Available online at www.sciencedirect.com

TAL effectors are remote controls for gene activation Heidi Scholze and Jens Boch

TAL (transcription activator-like) effectors constitute a novel typically 34 amino acids (aa) long, but also repeat types of class of DNA-binding proteins with predictable specificity. They 30–42 aa can be found ([2], Figure 2). The last repeat is are employed by Gram-negative plant-pathogenic bacteria of only a half repeat. Repeat-to-repeat variations are limited the genus Xanthomonas which translocate a cocktail of to a few aa positions including two hypervariable residues different effector proteins via a type III secretion system (T3SS) at positions 12 and 13 per repeat. Typically, TALs differ into plant cells where they serve as virulence determinants. in their number of repeats while most contain 15.5–19.5 Inside the plant cell, TALs localize to the nucleus, bind to target repeats [2]. The repeat domain determines the specificity promoters, and induce expression of plant genes. DNA-binding of TALs which is mediated by selective DNA binding specificity of TALs is determined by a central domain of tandem [7,8]. TAL repeats constitute a novel type of DNA- repeats. Each repeat confers recognition of one base pair (bp) binding domain [7] distinct from classical zinc finger, in the DNA. Rearrangement of repeat modules allows design of helix–turn–helix, and leucine zipper motifs. proteins with desired DNA-binding specificities. Here, we summarize how TAL specificity is encoded, first structural data The recent understanding of the DNA-recognition speci- and first data on site-specific TAL nucleases. ficity of TALs [9,10] has brought TAL studies to a new level, in regard to both functional studies and applied Address science. In this review we summarize the DNA-recog- Department of Genetics, Institute of Biology, Martin-Luther-University nition code of TALs, novel structural insights, and pro- Halle-Wittenberg, 06099 Halle (Saale), Germany gress in biotechnological applications. As TAL virulence Corresponding author: Boch, Jens ([email protected]) targets in plants and plant resistance against TALs were recently reviewed [2,3,5,6] these aspects will not be discussed here. Current Opinion in Microbiology 2011, 14:47–53

This review comes from a themed issue on TALs function as transcription factors Host-microbe interactions: bacteria TAL frequency Edited by Brett Finlay and Ulla Bonas Proteins with similarity to TALs have only been ident- ified in the plant-pathogenic bacteria Xanthomonas spp. Available online 5th January 2011 and Ralstonia solanacearum. The presence, number, and 1369-5274/$ – see front matter importance of TALs vary in different strains of Xantho- # 2010 Elsevier Ltd. All rights reserved. monas. Whereas some Xanthomonas pathovars carry none or only few TAL genes, others contain up to 28 TAL genes DOI 10.1016/j.mib.2010.12.001 (including pseudogenes) per strain (Table 1). A few TALs have been shown to be essential for virulence, but the contribution of others is minor or not detectable. Introduction It is unclear whether TALs with no effect are first, Plant-pathogenic Xanthomonas bacteria translocate a nonfunctional, but possibly serve as recombinatory reser- cocktail of 30–40 different effector proteins via a type voir, second, only functional in specific host plants, or III secretion system (T3SS) into plant cells to manipulate third, fulfill a task in virulence that has not been detected eukaryotic cellular pathways [1]. Members of the TAL so far. (transcription activator-like) effector family include important virulence factors which function as transcrip- Many R. solanacearum strains carry homologs of Xantho- tion factors for target plant genes [2–6]. This is believed monas TAL genes (Table 1;[11,12]). However, the N- to support bacterial proliferation, but the role of most terminal and C-terminal regions of R. solanacearum TALs TALs in virulence is still unknown. show low similarity to Xanthomonas TALs, and the repeats differ mainly in their C-terminal part (Figure 2). Because The N-terminal and C-terminal regions are highly con- of their similar overall repeat-architecture, it is possible served in TALs. The N-terminal region contains the that R. solanacearum TALs bind to nucleic acids, too. secretion and translocation signal for the T3SS, whereas the C-terminal region contains nuclear localization signals DNA-recognition specificity of TALs and a transcriptional activation domain, both of which are TAL-specific effects are due to specific sequences loca- important for protein activity ([2], Figure 1). The most lized in promoter regions of induced plant genes prominent feature of TALs is their central domain that [7,8,13,14,15]. Characterization of different AvrBs3- consists of tandem nearly identical repeats. Each repeat is induced genes in pepper revealed the presence of a www.sciencedirect.com Current Opinion in Microbiology 2011, 14:47–53 48 Host-microbe interactions: bacteria

Figure 1

(a) 0 Repeat domain NLS AD NC T 1 12/13 34 LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHG

