, both new TALB variants retained the ability to induce one or the other of the two susceptibility genes. These results reveal molecular and functional differences in TAL repertoires and will be important for the sustainable deployment of broad-spectrum and durable resistance to BLB in West Africa.

  • Efficient enrichment cloning of TAL Effector genes from Xanthomonas
    2018
    Co-Authors: Tuan Tu Tran, Mathilde Hutin, Boris Szurek, Sebastien Cunnac, Hinda Doucoure, Ralf Koebnik
    Abstract:

    Many plant-pathogenic xanthomonads use a type III secretion system to translocate Transcription Activator-Like (TAL) Effectors into eukaryotic host cells where they act as transcription factors. Target genes are induced upon binding of a TAL Effector to double-stranded DNA in a sequence-specific manner. DNA binding is governed by a highly repetitive protein domain, which consists of an array of nearly identical repeats of ca. 102 base pairs. Many species and pathovars of Xanthomonas, including pathogens of rice, cereals, cassava, citrus and cotton, encode multiple TAL Effectors in their genomes. Some of the TAL Effectors have been shown to act as key pathogenicity factors, which induce the expression of susceptibility genes to the benefit of the pathogen. However, due to the repetitive character and the presence of multiple gene copies, high-throughput cloning of TAL Effector genes remains a challenge. In order to isolate complete TAL Effector gene repertoires, we developed an enrichment cloning strategy based on (i) genome-informed in silico optimization of restriction digestions, (ii) selective restriction digestion of genomic DNA, and (iii) size fractionation of DNA fragments. Our rapid, cheap and powerful method allows efficient cloning of TAL Effector genes from xanthomonads, as demonstrated for two rice-pathogenic strains of Xanthomonas oryzae from Africa.

  • Image_4_Functional and Genome Sequence-Driven Characterization of TAL Effector Gene Repertoires Reveals Novel Variants With Altered Specificities in Closely Related Malian Xanthomonas oryzae pv. oryzae Strains.TIFF
    2018
    Co-Authors: Hinda Doucoure, Ralf Koebnik, Boris Szurek, Cheick Tekete, Alvaro L. Pérez-quintero, Ganna Reshetnyak, Florence Auguy, Emilie Thomas, Ousmane Koita, Valerie Verdier
    Abstract:

    Rice bacterial leaf blight (BLB) is caused by Xanthomonas oryzae pv. oryzae (Xoo) which injects Transcription Activator-Like Effectors (TALEs) into the host cell to modulate the expression of target disease susceptibility genes. Xoo major-virulence TALEs universally target susceptibility genes of the SWEET sugar transporter family. TALE-unresponsive alleles of OsSWEET genes have been identified in the rice germplasm or created by genome editing and confer resistance to BLB. In recent years, BLB has become one of the major biotic constraints to rice cultivation in Mali. To inform the deployment of alternative sources of resistance in this country, rice lines carrying alleles of OsSWEET14 unresponsive to either TALF (formerly TAL5) or TALC, two important TALEs previously identified in West African Xoo, were challenged with a panel of strains recently isolated in Mali and were found to remain susceptible to these isolates. The characterization of TALE repertoires revealed that TALF and TALC specific molecular markers were simultaneously present in all surveyed Malian strains, suggesting that the corresponding TALEs are broadly deployed by Malian Xoo to redundantly target the OsSWEET14 gene promoter. Consistent with this, the capacity of most Malian Xoo to induce OsSWEET14 was unaffected by either TALC- or TALF-unresponsive alleles of this gene. Long-read sequencing and assembly of eight Malian Xoo genomes confirmed the widespread occurrence of active TALF and TALC variants and provided a detailed insight into the diversity of TALE repertoires. All sequenced strains shared nine evolutionary related TAL Effector genes. Notably, a new TALF variant that is unable to induce OsSWEET14 was identified. Furthermore, two distinct TALB variants were shown to have lost the ability to simultaneously induce two susceptibility genes as previously reported for the founding members of this group from strains MAI1 and BAI3. Yet, both new TALB variants retained the ability to induce one or the other of the two susceptibility genes. These results reveal molecular and functional differences in TAL repertoires and will be important for the sustainable deployment of broad-spectrum and durable resistance to BLB in West Africa.

