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Transcription activator-like effectors (TALEs) are modular proteins derived from the plant Xanthomonas sp. pathogen that can be designed to target unique DNA sequences following a simple cipher. Customized TALE proteins can be used in a variety of molecular applications that include gene editing and transcriptional modulation. Presently, we provide a brief primer on the design and construction of TALEs. TALE proteins can be fused to a variety of different effector domains that alter the function of the TALE upon binding. This flexibility of TALE design and downstream effect may offer therapeutic applications that are discussed in this section. Finally, we provide a future perspective on TALE technology and what challenges remain for successful translation of gene-editing strategies to the clinic.

Manipulation of gene expression can be facilitated by editing the genome or the epigenome. Precise genome editing is traditionally achieved by using designer nucleases which are generally exploited to eliminate a specific gene product. Upon the introduction of a site-specific DNA double-strand break (DSB) by the nuclease, endogenous DSB repair mechanisms are in turn harnessed to induce DNA sequence changes that can result in target gene inactivation. Minimal off-target effects can be obtained by endowing designer nucleases with the highly specific DNA-binding domain (DBD) derived from transcription activator-like effectors (TALEs). In contrast, epigenome editing allows gene expression control without inducing changes in the DNA sequence by specifically altering epigenetic marks, as histone tails modifications or DNA methylation patterns within promoter or enhancer regions. Importantly, this approach allows both up- and downregulation of the target gene expression, and the effect is generally reversible. TALE-based designer epigenome modifiers combine the high specificity of TALE-derived DBDs with the power of epigenetic modifier domains to induce fast and long-lasting changes in the epigenetic landscape of a target gene and control its expression. Here we provide a detailed description for the generation of TALE-based designer epigenome modifiers and of a suitable reporter cell line to easily monitor their activity.

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 Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Xanthomonas spp. reduce crop yields and quality worldwide. During infection of their plant hosts, many strains secrete transcription activator-like (TAL) effectors, which enter the host cell nucleus and activate specific corresponding host genes at effector binding elements (EBEs) in the promoter. TAL effectors may contribute to disease by activating the expression of susceptibility genes or trigger resistance associated with the hypersensitive reaction (HR) by activating an executor resistance (R) gene. The rice bacterial leaf streak pathogen X. oryzae pv. oryzicola (Xoc) is known to suppress host resistance, and no host R gene has been identified against it, despite considerable effort. To further investigate Xoc suppression of host resistance, we conducted a screen of effectors from BLS256 and identified Tal2a as an HR elicitor in rice when delivered heterologously by a strain of the closely related rice bacterial blight pathogen X. oryzae pv. oryzae (Xoo) or by the soybean pathogen X. axonopodis pv. glycines. The HR required the Tal2a activation domain, suggesting an executor R gene. Tal2a activity was differentially distributed among geographically diverse Xoc isolates, being largely conserved among Asian isolates. We identified four genes induced by Tal2a in next-generation RNA sequencing experiments and confirmed them using quantitative real-time reverse transcription-polymerase chain reaction (qPCR). However, neither individual nor collective activation of these genes by designer TAL effectors resulted in HR. A tal2a knockout mutant of BLS256 showed virulence comparable with the wild-type, but plasmid-based overexpression of tal2a at different levels in the wild-type reduced virulence in a directly corresponding way. Overall, the results reveal that host resistance suppression by Xoc plays a critical role in pathogenesis. Further, the dose-dependent avirulence activity of Tal2a and the apparent lack of a single canonical target that accounts for HR point to a novel, activation domain-dependent mode of action, which might involve, for example, a non-coding gene or a specific pattern of activation across multiple targets., (© 2016 BSPP and John Wiley & Sons Ltd.)