Repeat Variable Diresidue Research Articles

Complete Genome Sequence Resource for Xanthomonas translucens pv. undulosa MAI5034, a Wheat Pathogen from Uruguay.

Complete Genome Sequence Resource for Xanthomonas translucens pv. undulosa MAI5034, a Wheat Pathogen from Uruguay.

A varied AvrXa23-like TALE enables the bacterial blight pathogen to avoid being trapped by Xa23 resistance gene in rice

IntroductionXa23 as an executor mediates broad-spectrum resistance to Xanthomonas oryzae pv. oryzae (Xoo), which contains a matching avirulence gene avrXa23, in rice for bacterial leaf blight (BLB). avrXa23 encodes a transcription activator-like effector (TALE) protein which binds to the EBE (effector-binding element) of the Xa23 promoter. It is unclear whether the considerable pressure of Xa23 leads to an emerging Xoo strain that overcomes Xa23 resistance. ObjectivesThis study aimed to uncover new Xoo isolate(s) that overcome Xa23-mediated resistance and to investigate how the pathogen evades the resistance. MethodsTotally 185 Xoo isolates were used to screen possibly compatible strain(s) with Xa23-containing rice CBB23 by pathogenicity test. Genome Sequencing, Southern blot, tal gene cloning, Western blot, qRT-PCR and electrophoretic mobility shift assays (EMSA) were conducted to determine the mechanism of one Xoo isolate being compatible with Xa23-containing rice. ResultsOne isolate AH28 from Anhui province is compatible with CBB23. AH28 strain contains an ortholog of avrXa23, tal7b and has 17 tal genes. The 4th RVD (repeat-variable diresidue) in Tal7b are missed and the 5th and 8th RVDs changed from NG and NS to NS and S*, respectively. These alternations made Tal7b unable to bind to the EBE of Xa23 promoter to activate the expression of Xa23 in rice. The ectopic expression of tal7b in a tal-free mutant PH of PXO99A did not alter the virulence of the strain PH, whereas avrXa23 made AH28 from compatibility to incompatibility with Xa23 rice. ConclusionBest to our knowledge, this is the first insight of a naturally-emerging Xoo isolate that overcomes the broad-spectrum resistance of Xa23 by the variable AvrXa23-like TALE Tal7b. The RVD alteration in AvrXa23 may be a common strategy for the pathogen evolution to avoid being “trapped” by the executor R gene.

Pan-Genome Analysis of Effectors in Korean Strains of the Soybean Pathogen Xanthomonas citri pv. glycines.

Xanthomonas citri pv. glycines is a major pathogen of soybean in Korea. Here, we analyzed pathogenicity genes based on a comparative genome analysis of five Korean strains and one strain from the United States, 8ra. Whereas all six strains had nearly identical profiles of carbohydrate-active enzymes, they varied in diversity and number of candidate type III secretion system effector (T3SE) genes. The five Korean strains were similar in their effectors, but differed from the 8ra strain. Across the six strains, transcription activator-like effectors (TALEs) showed diverse repeat sizes and at least six forms of the repeat variable di-residue (RVD) sequences, with differences not correlated with the origin of the strains. However, a phylogenetic tree based on the alignment of RVD sequences showed two distinct clusters with 17.5 repeats, suggesting that two distinct 17.5 RVD clusters have evolved, potentially to adapt Xcg to growth on distinct soybean cultivars. The predicted effector binding elements of the TALEs fell into six groups and were strongly overlapping in sequence, suggesting evolving target specificity of the binding domains in soybean cultivars. Our findings reveal the variability and adaptability of T3SEs in the Xcg strains and enhance our understanding of Xcg pathogenicity in soybean.

Genomics-Enabled Novel Insight Into the Pathovar-Specific Population Structure of the Bacterial Leaf Streak Pathogen Xanthomonas translucens in Small Grain Cereals.

