TAL effectors: highly adaptable phytobacterial virulence factors and readily engineered DNA-targeting proteins

Abstract

Transcription activator-like (TAL) effectors are transcription factors injected into plant cells by pathogenic bacteria of the genus Xanthomonas. They function as virulence factors by activating host genes important for disease, or as avirulence factors by turning on genes that provide resistance. DNA-binding specificity is encoded by polymorphic repeats in each protein that correspond one-to-one with different nucleotides. This code has facilitated target identification and opened new avenues for engineering disease resistance. It has also enabled TAL effector customization for targeted gene control, genome editing, and other applications. This article reviews the structural basis for TAL effector-DNA specificity, the impact of the TAL effector-DNA code on plant pathology and engineered resistance, and recent accomplishments and future challenges in TAL effector-based DNA targeting.

Keywords: DNA targeting; TAL effector nucleases; crystal structure; gene therapy; plant disease; resistance genes; susceptibility genes.

Figure 1. Structure of the TAL effector-DNA association and the basis of specificity

At top, the structure of PthXo1 bound to its DNA target site [] is shown (left) from the side of the DNA duplex and (right) looking down the axis of the DNA. The effector contains 22.5 repeat modules each colored separately. In the side view, the N-terminal end of the protein is leftmost. It contains two cryptic repeats that engage the DNA backbone via a series of basic residues, and that also capture the strongly conserved thymine (5’ T) at position ‘zero’ of the binding site. The labeled repeats (14, 15, and 16) are shown in detail at bottom. Bottom left illustrates the contacts made by the ‘HD’ RVD (residues 12 and 13) in repeat number 14. The histidine at position 12 in the repeat forms a hydrogen bond to the backbone carbonyl oxygen of residue 8 in the first alpha-helix, while the aspartate at position 13 forms a hydrogen bond to the extracyclic amino nitrogen of the cytosine base. Bottom right shows repeats 14, 15, and 16 interacting with the DNA, illustrating that consecutive RVDs (HD, NG and NN, respectively in these repeats) contact consecutive bases (cytosine, thymine, and guanine in this case) on the same DNA strand.

Figure 2. TAL effectors in plant disease, natural disease resistance, and engineered resistance

Following delivery into the host plant cytoplasm via the bacterial type III secretion system (T3SS) and translocation to the nucleus, TAL effectors bind to RVD-specified sequences in host DNA (matched colors). Binding leads to the activation of host susceptibility (S) genes, top left, that contribute to disease. Resistant plants may harbor an S gene promoter mutation, top middle (red DNA), that blocks binding and activation by the TAL effector, or they may harbor an S gene mimic called an executor resistance (R) gene, top right, that has a TAL effector binding site in its promoter but encodes a protein that mediates localized cell death and limits the infection when activated. Strategies for engineered resistance to pathogens that depend on TAL effectors as virulence factors include, bottom left, site-directed mutagenesis (red DNA) of TAL effector binding sites in major S genes using engineered nucleases such as TALENs (see also Figure 3); bottom middle, R genes with an artificial promoter containing multiple TAL effector binding sites so that pathogen loss of a single effector will not make the gene ineffective, and so the gene can be effective against diverse strains; and bottom right, an S gene silencing construct consisting of a short hairpin RNA (shRNA) driven by one or more TAL effectors. In this last example, the context (grey DNA) for the TAL effector binding site in the silencing construct should be different from that of the S gene to avoid silencing any endogenous expression of the S gene in the absence of the TAL effector.

Figure 3. TALEN-mediated genome editing

Double strand breaks introduced by TALENs are repaired by non-homologous end joining (NHEJ), leading to short insertions or deletions, or by homologous recombination (HR), which can be used to replace or insert new DNA. TALENs are shown as TAL effector fusions to the catalytic domain of the type IIS restriction endonuclease FokI, which cuts as a dimer.

Figure 4. Applications of custom TAL effectors

FokI, FokI endonuclease catalytic domain. AD, activation domain. RD, repressor domain. HDAC, histone deacytelase. DNMT, DNA methyltransferase. Gin, Gin recombinase. CD, cytosine deaminase.