TAL effectors: function, structure, engineering and applications

Abstract

TAL effectors are proteins secreted by bacterial pathogens into plant cells, where they enter the nucleus and activate expression of individual genes. TAL effectors display a modular architecture that includes a central DNA-binding region comprising a tandem array of nearly identical repeats that are almost all 34 residues long. Residue number 13 in each TAL repeat (one of two consecutive polymorphic amino acids that are termed 'repeat variable diresidues', or 'RVDs') specifies the identity of a single base; collectively the sequential repeats and their RVDs dictate the recognition of sequential bases along one of the two DNA strands. The modular architecture of TAL effectors has facilitated their extremely rapid development and application as artificial gene targeting reagents, particularly in the form of site-specific nucleases. Recent crystallographic and biochemical analyses of TAL effectors have established the structural basis of their DNA recognition properties and provide clear directions for future research.

Figure 1. Domain organization and activity of TAL effectors

a: TAL effectors contain N-terminal signals for bacterial type III secretion, variable numbers of tandem repeats that specify the target nucleotide sequence, nuclear localization signals, and a C-terminal region that is required for transcriptional activation. PthXo1 (schematized in this figure) contains 23.5 canonical repeats (color coded to match Figure 2) that contact the DNA target found in the promoter of the rice Os8N3 gene [20]. Blue bases correspond to positions in the target where the match between protein and DNA differs from the optimal match specified by the recognition code. The sequence of a representative repeat (#14) is shown; RVD residues (HD) that recognize cytosine are red. b: TAL effectors are translocated into the plant nucleus, where they bind to target sites (termed ‘UPregulated by TAL’ or ‘UPT’ boxes, or ‘EBEs’ for ‘Effector Binding Elements’) that are located in the 5′ promoter regions of genes that are subsequently activated (Termed ‘S’ for a gene which confers susceptibility to infection as a result of activation, or ‘R’ for a gene which confers resistance to infection). The C-terminal region of the TAL effector interacts with plant transcriptional machinery as part of the gene activation mechanism. Plants can acquire resistance traits against bacterial infection through at least three separate mechanisms: acquisition of mutations in the EBE that reduce DNA binding affinity, acquisition of mutations in transcription factors that ostensibly inhibit protein-protein association with the TAL effector acidic activation region, or by coupling the sequence of the EBE box to the promoter region of a resistance gene thereby leading to an avirulence phenotype upon infection.

Figure 2. Crystal structures of dHAX3 and pthXo1 TAL effectors

a: The structure of PthXo1 bound to its DNA target site [28]. The effector contains 22.5 repeat modules each colored separately as shown in Figure 1. The protein-DNA complex is shown from the side of the DNA duplex (left) and looking down the axis of the DNA (right). In the left panel, the N-terminal end of the protein (containing two cryptic repeats that engage the DNA backbone via a series of basic residues, and that also capture the strongly conserved thymine at position ‘zero’ of the EBE is at the top of the complex (see also Figure 3b). b: The structure of the artificial dHax3 TAL effector bound to its corresponding DNA target (left) and in the absence of bound DNA (right) [27]. The protein is displayed with the N-terminal region at the top of both structures. N-terminal cryptic repeat 0, and a truncated portion of repeat −1, is shown as grey ribbon in the left panel. Because a portion of the N-terminal helix of repeat −1 is missing in the construct, the full set of contacts extending to ‘thymine zero’ in the target site are not entirely formed. Superposition of the DNA bound structures of dHAX3 and PthXo1 yields an overall rmsd for superimposed backbone atoms of approximately 0.9 Å.

Figure 3. DNA contacts and recognition by TAL effectors

a: Left: Interactions formed between four common RVD types (HD, NG, NN and NI) against their cognate nucleotide bases from the PthXo1 target site (cytosine, thymine, purine and adenine, respectively). Whereas the first three RVDs form highly complementary combinations of atomic interactions, NI appears to make desolvating interactions with the neighboring nucleotide and to influence specificity at least in part through steric exclusion, rather than through highly complementary, sequence-specific hydrogen bonds or van der Waals contacts. Recent studies of the overall strength of TAL repeat/RVD interactions to cognate bases indicates that NI is associated with considerably weaker interactions than either HD or NN repeats [33]. b: Sequence and structural contacts made by residues immediately N-terminal to the central (or ‘canonical’) TAL repeats in the PthXo1 structure. This region is highly basic (denoted by blue lysine and arginine residues) as compared to the rest of the DNA binding region, and displays limited sequence homology to the C-terminal TAL repeats (indicated by rectangles). The structure of PthXo1 bound to DNA (bottom) demonstrates that residues 220 to 289 form two cryptic repeats (numbered ‘0’ and ‘−1’) that harbor the same left-handed two-helix bundle as a canonical repeat. These two repeats present a series of basic residues to the DNA backbone (K262, K265 and R266 from the ‘0’ repeat) as well as a single tryptophan residue (W232 from the ‘−1’ repeat) that contacts the ‘thymine zero’ nucleotide (which is strongly conserved in TAL recognition sites and required for effector activity). The more distal N-terminal region of PthXo1 (and other similar TAL effectors) was not observed in the crystal structure, but appears to contain sequence elements that could form two additional cryptic repeats (numbered ‘−3’ and ‘−2 and indicated by light grey and light blue fonts and dashed arrow, to distinguish from the region that has been observed in the crystal structure).