The Gram-negative bacterium Xanthomonas translucens infects a wide range of gramineous plants with a notable impact on small grain cereals. However, genomics-informed intra-species population structure and virulence repertories of the pathogen have rarely been investigated. In this study, the complete genome sequences of seven X. translucens strains representing an entire set of genetic diversity of two pathovars X. translucens pv. undulosa and X. translucens pv. translucens is provided and compared with those of seven publicly available complete genomes of the pathogen. Organization of the 25 type III secretion system genes in all the 14 X. translucens strains was exactly the same, while TAL effector genes localized singly or in clusters across four loci in X. translucens pv. translucens and five to six loci in X. translucens pv. undulosa. Beside two previously unreported endogenous plasmids in X. translucens pv. undulosa, and variations in repeat variable diresidue (RVD) of the 14 strains, tal1a of X. translucens pv. translucens strain XtKm8 encode the new RVDs HE and YI which have not previously been reported in xanthomonads. Further, a number of truncated tal genes were predicted among the 14 genomes lacking conserved BamHI site at N-terminus and SphI site at C-terminus. Our data have doubled the number of complete genomes of X. translucens clarifying the population structure and genomics of the pathogen to pave the way in the small grain cereals industry for disease resistance breeding in the 21st century’s agriculture.

Context and number of noncanonical repeat variable diresidues impede the design of TALE proteins with improved DNA targeting.

Transcription activator-like effector (TALE) proteins have been used extensively for targeted binding of fusion proteins to loci of interest in (epi)genome engineering. Such approaches typically utilize four canonical TALE repeat variable diresidue (RVD) types, corresponding to the identities of two key amino acids, to target each nucleotide. Alternate RVDs with improved specificity are desired. Here, we focused on seven noncanonical RVDs that have been suggested to have improved specificity for their target nucleotides. We used custom protein binding microarrays to characterize the DNA-binding activity of 65 TALEs containing these alternate or corresponding canonical RVDs at multiple positions to ~5,000 unique DNA sequences per protein. We found that none of the noncanonical thymine-targeting RVDs displayed stronger preference for thymine than did the canonical RVD. Of the noncanonical RVDs with putatively improved specificity for guanine, only EN and NH showed greater discrimination of guanine over adenine. This improved specificity, however, comes at a cost: more substitutions of a noncanonical RVD for a canonical RVD generally decreased the protein's DNA-binding activity. Our results highlight the need to investigate RVD-nucleotide specificities in multiple protein contexts and suggest that a balance between canonical and noncanonical RVDs is needed to build TALEs with improved specificity.

Deciphering TAL effectors for 5-methylcytosine and 5-hydroxymethylcytosine recognition

DNA recognition by transcription activator-like effector (TALE) proteins is mediated by tandem repeats that specify nucleotides through repeat-variable diresidues. These repeat-variable diresidues form direct and sequence-specific contacts to DNA bases; hence, TALE–DNA interaction is sensitive to DNA chemical modifications. Here we conduct a thorough investigation, covering all theoretical repeat-variable diresidue combinations, for their recognition capabilities for 5-methylcytosine and 5-hydroxymethylcytosine, two important epigenetic markers in higher eukaryotes. We identify both specific and degenerate repeat-variable diresidues for 5-methylcytosine and 5-hydroxymethylcytosine. Utilizing these novel repeat-variable diresidues, we achieve methylation-dependent gene activation and genome editing in vivo; we also report base-resolution detection of 5hmC in an in vitro assay. Our work deciphers repeat-variable diresidues for 5-methylcytosine and 5-hydroxymethylcytosine, and provides tools for TALE-dependent epigenome recognition.

Evolution of Transcription Activator-Like Effectors in Xanthomonas oryzae.

