Generating and applying new knowledge from the wealth of available genomic information is hindered, in part, by the difficulty of altering nucleotide sequences and expression of genes in living cells ...in a targeted fashion. Progress has been made in engineering DNA binding domains to direct proteins to particular sequences for mutagenesis or manipulation of transcription; however, achieving the requisite specificities has been challenging. Transcription activator—like (TAL) effectors of plant pathogenic bacteria contain a modular DNA binding domain that appears to overcome this challenge. Comprising tandem, polymorphic amino acid repeats that individually specify contiguous nucleotides in DNA, this domain is being deployed in DNA targeting for applications ranging from understanding gene function in model organisms to improving traits in crop plants to treating genetic disorders in people.
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.
Transcription activator like effectors (TALEs) are injected via the type III secretion pathway of many plant pathogenic
Xanthomonas spp. into plant cells where they contribute to disease or trigger ...resistance by binding to DNA and turning on TALE-specific host genes. Advances in our understanding of TALEs and their targets have yielded new models for pathogen recognition and defense. Similarly, we have gained insight into plant molecules and processes that can be co-opted to promote infection. Recent elucidation of the basis for specificity in DNA binding by TALEs expedites further discovery and opens the door to biotechnological applications. This article reviews the most significant findings in TALE research, with a focus on recent advances, and discusses future prospects including pressing questions yet to be answered.
TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in ...pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online.
Crossovers (COs), that drive genetic exchange between homologous chromosomes, are strongly biased toward subtelomeric regions in plant species. Manipulating the rate and positions of COs to increase ...the genetic variation accessible to breeders is a longstanding goal. Use of genome editing reagents that induce double-stranded breaks (DSBs) or modify the epigenome at desired sites of recombination, and manipulation of CO factors, are increasingly applicable approaches for achieving this goal. These strategies for ‘controlled recombination’ have potential to reduce the time and expense associated with traditional breeding, reveal currently inaccessible genetic diversity, and increase control over the inheritance of preferred haplotypes. Considerable challenges to address include translating knowledge from models to crop species and determining the best stages of the breeding cycle at which to control recombination.
The genetic diversity accessible to plant breeders has traditionally been limited by chromosomal COs, but recent advances in targeted DNA cleavage and epigenetic modification are increasing access.Overcoming the low frequency and uneven distribution of COs in plants can reveal allelic diversity and may increase control over the inheritance of preferred haplotypes.The frequency and location of COs can be altered with manipulation of pro- and anti-CO factors, site-directed nucleases, or epigenetic modifiers; we refer to such alteration as ‘controlled recombination’.Epigenetic modifiers can induce COs near centromeres, which are otherwise very low-frequency CO regions.Controlled recombination may enable breeders and geneticists to unlock otherwise inaccessible genetic diversity.
Transcription activator-like (TAL) effectors are repeat-containing proteins used by plant pathogenic bacteria to manipulate host gene expression. Repeats are polymorphic and individually specify ...single nucleotides in the DNA target, with some degeneracy. A TAL effector-nucleotide binding code that links repeat type to specified nucleotide enables prediction of genomic binding sites for TAL effectors and customization of TAL effectors for use in DNA targeting, in particular as custom transcription factors for engineered gene regulation and as site-specific nucleases for genome editing. We have developed a suite of web-based tools called TAL Effector-Nucleotide Targeter 2.0 (TALE-NT 2.0; https://boglab.plp.iastate.edu/) that enables design of custom TAL effector repeat arrays for desired targets and prediction of TAL effector binding sites, ranked by likelihood, in a genome, promoterome or other sequence of interest. Search parameters can be set by the user to work with any TAL effector or TAL effector nuclease architecture. Applications range from designing highly specific DNA targeting tools and identifying potential off-target sites to predicting effector targets important in plant disease.
The ability to precisely engineer plant genomes offers much potential for advancing basic and applied plant biology. Here, we describe methods for the targeted modification of plant genomes using ...transcription activator-like effector nucleases (TALENs). Methods were optimized using tobacco (Nicotiana tabacum) protoplasts and TALENs targeting the acetolactate synthase (ALS) gene. Optimal TALEN scaffolds were identified using a protoplast-based single-strand annealing assay in which TALEN cleavage creates a functional yellow fluorescent protein gene, enabling quantification of TALEN activity by flow cytometry. Single-strand annealing activity data for TALENs with different scaffolds correlated highly with their activity at endogenous targets, as measured by high-throughput DNA sequencing of polymerase chain reaction products encompassing the TALEN recognition sites. TALENs introduced targeted mutations in ALS in 30% of transformed cells, and the frequencies of targeted gene insertion approximated 14%. These efficiencies made it possible to recover genome modifications without selection or enrichment regimes: 32% of tobacco calli generated from protoplasts transformed with TALEN-encoding constructs had TALEN-induced mutations in ALS, and of 16 calli characterized in detail, all had mutations in one allele each of the duplicate ALS genes (SurA and SurB). In calli derived from cells treated with a TALEN and a 322-bp donor molecule differing by 6 bp from the ALS coding sequence, 4% showed evidence of targeted gene replacement. The optimized reagents implemented in plant protoplasts should be useful for targeted modification of cells from diverse plant species and using a variety of means for reagent delivery.
DNA recognition by TAL effectors is mediated by tandem repeats, each 33 to 35 residues in length, that specify nucleotides via unique repeat-variable diresidues (RVDs). The crystal structure of ...PthXo1 bound to its DNA target was determined by high-throughput computational structure prediction and validated by heavy-atom derivatization. Each repeat forms a left-handed, two-helix bundle that presents an RVD-containing loop to the DNA. The repeats self-associate to form a right-handed superhelix wrapped around the DNA major groove. The first RVD residue forms a stabilizing contact with the protein backbone, while the second makes a base-specific contact to the DNA sense strand. Two degenerate amino-terminal repeats also interact with the DNA. Containing several RVDs and noncanonical associations, the structure illustrates the basis of TAL effector-DNA recognition.
Engineered nucleases that cleave specific DNA sequences in vivo are valuable reagents for targeted mutagenesis. Here we report a new class of sequence-specific nucleases created by fusing ...transcription activator-like effectors (TALEs) to the catalytic domain of the FokI endonuclease. Both native and custom TALE-nuclease fusions direct DNA double-strand breaks to specific, targeted sites.
Many disease resistance (R) proteins of plants detect the presence of disease-causing bacteria, viruses, or fungi by recognizing specific pathogen effector molecules that are produced during the ...infection process. Effectors are often pathogen proteins that probably evolved to subvert various host processes for promotion of the pathogen life cycle. Five classes of effector-specific R proteins are known, and their sequences suggest roles in both effector recognition and signal transduction. Although some R proteins may act as primary receptors of pathogen effector proteins, most appear to play indirect roles in this process. The functions of various R proteins require phosphorylation, protein degradation, or specific localization within the host cell. Some signaling components are shared by many R gene pathways whereas others appear to be pathway specific. New technologies arising from the genomics and proteomics revolution will greatly expand our ability to investigate the role of R proteins in plant disease resistance.
PDF