  • Table_1_Functional and Genome Sequence-Driven Characterization of TAL Effector Gene Repertoires Reveals Novel Variants With Altered Specificities in Closely Related Malian Xanthomonas oryzae pv. oryzae Strains.XLSX
    2018
    Co-Authors: Hinda Doucoure, Ralf Koebnik, Boris Szurek, Cheick Tekete, Alvaro L. Pérez-quintero, Ganna Reshetnyak, Florence Auguy, Emilie Thomas, Ousmane Koita, Valerie Verdier
    Abstract:

    Rice bacterial leaf blight (BLB) is caused by Xanthomonas oryzae pv. oryzae (Xoo) which injects Transcription Activator-Like Effectors (TALEs) into the host cell to modulate the expression of target disease susceptibility genes. Xoo major-virulence TALEs universally target susceptibility genes of the SWEET sugar transporter family. TALE-unresponsive alleles of OsSWEET genes have been identified in the rice germplasm or created by genome editing and confer resistance to BLB. In recent years, BLB has become one of the major biotic constraints to rice cultivation in Mali. To inform the deployment of alternative sources of resistance in this country, rice lines carrying alleles of OsSWEET14 unresponsive to either TALF (formerly TAL5) or TALC, two important TALEs previously identified in West African Xoo, were challenged with a panel of strains recently isolated in Mali and were found to remain susceptible to these isolates. The characterization of TALE repertoires revealed that TALF and TALC specific molecular markers were simultaneously present in all surveyed Malian strains, suggesting that the corresponding TALEs are broadly deployed by Malian Xoo to redundantly target the OsSWEET14 gene promoter. Consistent with this, the capacity of most Malian Xoo to induce OsSWEET14 was unaffected by either TALC- or TALF-unresponsive alleles of this gene. Long-read sequencing and assembly of eight Malian Xoo genomes confirmed the widespread occurrence of active TALF and TALC variants and provided a detailed insight into the diversity of TALE repertoires. All sequenced strains shared nine evolutionary related TAL Effector genes. Notably, a new TALF variant that is unable to induce OsSWEET14 was identified. Furthermore, two distinct TALB variants were shown to have lost the ability to simultaneously induce two susceptibility genes as previously reported for the founding members of this group from strains MAI1 and BAI3. Yet, both new TALB variants retained the ability to induce one or the other of the two susceptibility genes. These results reveal molecular and functional differences in TAL repertoires and will be important for the sustainable deployment of broad-spectrum and durable resistance to BLB in West Africa.

  • Two ancestral genes shaped the Xanthomonas campestris TAL Effector gene repertoire
    New Phytologist, 2018
    Co-Authors: Nicolas Denancé, Boris Szurek, Erin Doyle, Emmanuelle Lauber, Lisa Fontaine-bodin, Sebastien Carrère, Endrick Guy, Ahmed Hajri, Aude Cerutti, Tristan Boureau
    Abstract:

    Xanthomonas transcription activator-like Effectors (TALEs) are injected inside plant cells to promote host susceptibility by enhancing transcription of host susceptibility genes. TALE-encoding (TAL) genes were thought to be absent from Brassicaceae-infecting Xanthomonas campestris (Xc) genomes based on four reference genomic sequences. We discovered TAL genes in 26 of 49 Xc strains isolated worldwide and used a combination of single molecule real time (SMRT) and TAL amplicon sequencing to yield a near-complete description of the TALEs found in Xc (Xc TALome). The 53 sequenced TAL genes encode 21 distinct DNA binding domains that sort into seven major DNA binding specificities. In silico analysis of the Brassica rapa promoterome identified a repertoire of predicted TALE targets, five of which were experimenTALly validated using quantitative reverse transcription polymerase chain reaction. The Xc TALome shows multiple signs of DNA rearrangements that probably drove its evolution from two ancestral TAL genes. We discovered that TAL12a and TAL15a of Xcc strain Xca5 contribute together in the development of disease symptoms on susceptible B. oleracea var. botrytis cv Clovis. This large and polymorphic repertoire of TALEs opens novel perspectives for elucidating TALE-mediated susceptibility of Brassicaceae to black rot disease and for understanding the molecular processes underlying TALE evolution.

Jens Boch - One of the best experts on this subject based on the ideXlab platform.