Transcription activator-like effectors (TALEs) are secreted by plant–pathogenic Xanthomonas bacteria into plant cells where they act as transcriptional activators and, hence, are major drivers in reprogramming the plant for the benefit of the pathogen. TALEs possess a highly repetitive DNA-binding domain of typically 34 amino acid (AA) tandem repeats, where AA 12 and 13, termed repeat variable di-residue (RVD), determine target specificity. Different Xanthomonas strains possess different repertoires of TALEs. Here, we study the evolution of TALEs from the level of RVDs determining target specificity down to the level of DNA sequence with focus on rice-pathogenic Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc) strains. We observe that codon pairs coding for individual RVDs are conserved to a similar degree as the flanking repeat sequence. We find strong indications that TALEs may evolve 1) by base substitutions in codon pairs coding for RVDs, 2) by recombination of N-terminal or C-terminal regions of existing TALEs, or 3) by deletion of individual TALE repeats, and we propose possible mechanisms. We find indications that the reassortment of TALE genes in clusters is mediated by an integron-like mechanism in Xoc. We finally study the effect of the presence/absence and evolutionary modifications of TALEs on transcriptional activation of putative target genes in rice, and find that even single RVD swaps may lead to considerable differences in activation. This correlation allowed a refined prediction of TALE targets, which is the crucial step to decipher their virulence activity.

The N6-Position of Adenine Is a Blind Spot for TAL-Effectors That Enables Effective Binding of Methylated and Fluorophore-Labeled DNA.

Transcription-activator-like effectors (TALEs) are programmable DNA binding proteins widely used for genome targeting. TALEs consist of multiple concatenated repeats, each selectively recognizing one nucleobase via a defined repeat variable diresidue (RVD). Effective use of TALEs requires knowledge about their binding ability to epigenetic and other modified nucleobases occurring in target DNA. However, aside from epigenetic cytosine-5 modifications, the binding ability of TALEs to modified DNA is unknown. We here study the binding of TALEs to the epigenetic nucleobase N6-methyladenine (6mA) found in prokaryotic and recently also eukaryotic genomes. We find that the natural, adenine (A)-binding RVD NI is insensitive to 6mA. Model-assisted structure-function studies reveal accommodation of 6mA by RVDs with altered hydrophobic surfaces and abilities of hydrogen bonding to the N6-amino group or N7 atom of A. Surprisingly, this tolerance of N6 substitution was transferrable to bulky N6-alkynyl substituents usable for click chemistry and even to a large rhodamine dye, establishing the N6 position of A as the first site of DNA that offers label introduction within TALE target sites without interference. These findings will guide future in vivo studies with TALEs and expand their applicability as DNA capture probes for analytical applications in vitro.

Non-RVD mutations that enhance the dynamics of the TAL repeat array along the superhelical axis improve TALEN genome editing efficacy.

Transcription activator-like effector (TALE) nuclease (TALEN) is widely used as a tool in genome editing. The DNA binding part of TALEN consists of a tandem array of TAL-repeats that form a right-handed superhelix. Each TAL-repeat recognises a specific base by the repeat variable diresidue (RVD) at positions 12 and 13. TALEN comprising the TAL-repeats with periodic mutations to residues at positions 4 and 32 (non-RVD sites) in each repeat (VT-TALE) exhibits increased efficacy in genome editing compared with a counterpart without the mutations (CT-TALE). The molecular basis for the elevated efficacy is unknown. In this report, comparison of the physicochemical properties between CT- and VT-TALEs revealed that VT-TALE has a larger amplitude motion along the superhelical axis (superhelical motion) compared with CT-TALE. The greater superhelical motion in VT-TALE enabled more TAL-repeats to engage in the target sequence recognition compared with CT-TALE. The extended sequence recognition by the TAL-repeats improves site specificity with limiting the spatial distribution of FokI domains to facilitate their dimerization at the desired site. Molecular dynamics simulations revealed that the non-RVD mutations alter inter-repeat hydrogen bonding to amplify the superhelical motion of VT-TALE. The TALEN activity is associated with the inter-repeat hydrogen bonding among the TAL repeats.

