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The Xo1 locus in the heirloom rice variety Carolina Gold Select confers resistance to bacterial leaf streak and bacterial blight, caused by Xanthomonas oryzae pvs. oryzicola and oryzae, respectively. Resistance is triggered by pathogen-delivered transcription activator-like effectors (TALEs) independent of their ability to activate transcription, and is suppressed by variants called truncTALEs common among Asian strains. By transformation of the susceptible variety Nipponbare, we show that one of 14 nucleotide-binding, leucine-rich repeat (NLR) protein genes at the locus, with a zfBED domain, is the Xo1 gene. Analyses of published transcriptomes revealed that the Xo1-mediated response is similar to those of NLR resistance genes Pia and Rxo1 and distinct from that associated with induction of the executor resistance gene Xa23, and that a truncTALE dampens or abolishes activation of defense-associated genes by Xo1. In Nicotiana benthamiana leaves, fluorescently-tagged Xo1 protein, like TALEs and truncTALEs, localized to the nucleus. And, endogenous Xo1 specifically co-immunoprecipitated from rice leaves with a pathogen-delivered, epitope-tagged truncTALE. These observations suggest that suppression of Xo1-function by truncTALEs occurs through direct or indirect physical interaction. They further suggest that effector co-immunoprecipitation may be effective for identifying or characterizing other resistance genes.

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.

Understanding how gene transcription is spatially regulated by cis-regulatory elements (CREs) is both genetically and biomedically important. Cis-regulatory elements can be located thousands to millions of base pairs away from the promoter site, yet they somehow control and regulate transcription. Here we outline a new live cell, high resolution imaging methodology that allows for the visualization and quantification of these critical regulatory interactions. The method is based on the insertion of DNA sequences highly specific for a class of DNA binding proteins, known as Transcription Activator-Like Effectors (TALEs) into the human genome. TALEs are known to have high binding affinity and specificity to their target sites, and through conditional expression of fluorescently labeled TALEs, we are able to image specific chromosomal loci with improved signal-to-background and excellent localization precision. By using multicolor enhancer and promoter targeted TALEs, the technique can be coupled with super-resolution microscopy to detect and quantify subtle spatial changes occurring in the nucleus prior to or during transcriptional activation. This new methodology will help answer important questions on when, where and how genes are regulated during the process of cell differentiation or from external stresses and stimuli.

Transcription activator-like effector (TALE) proteins are a set of regulatory proteins that can be programmed to bind specifically any DNA sequence of choice. Despite the obvious potential of TALE proteins as fluorescence imaging and site specific genome editing tools, very little is known about their binding kinetics and affinity in the presence of other DNA-binding proteins that may be competing for the same or nearby sites. Utilizing a polyethylene glycol (PEG) coated flow cell we are able to observe TALE protein binding to a tethered target DNA. We compare the binding kinetics both on and off target to study TALE protein binding specificity. To investigate the usefulness of TALE proteins for live cell imaging of targeted loci we examined TALE binding kinetics in the presence of competition and interactions with DNA binding proteins.

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.