  • TAL Effector DNA-Binding Principles and Specificity
    Methods in molecular biology (Clifton N.J.), 2016
    Co-Authors: Annekatrin Richter, Jana Streubel, Jens Boch
    Abstract:

    Transcription activator-like Effectors (TALEs) are proteins with a unique DNA-binding domain that confers both a predictable and programmable specificity. The DNA-binding domain consists typically of 34-amino acid near-identical repeats. The repeats form a right-handed superhelical structure that wraps around the DNA double helix and exposes the variable amino acids at position 13 of each repeat to the sense strand DNA bases. Each repeat binds one base in a highly specific, non-overlapping, and comma-free fashion. Although TALE specificities are encoded in a simple way, sophisticated rules can be taken into account to build highly efficient DNA-binding modules for biotechnological use.

  • A TAL Effector repeat architecture for frameshift binding
    Nature communications, 2014
    Co-Authors: Annekatrin Richter, Jana Streubel, Boris Szurek, Jan Grau, Maik Reschke, Christina Blücher, Jens Boch
    Abstract:

    Transcription activator-like Effectors (TALEs) are important Xanthomonas virulence factors that bind DNA via a unique tandem 34-amino-acid repeat domain to induce expression of plant genes. So far, TALE repeats are described to bind as a consecutive array to a consecutive DNA sequence, in which each repeat independently recognizes a single DNA base. This modular protein architecture enables the design of any desired DNA-binding specificity for biotechnology applications. Here we report that natural TALE repeats of unusual amino-acid sequence length break the strict one repeat-to-one base pair binding mode and introduce a local flexibility to TALE-DNA binding. This flexibility allows TALEs and TALE nucleases to recognize target sequence variants with single nucleotide deletions. The flexibility also allows TALEs to activate transcription at allelic promoters that otherwise confer resistance to the host plant.

  • five phylogenetically close rice sweet genes confer TAL Effector mediated susceptibility to xanthomonas oryzae pv oryzae
    New Phytologist, 2013
    Co-Authors: Jana Streubel, Mathilde Hutin, Ralf Koebnik, Jens Boch, Celine Pesce, Boris Szurek
    Abstract:

    Bacterial plant-pathogenic Xanthomonas strains translocate transcription activator-like (TAL) Effectors into plant cells to function as specific transcription factors. Only a few plant target genes of TAL Effectors have been identified, so far. Three plant SWEET genes encoding putative sugar transporters are known to be induced by TAL Effectors from rice-pathogenic Xanthomonas oryzae pv. oryzae (Xoo). We predict and validate that expression of OsSWEET14 is induced by a novel TAL Effector, TAL5, from an African Xoo strain. Artificial TAL Effectors (ArtTALs) were constructed to individually target 20 SWEET orthologs in rice. They were used as designer virulence factors to study which rice SWEET genes can support Xoo virulence. The TAL5 target box differs from those of the already known TAL Effectors TALC, AvrXa7 and PthXo3, which also induce expression of OsSWEET14, suggesting evolutionary convergence on key targets. ArtTALs efficiently complemented an Xoo TALC mutant, demonstrating that specific induction of OsSWEET14 is the key target of TALC. ArtTALs that specifically target individual members of the rice SWEET family revealed three known and two novel SWEET genes to support bacterial virulence. Our results demonstrate that five phylogenetically close SWEET proteins, which presumably act as sucrose transporters, can support Xoo virulence. © 2013 No claim to French Government works. © 2013 New Phytologist Trust.

  • five phylogenetically close rice sweet genes confer TAL Effector mediated susceptibility to xanthomonas oryzae pv oryzae
    New Phytologist, 2013
    Co-Authors: Jana Streubel, Mathilde Hutin, Ralf Koebnik, Jens Boch, Celine Pesce, Boris Szurek
    Abstract:

    Bacterial plant-pathogenic Xanthomonas strains translocate transcription activator-like (TAL) Effectors into plant cells to function as specific transcription factors. Only a few plant target genes of TAL Effectors have been identified, so far. Three plant SWEET genes encoding putative sugar transporters are known to be induced by TAL Effectors from rice-pathogenic Xanthomonas oryzae pv. oryzae (Xoo). We predict and validate that expression of OsSWEET14 is induced by a novel TAL Effector, TAL5, from an African Xoo strain. Artificial TAL Effectors (ArtTALs) were constructed to individually target 20 SWEET orthologs in rice. They were used as designer virulence factors to study which rice SWEET genes can support Xoo virulence. The TAL5 target box differs from those of the already known TAL Effectors TALC, AvrXa7 and PthXo3, which also induce expression of OsSWEET14, suggesting evolutionary convergence on key targets. ArtTALs efficiently complemented an Xoo TALC mutant, demonstrating that specific induction of OsSWEET14 is the key target of TALC. ArtTALs that specifically target individual members of the rice SWEET family revealed three known and two novel SWEET genes to support bacterial virulence. Our results demonstrate that five phylogenetically close SWEET proteins, which presumably act as sucrose transporters, can support Xoo virulence. © 2013 No claim to French Government works. © 2013 New Phytologist Trust.