TALE-Like Effectors Are an Ancestral Feature of the Ralstonia solanacearum Species Complex and Converge in DNA Targeting Specificity.

Ralstonia solanacearum, a species complex of bacterial plant pathogens divided into four monophyletic phylotypes, causes plant diseases in tropical climates around the world. Some strains exhibit a broad host range on solanaceous hosts, while others are highly host-specific as for example some banana-pathogenic strains. Previous studies showed that transcription activator-like (TAL) effectors from Ralstonia, termed RipTALs, are capable of activating reporter genes in planta, if these are preceded by a matching effector binding element (EBE). RipTALs target DNA via their central repeat domain (CRD), where one repeat pairs with one DNA-base of the given EBE. The repeat variable diresidue dictates base repeat specificity in a predictable fashion, known as the TALE code. In this work, we analyze RipTALs across all phylotypes of the Ralstonia solanacearum species complex. We find that RipTALs are prevalent in phylotypes I and IV but absent from most phylotype III and II strains (10/12, 8/14, 1/24, and 1/5 strains contained a RipTAL, respectively). RipTALs originating from strains of the same phylotype show high levels of sequence similarity (>98%) in the N-terminal and C-terminal regions, while RipTALs isolated from different phylotypes show 47–91% sequence similarity in those regions, giving rise to four RipTAL classes. We show that, despite sequence divergence, the base preference for guanine, mediated by the N-terminal region, is conserved across RipTALs of all classes. Using the number and order of repeats found in the CRD, we functionally sub-classify RipTALs, introduce a new simple nomenclature, and predict matching EBEs for all seven distinct RipTALs identified. We experimentally study RipTAL EBEs and uncover that some RipTALs are able to target the EBEs of other RipTALs, referred to as cross-reactivity. In particular, RipTALs from strains with a broad host range on solanaceous hosts cross-react on each other’s EBEs. Investigation of sequence divergence between RipTAL repeats allows for a reconstruction of repeat array biogenesis, for example through slipped strand mispairing or gene conversion. Using these studies we show how RipTALs of broad host range strains evolved convergently toward a shared target sequence. Finally, we discuss the differences between TALE-likes of plant pathogens in the context of disease ecology.

BioSpotlight / Citations

BioSpotlight / Citations

Rapid and accurate synthesis of TALE genes from synthetic oligonucleotides.

Custom synthesis of transcription activator-like effector (TALE) genes has relied upon plasmid libraries of pre-fabricated TALE-repeat monomers or oligomers. Here we describe a novel synthesis method that directly incorporates annealed synthetic oligonucleotides into the TALE-repeat units. Our approach utilizes iterative sets of oligonucleotides and a translational frame check strategy to ensure the high efficiency and accuracy of TALE-gene synthesis. TALE arrays of more than 20 repeats can be constructed, and the majority of the synthesized constructs have perfect sequences. In addition, this novel oligonucleotide-based method can readily accommodate design changes to the TALE repeats. We demonstrated an increased gene targeting efficiency against a genomic site containing a potentially methylated cytosine by incorporating non-conventional repeat variable di-residue (RVD) sequences.

Investigating the Kinetics and Specificity of Transcription Activator-Like Effector Binding using High Throughput Single Molecule Imaging

Transcription activator-like effectors (TALEs) are proteins secreted by Xanthomonas bacteria which bind to specific sequences of DNA. The TALE DNA binding domain is composed of repeats of 33-34 highly conserved amino acids with two variable amino acid residues at positions 12 and 13, known as the repeat variable diresidue (RVD). By varying the number of amino acid sequence repeats and the two critical amino acids of each RVD, any DNA sequence can be targeted with high affinity using TALEs. This flexibility makes these designer TALEs (dTALEs) useful for locating and editing sites in the genome and for fluorescently labeling specific loci in the nuclei of live cells. To better understand the binding kinetics, target specificity and site search mechanisms used by TALEs, we have applied a DNA flow cell device to trap and extend DNA molecules under a hydrodynamic force in the presence of engineered TALEs. Known as DNA curtains, these devices provide a high throughput method of imaging TALE protein-DNA interactions at the single molecule level. Using this technique, we are able to monitor TALE binding, site occupation probabilities and protein off-rates.

