Transcription Activator-like Proteins Research Articles

Transcription activator-like effector protects bacterial endosymbionts from entrapment within fungal hyphae

As an endosymbiont of the ecologically and medically relevant fungus Rhizopus microsporus, the toxin-producing bacterium Mycetohabitans rhizoxinica faces myriad challenges, such as evading the host's defense mechanisms. However, the bacterial effector(s) that facilitate the remarkable ability of M.rhizoxinica to freely migrate within fungal hyphae have thus far remained unknown. Here, we show that a transcription activator-like (TAL) effector released by endobacteria is an essential symbiosis factor. By combining microfluidics with fluorescence microscopy, we observed enrichment of TAL-deficient M.rhizoxinica in side hyphae. High-resolution live imaging showed the formation of septa at the base of infected hyphae, leading to the entrapment of endobacteria. Using a LIVE/DEAD stain, we demonstrate that the intracellular survival of trapped TAL-deficient bacteria is significantly reduced compared with wild-type M.rhizoxinica, indicative of a protective host response in the absence of TAL proteins. Subversion of host defense in TAL-competent endobacteria represents an unprecedented function of TAL effectors. Our data illustrate an unusual survival strategy of endosymbionts in the host and provide deeper insights into the dynamic interactions between bacteria and eukaryotes.

Silencing of an aquaporin gene diminishes bacterial blight disease in rice

The translocation of type III effectors of plant-pathogenic bacteria is required both for the expression of their role in virulence, and for the triggering of plant defenses. The effector translocation needs molecular associations between bacterial translocators and their recognition compounds situated in plant plasma membranes. We show here that rice aquaporin PIP1;3, a member of the plasma membrane intrinsic protein family, is implicated in translocation of a transcription activator-like (TAL) effector from the bacterial blight pathogen into the cytosol of rice cells. The TAL protein PthXo1 targets rice disease-susceptible gene SWEET11 to determine virulence of the bacterial strain PXO99A on susceptible rice variety Nipponbare. In Nipponbare, post-transcriptional gene silencing of PIP1;3 results in highly alleviated susceptibility and concomitantly decreased expression of SWEET11 as compared to levels of the bacterial virulence and the plant SWEET11 expression in the wild-type plant with normal transcription of PIP1;3. In coincidence, the efficiency in PthXo1 translocation is substantially reduced in PIP1;3-silenced rice lines in contrast to the wild-type plant. In the case of PIP1;3 silencing, moreover, the bacterial type III translocator Hpa1 loses the ability to mediate PthXo1 translocation from the bacterial cells into the cytosol of rice cells. By contrast, PIP1;3 silencing does not affect the performance of isolated Hpa1 as a pattern molecule to induce immune responses in rice. Our data suggest that rice aquaporin PIP1;3 is a candidate of disease-susceptible factor with functional relevance to PthXo1 translocation as a prerequisite of the bacterial virulence on the susceptible rice variety.

Isolation of Specific Genomic Regions and Identification of Their Associated Molecules by Engineered DNA-Binding Molecule-Mediated Chromatin Immunoprecipitation (enChIP) Using the CRISPR System and TAL Proteins

Comprehensive understanding of genome functions requires identification of molecules (proteins, RNAs, genomic regions, etc.) bound to specific genomic regions of interest in vivo. To perform biochemical and molecular biological analysis of specific genomic regions, we developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) to purify genomic regions of interest. In enChIP, specific genomic regions are tagged for biochemical purification using engineered DNA-binding molecules, such as transcription activator-like (TAL) proteins and a catalytically inactive form of the clustered regularly interspaced short palindromic repeats (CRISPR) system. enChIP is a comprehensive approach that emphasizes non-biased search using next-generation sequencing (NGS), microarrays, mass spectrometry (MS), and other methods. Moreover, this approach is not restricted to cultured cell lines and can be easily extended to organisms. In this review, we discuss applications of enChIP to elucidating the molecular mechanisms underlying genome functions.

Identification of non-coding RNAs associated with telomeres using a combination of enChIP and RNA sequencing.