  • Five phylogenetically close rice SWEET genes confer TAL Effector‐mediated susceptibility to Xanthomonas oryzae pv. oryzae
    The New phytologist, 2013
    Co-Authors: Jana Streubel, Mathilde Hutin, Ralf Koebnik, Jens Boch, Celine Pesce, Boris Szurek
    Abstract:

    Bacterial plant-pathogenic Xanthomonas strains translocate transcription activator-like (TAL) Effectors into plant cells to function as specific transcription factors. Only a few plant target genes of TAL Effectors have been identified, so far. Three plant SWEET genes encoding putative sugar transporters are known to be induced by TAL Effectors from rice-pathogenic Xanthomonas oryzae pv. oryzae (Xoo). We predict and validate that expression of OsSWEET14 is induced by a novel TAL Effector, TAL5, from an African Xoo strain. Artificial TAL Effectors (ArtTALs) were constructed to individually target 20 SWEET orthologs in rice. They were used as designer virulence factors to study which rice SWEET genes can support Xoo virulence. The TAL5 target box differs from those of the already known TAL Effectors TALC, AvrXa7 and PthXo3, which also induce expression of OsSWEET14, suggesting evolutionary convergence on key targets. ArtTALs efficiently complemented an Xoo TALC mutant, demonstrating that specific induction of OsSWEET14 is the key target of TALC. ArtTALs that specifically target individual members of the rice SWEET family revealed three known and two novel SWEET genes to support bacterial virulence. Our results demonstrate that five phylogenetically close SWEET proteins, which presumably act as sucrose transporters, can support Xoo virulence. © 2013 No claim to French Government works. © 2013 New Phytologist Trust.

Daniel F Voytas - One of the best experts on this subject based on the ideXlab platform.

  • non transgenic plant genome editing using purified sequence specific nucleases
    Molecular Plant, 2015
    Co-Authors: Song Luo, Thomas Stoddard, Nicholas J Baltes, Zachary L Demorest, Benjamin M Clasen, Andrew Coffman, Adam Retterath, Luc Mathis, Daniel F Voytas, Feng Zhang
    Abstract:

    Sequence-specific nucleases, including zinc-finger nucleases, meganucleases, TAL Effector nucleases (TALENs), and CRISPR/Cas systems, have been used to introduce targeted mutations in a wide range of plant species (Voytas, 2013; Baltes and Voytas, 2015). However, delivery of these nucleases using traditional transformation methods (e.g., particle bombardment, Agrobacterium or protoplast transformation) may result in undesired genetic alterations due to random insertion of nuclease-encoding DNA into the host genome.

  • Engineered TAL Effector Proteins: Versatile Reagents for Manipulating Plant Genomes
    Advances in New Technology for Targeted Modification of Plant Genomes, 2015
    Co-Authors: Michelle Christian, Daniel F Voytas
    Abstract:

    Transcription activator-like (TAL) Effectors are proteins produced by plant pathogens of the genus Xanthomonas. They are delivered to plant cells during infection and bind to specific plant gene promoters to activate transcription and promote bacterial infection. DNA binding by TAL Effectors is mediated by an array of typically 14–24 repeats; each repeat is 34 amino acids in length and folds into a hairpin-like structure that contacts a single base in the target DNA. The TAL Effector DNA-binding motif has proven highly modular, and custom TAL Effector arrays can be made to recognize virtually any site in a plant genome, thereby providing a valuable reagent for genome manipulation. In particular, when TAL Effector arrays are fused to a nuclease, they can create targeted double-strand breaks at a locus of interest. The repair of the breaks can be directed to achieve a variety of targeted genome modifications, with applications ranging from understanding plant gene function to creating novel traits in agronomically important crop species.