Megatal, Crispr/Cas9, and Talen T-Cell Receptor Gene Editing

T-cell based immunotherapy holds great promise for targeted tumor therapies and includes the use of tumor infiltrating lymphocytes or cells engineered to express chimeric antigen receptors. Each have resulted in promising therapeutic responses; however, they are largely reliant on autologous, ex vivo cellular expansion, manipulation, and reinfusion. This individualized process may result in non-uniformity and may be limiting for urgent and widespread use. An attractive strategy would be to generate a universal donor pool of cells at sufficient scale to be broadly applicable. Allogeneic approaches to this hold the risk of off tumor reactivity toward host tissue as part of graft versus host disease (GVHD), a pathogenic process mediated by the T-cell receptor (TCR). The TCR is a heterodimeric complex comprised of alpha and beta chains encoded by the TRAC and TRBC genes, respectively. Because the alpha chain is encoded by a single gene, while the beta chain is encoded by two, it has become a prime candidate for genome editing strategies aimed at disrupting the formation of the TCR and diminishing the risk of GVHD.Four major classes of gene editing proteins exist that share the commonality of binding a target sequence of DNA and generating a double stranded DNA break (DSB). The DSB is subsequently repaired by non- homologous endjoining an error prone pathway resulting in insertions/deletions that can disrupt open reading frames. Zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) are heterodimeric arrays that are tethered to the Fok I nuclease domain that co-localize at a target DNA site. ZFNs are comprised of 3-6 finger subunits that bind DNA and TALENs are comprised of repeating DNA binding units containing a hypervariable two amino acid sequence (repeat variable diresidue; RVD) that governs DNA base recognition. Meganucleases are monomeric proteins with innate nuclease activity that are derived from bacterial homing endonucleases that have been fused with TAL RVDs to generate megaTAL (MT) proteins. The clustered regularly interspaced short palindromic repeats (CRISPR) and associated Cas9 nuclease platform is comprised of a small guide RNA (gRNA) transcript that contacts a target DNA sequence via Watson-Crick base pairing and the Cas9 nuclease that cleaves the DNA.No evaluation has been performed between the different platforms for TCR disruption in the context of a translatable manufacturing process. Critical components of such an evaluation are reagent optimization, interrogation of off-target (OT) effects, and scalable expansion of gene edited cell populations. Utilizing Jurkat cells we observed that polyadenylated TALEN and MT mRNA and Cas9 with a gRNA encoded on a plasmid were the most effective in TRAC gene disruption (Fig 1A). A comparison of the optimized expression platforms in primary human T-cells revealed maximal editing rates were achieved with the MT reagent (Fig 1B). We then sorted the TCR negative cells to purity and re-introduced the TRAC gene mRNA that resulted in transient reconstitution of the CD3 complex that allowed for CD3/CD28 bead stimulation and a 5-fold expansion (Fig 1C). This represents a pool of TCR null cells for archival. Finally, we performed a genome level assessment of the potential off target (OT) activity of the nuclease candidates. Surprisingly, the CRISPR/Cas9 and TALEN reagents did not display OT effects (Fig 2A). At saturation levels of MT treatment we observed one bona fide OT site in both Jurkats and primary T-cells (Fig 2B); importantly, this occurred within an intron and did not impact proper gene splicing or cellular viability.In summary, we evaluated three different TRAC -targeting nucleases, and developed an optimized process for generating and expanding TCR/CD3-deficient cells. Our study demonstrates the utility of performing unbiased genome wide assessment of editing for identification of off target cleavage, and the importance of achieving a high efficiency of gene editing and cellular expansion to facilitate clinical translation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

QueTAL: a suite of tools to classify and compare TAL effectors functionally and phylogenetically.