Accumulating evidence suggests that RNAs interacting with genomic regions play important roles in the regulation of genome functions, including X chromosome inactivation and gene expression. However, to our knowledge, no non-biased methods of identifying RNAs that interact with a specific genomic region have been reported. Here, we used enChIP-RNA-Seq, a combination of engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) and RNA sequencing (RNA-Seq), to perform a non-biased search for RNAs interacting with telomeres. In enChIP-RNA-Seq, the target genomic regions are captured using an engineered DNA-binding molecule such as a transcription activator-like protein. Subsequently, RNAs that interact with the target genomic regions are purified and sequenced. The RNAs detected by enChIP-RNA-Seq contained known telomere-binding RNAs, including the telomerase RNA component (Terc), the RNA component of mitochondrial RNA processing endoribonuclease (Rmrp), and Cajal body-specific RNAs. In addition, a number of novel telomere-binding non-coding RNAs were also identified. Binding of two candidate non-coding RNAs to telomeres was confirmed by immunofluorescence microscopy and RNA fluorescence in situ hybridization (RNA-FISH) analyses. The novel telomere-binding non-coding RNAs identified here may play important roles in telomere functions. To our knowledge, this study is the first non-biased identification of RNAs associated with specific genomic regions. The results presented here suggest that enChIP-RNA-Seq analyses are useful for the identification of RNAs interacting with specific genomic regions, and may help to contribute to current understanding of the regulation of genome functions.

Identification of telomere-associated molecules by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP)

Biochemical analysis of molecular interactions in specific genomic regions requires their isolation while retaining molecular interactions in vivo. Here, we report isolation of telomeres by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using a transcription activator-like (TAL) protein recognizing telomere repeats. Telomeres recognized by the tagged TAL protein were immunoprecipitated with an antibody against the tag and subjected to identification of telomere-binding molecules. enChIP-mass spectrometry (enChIP-MS) targeting telomeres identified known and novel telomere-binding proteins. The data have been deposited to the ProteomeXchange with identifier PXD000461. In addition, we showed that RNA associated with telomeres could be isolated by enChIP. Identified telomere-binding molecules may play important roles in telomere biology. enChIP using TAL proteins would be a useful tool for biochemical analysis of specific genomic regions of interest.

Predicting promoters targeted by TAL effectors in plant genomes: from dream to reality

OPINION article Front. Plant Sci., 03 September 2013Sec. Plant Pathogen Interactions Volume 4 - 2013 | https://doi.org/10.3389/fpls.2013.00333

Transcriptional Activators of Human Genes with Programmable DNA-Specificity

TAL (transcription activator-like) effectors are translocated by Xanthomonas bacteria into plant cells where they activate transcription of target genes. DNA target sequence recognition occurs in a unique mode involving a central domain of tandem repeats. Each repeat recognizes a single base pair in a contiguous DNA sequence and a pair of adjacent hypervariable amino acid residues per repeat specifies which base is bound. Rearranging the repeats allows the design of novel TAL proteins with predictable DNA-recognition specificities. TAL protein-based transcriptional activation in plant cells is mediated by a C-terminal activation domain (AD). Here, we created synthetic TAL proteins with designed repeat compositions using a novel modular cloning strategy termed “Golden TAL Technology”. Newly programmed TAL proteins were not only functional in plant cells, but also in human cells and activated targeted expression of exogenous as well as endogenous genes. Transcriptional activation in different human cell lines was markedly improved by replacing the TAL-AD with the VP16-AD of herpes simplex virus. The creation of TAL proteins with potentially any desired DNA-recognition specificity allows their versatile use in biotechnology.

Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors

The pathogenicity of many bacteria depends on the injection of effector proteins via type III secretion into eukaryotic cells in order to manipulate cellular processes. TAL (transcription activator-like) effectors from plant pathogenic Xanthomonas are important virulence factors that act as transcriptional activators in the plant cell nucleus, where they directly bind to DNA via a central domain of tandem repeats. Here, we show how target DNA specificity of TAL effectors is encoded. Two hypervariable amino acid residues in each repeat recognize one base pair in the target DNA. Recognition sequences of TAL effectors were predicted and experimentally confirmed. The modular protein architecture enabled the construction of artificial effectors with new specificities. Our study describes the functionality of a distinct type of DNA binding domain and allows the design of DNA binding domains for biotechnology.

A Simple Cipher Governs DNA Recognition by TAL Effectors

TAL effectors of plant pathogenic bacteria in the genus Xanthomonas bind host DNA and activate genes that contribute to disease or turn on defense. Target specificity depends on an effector-variable number of typically 34 amino acid repeats, but the mechanism of recognition is not understood. We show that a repeat-variable pair of residues specifies the nucleotides in the target site, one pair to one nucleotide, with no apparent context dependence. Our finding represents a previously unknown mechanism for protein-DNA recognition that explains TAL effector specificity, enables target site prediction, and opens prospects for use of TAL effectors in research and biotechnology.