  • Schematics of the four classes of sequence-specific nucleases.
    2014
    Co-Authors: Daniel F Voytas, Caixia Gao
    Abstract:

    (A) The meganuclease, I-SceI, is shown bound to its DNA target. The caTALytic domain, which also determines DNA sequence specificity, is shown in red. (B) A ZFN dimer is illustrated bound to DNA. ZFN targets are bound by two zinc-finger DNA binding domains (dark blue) separated by a 5–7-bp spacer sequence. FokI cleavage occurs within the spacer. Each zinc finger typically recognizes 3 bp. (C) Depicted is a TALEN dimer bound to DNA. The DNA binding domains are in dark blue. The two TALEN target sites are typically separated by a 15–20-bp spacer sequence. Like ZFNs, the TAL Effector repeat arrays are fused to FokI. Each TAL Effector motif recognizes one base. (D) The CRISPR/Cas9 system recognizes DNA through base pairing between DNA sequences at the target site and a CRISPR-based guide RNA (gRNA). Cas9 has two nuclease domains (shown by red arrowheads) that each cleave one strand of double-stranded DNA.

  • TAL Effector specificity for base 0 of the DNA target is altered in a complex, Effector- and assay-dependent manner by substitutions for the tryptophan in cryptic repeat -1.
    PloS one, 2013
    Co-Authors: Erin L Doyle, Zachary L Demorest, Daniel F Voytas, Aaron W. Hummel, Colby G. Starker, Philip Bradley, Adam J Bogdanove
    Abstract:

    TAL Effectors are re-targetable transcription factors used for tailored gene regulation and, as TAL Effector-nuclease fusions (TALENs), for genome engineering. Their hallmark feature is a customizable central string of polymorphic amino acid repeats that interact one-to-one with individual DNA bases to specify the target. Sequences targeted by TAL Effector repeats in nature are nearly all directly preceded by a thymine (T) that is required for maximal activity, and target sites for custom TAL Effector constructs have typically been selected with this constraint. Multiple crysTAL structures suggest that this requirement for T at base 0 is encoded by a tryptophan residue (W232) in a cryptic repeat N-terminal to the central repeats that exhibits energetically favorable van der Waals contacts with the T. We generated variants based on TAL Effector PthXo1 with all single amino acid substitutions for W232. In a transcriptional activation assay, many substitutions altered or relaxed the specificity for T and a few were as active as wild type. Some showed higher activity. However, when replicated in a different TAL Effector, the effects of the substitutions differed. Further, the effects differed when tested in the context of a TALEN in a DNA cleavage assay, and in a TAL Effector-DNA binding assay. Substitution of the N-terminal region of the PthXo1 construct with that of one of the TAL Effector-like proteins of Ralstonia solanacearum, which have arginine in place of the tryptophan, resulted in specificity for guanine as the 5’ base but low activity, and several substitutions for the arginine, including tryptophan, destroyed activity altogether. Thus, the effects on specificity and activity generated by substitutions at the W232 (or equivalent) position are complex and context dependent. Generating TAL Effector scaffolds with high activity that robustly accommodate sites without a T at position 0 may require larger scale re-engineering.

  • Targeted Mutagenesis of Arabidopsis thaliana Using Engineered TAL Effector Nucleases
    G3 (Bethesda Md.), 2013
    Co-Authors: Michelle Christian, Yong Zhang, Daniel F Voytas
    Abstract:

    Custom TAL Effector nucleases (TALENs) are increasingly used as reagents to manipulate genomes in vivo. Here, we used TALENs to modify the genome of the model plant, Arabidopsis thaliana. We engineered seven TALENs targeting five Arabidopsis genes, namely ADH1, TT4, MAPKKK1, DSK2B, and NATA2. In pooled seedlings expressing the TALENs, we observed somatic mutagenesis frequencies ranging from 2–15% at the intended targets for all seven TALENs. Somatic mutagenesis frequencies as high as 41–73% were observed in individual transgenic plant lines expressing the TALENs. Additionally, a TALEN pair targeting a tandemly duplicated gene induced a 4.4-kb deletion in somatic cells. For the most active TALEN pairs, namely those targeting ADH1 and NATA2, we found that TALEN-induced mutations were transmitted to the next generation at frequencies of 1.5–12%. Our work demonstrates that TALENs are useful reagents for achieving targeted mutagenesis in this important plant model.