Transcription Activator-Like (TAL) effectors from Xanthomonas plant pathogenic bacteria can bind to the promoter region of plant genes and induce their expression. DNA-binding specificity is governed by a central domain made of nearly identical repeats, each determining the recognition of one base pair via two amino acid residues (a.k.a. Repeat Variable Di-residue, or RVD). Knowing how TAL effectors differ from each other within and between strains would be useful to infer functional and evolutionary relationships, but their repetitive nature precludes reliable use of traditional alignment methods. The suite QueTAL was therefore developed to offer tailored tools for comparison of TAL effector genes. The program DisTAL considers each repeat as a unit, transforms a TAL effector sequence into a sequence of coded repeats and makes pair-wise alignments between these coded sequences to construct trees. The program FuncTAL is aimed at finding TAL effectors with similar DNA-binding capabilities. It calculates correlations between position weight matrices of potential target DNA sequence predicted from the RVD sequence, and builds trees based on these correlations. The programs accurately represented phylogenetic and functional relationships between TAL effectors using either simulated or literature-curated data. When using the programs on a large set of TAL effector sequences, the DisTAL tree largely reflected the expected species phylogeny. In contrast, FuncTAL showed that TAL effectors with similar binding capabilities can be found between phylogenetically distant taxa. This suite will help users to rapidly analyse any TAL effector genes of interest and compare them to other available TAL genes and should improve our understanding of TAL effectors evolution. It is available at http://bioinfo-web.mpl.ird.fr/cgi-bin2/quetal/quetal.cgi.

334. Improved MegaTAL Endonuclease Engineering Strategy for Gene Modification Applications

MegaTALs (MTALs) comprise a novel rare cutting endonuclease platform that couples a transcription activator-like effector (TALE) DNA binding domain with a homing endonuclease (HE) cleavage domain. MTALs are well suited for clinical translation due to their compact size coupled with high cleavage efficacy and specificity in gene modification applications. However, neither the relative contribution of the TALE domain vs. HE specificity to MTAL in vivo cleavage activity, nor the optimal strategy for MTAL engineering, have been rigorously explored. In the current study, we engineered multiple HE I-Anil variants with different properties, targeted to recognize a unique site in the human Bruton's tyrosine kinase (BTK) gene using a yeast surface display-based high throughput selection system. These BTK-Ani variants were combined with a TALE DNA binding domain containing an 11 repeat variable diresidue (RVD) array recognizing a specific sequence upstream of the BTK target site. The in vivo cleavage efficacy of this series of BTK MTAL enzymes was tested in a mammalian cell line report system. Here, we demonstrate that the turnover rate (Kcat) of the HE represents the most important contributing factor for MTAL activity in cellulo. This finding contrasts with activity profiling of HEs alone, where HE binding affinity and in vitro cleavage activity represent the most important factors predicting in vivo activity. Based on this observation, we optimized our engineering strategy to directly select for HE variants with higher Kcat activity. This modification significantly improved the post-selection output leading to engineered BTK MTALs with higher in vivo enzyme activity. Our emerging data demonstrates that the MTAL platform facilitates high rates of homology-directed-repair in primary human hematopoietic cells. Thus, this improved engineering platform should facilitate rapid development of new reagents for clinical genome editing applications.

Improved specificity of TALE-based genome editing using an expanded RVD repertoire.

Transcription activator-like effector (TALE) proteins have gained broad appeal as a platform for targeted DNA recognition, largely owing to their simple rules for design. These rules relate the base specified by a single TALE repeat to the identity of two key residues (the repeat variable diresidue, or RVD) and enable design for new sequence targets via modular shuffling of these units. A key limitation of these rules is that their simplicity precludes options for improving designs that are insufficiently active or specific. Here we address this limitation by developing an expanded set of RVDs and applying them to improve the performance of previously described TALEs. As an extreme example, total conversion of a TALE nuclease to new RVDs substantially reduced off-target cleavage in cellular studies. By providing new RVDs and design strategies, these studies establish options for developing improved TALEs for broader application across medicine and biotechnology.

A multiplexed transcription activator-like effector system for detecting specific DNA sequences.

Transcription activator-like effectors (TALEs), originating from the Xanthomonas genus of bacteria, bind to specific DNA sequences based on amino acid sequence in the repeat-variable diresidue (RVD) positions of the protein. By altering these RVDs, it has been shown that a TALE protein can be engineered to bind virtually any DNA sequence of interest. The possibility of multiplexing TALEs for the purposes of identifying specific DNA sequences has yet to be explored. Here, we demonstrate a system in which a TALE protein bound to a nitrocellulose strip has been utilized to capture purified DNA, which is then detected using the binding of a second distinct TALE protein conjugated to a protein tag that is then detected by a dot blot. This system provides a signal only when both TALEs bind to their respective sequences, further demonstrating the specificity of the TALE binding.

Repeat 1 of TAL effectors affects target specificity for the base at position zero.

AvrBs3, the founding member of the Xanthomonas transcription-activator-like effectors (TALEs), is translocated into the plant cell where it localizes to the nucleus and acts as transcription factor. The DNA-binding domain of AvrBs3 consists of 17.5 nearly-identical 34 amino acid-repeats. Each repeat specifies binding to one base in the target DNA via amino acid residues 12 and 13 termed repeat variable diresidue (RVD). Natural target sequences of TALEs are generally preceded by a thymine (T0), which is coordinated by a tryptophan residue (W232) in a degenerated repeat upstream of the canonical repeats. To investigate the necessity of T0 and the conserved tryptophan for AvrBs3-mediated gene activation we tested TALE mutant derivatives on target sequences preceded by all possible four bases. In addition, we performed domain swaps with TalC from a rice pathogenic Xanthomonas because TalC lacks the tryptophan residue, and the TalC target sequence is preceded by cytosine. We show that T0 works best and that T0 specificity depends on the repeat number and overall RVD-composition. T0 and W232 appear to be particularly important if the RVD of the first repeat is HD (‘rep1 effect’). Our findings provide novel insights into the mechanism of T0 recognition by TALE proteins and are important for TALE-based biotechnological applications.

TAL Effectors Specificity Stems from Negative Discrimination

Transcription Activator-Like (TAL) effectors are DNA-binding proteins secreted by phytopathogenic bacteria that interfere with native cellular functions by binding to plant DNA promoters. The key element of their architecture is a domain of tandem-repeats with almost identical sequences. Most of the polymorphism is located at two consecutive amino acids termed Repeat Variable Diresidue (RVD). The discovery of a direct link between the RVD composition and the targeted nucleotide allowed the design of TAL-derived DNA-binding tools with programmable specificities that revolutionized the field of genome engineering. Despite structural data, the molecular origins of this specificity as well as the recognition mechanism have remained unclear. Molecular simulations of the recent crystal structures suggest that most of the protein-DNA binding energy originates from non-specific interactions between the DNA backbone and non-variable residues, while RVDs contributions are negligible. Based on dynamical and energetic considerations we postulate that, while the first RVD residue promotes helix breaks – allowing folding of TAL as a DNA-wrapping super-helix – the second provides specificity through a negative discrimination of matches. Furthermore, we propose a simple pharmacophore-like model for the rationalization of RVD-DNA interactions and the interpretation of experimental findings concerning shared affinities and binding efficiencies. The explanatory paradigm presented herein provides a better comprehension of this elegant architecture and we hope will allow for improved designs of TAL-derived biotechnological tools.