(b) Repeat type NI HD NG IG NK NN NS DNA specificity A C TTG G A A C G T

Current Opinion in Microbiology

TAL effector-DNA-binding code. (a) TALs contain nuclear localization signals (NLS, yellow) and an activation domain (AD, green) to function as transcriptional activators. A central tandem repeat domain (red) confers DNA-binding specificity. Each 34 amino acid (aa)-repeat binds to one base pair (bp) with specificity being determined by aa 12 and 13. One sample repeat is shown. Numbers refer to aa positions within the repeat. Repeat zero is indicated which specifies for T. (b) Repeat types have specificity for one or several DNA bp. Only bases of the DNA leading strand are shown. common promoter element, termed UPA (upregulated by rearranged to generate artificial TALs with novel and AvrBs3) box [7,8,16], which was essential for AvrBs3 predictable DNA-binding specificities [9,22]. recognition and whose function was sensitive to mutations [7,8,17,18]. TAL repeat structure PSIPRED predictions revealed a strong similarity of TAL The length of the UPA box roughly matches the number repeats with tetratrico peptide repeat (TPR) proteins and of repeats in AvrBs3, leading to a model to explain pentatrico peptide repeat (PPR) proteins [23,24]. TPRs AvrBs3 DNA-recognition specificity [9,10]. Accord- are 34 aa long, contain two alpha helices, and mediate ingly, one TAL repeat binds to one DNA base pair (bp), protein–protein interactions in prokaryotes and eukaryotes and the specificity of individual repeats is encoded in (e.g. 79 TPR proteins in Arabidopsis thaliana, 260 TPR the hypervariable residues at positions 12 and 13 in each proteins in humans) [25]. TPR proteins contain 3–16 repeat (Figure 2;[9,10]; also termed RVD, repeat- degenerated tandem repeats and are involved in diverse variable diresidue). Some repeat types are specific for a cellular processes [25]. In contrast, PPR proteins carry 2–26 particular DNA bp whereas others recognize more than either degenerated tandem 35 aa-repeats or tandem alter- one bp ([9,10]; Figure 1). Repeat positions other than nating 31 aa-repeats, 36 aa-repeats, and 35 aa-repeats the hypervariable residues do not exhibit a significant [26,27]. PPR proteins mediate RNA binding and are effect on repeat specificity [19]. The target DNA especially abundant in higher plants (e.g. >450 PPR elements of TALs have been termed differently, that proteins in A. thaliana, six PPR proteins in humans) [26]. is, UPA box [7], UPT box (upregulated by TAL, [20]) As no PPR-3D structures are known TAL repeats were or simply TAL box [9]. We have suggested to name modeled onto TPR protein structures [23,24]. In such this element TAL box (e.g. AvrBs3 box, Hax3 box; models, the specificity-determining aa residues 12 and 13 [9]), because the DNA sequence controls recognition of each TAL repeat are located next to another, oriented to and binding of the TAL, but not necessarily upregula- the inner face of the superhelix [23,24] which fits well to a tion of gene expression. TAL boxes all start with a ‘T’ possible TAL–DNA interaction (Figure 2). Typically, indicating that the N-terminal region preceding the first protein repeat units (e.g. TPR and PPR) are highly degen- repeat (termed repeat ‘zero’) contributes to DNA bind- erate and the consensus structure only forms a scaffold to ing ([9,21]; Figure 2). Furthermore, it was shown on present functional residues for interactions [25,28]. In the basis of artificial TALs containing 1.5–16.5 repeats contrast, TAL repeats show an extraordinarily high degree that a minimum of 6.5 repeats is necessary to induce of repeat-to-repeat identity. This probably generates a transcription and 10.5 repeats are sufficient for full highly symmetric framework to position the two speci- induction [9]. ficity-determining hypervariable residues to its highly symmetrical binding partner, the DNA double helix. The simple and modular way of how TALs bind to DNA has far-reaching consequences. DNA-binding specifici- Nuclear magnetic resonance (NMR) structural data of a ties of TALs can be predicted and repeat modules can be 1.5 TAL repeat polypeptide showed that the TAL repeat

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folds into a helix–turn–helix–turn structure reminiscent as well as endogenes in plants [9,19,22]. The position of of TPRs (Figure 2;[23]). The hypervariable residues a TAL box defines the TAL-dependent transcriptional were located at the end of the first helix. The two alpha start site at approximately 40–60 bp downstream of the helices of one repeat are arranged in antiparallel orien- box [7,17,18,20]. Obviously, TAL effectors control the tation and likely interact with each other via hydrophobic transcriptional start site analogous to the TATA box residues [23]. Possibly, the structure of the whole TAL binding protein which may indicate similar functions. repeat domain differs, because of repeat-to-repeat inter- In addition, different TAL boxes can be combined in a actions which could not be resolved in the short 1.5 repeat cis-regulatory element in one promoter to render the fragment. downstream gene inducible by more than one TAL [20]. This has consequences for biotechnology, because TAL–DNA interaction a gene of interest can be induced using different TALs TAL repeats specifically bind to DNA in vitro in a and these TALs can target DNA boxes at different places sequence-dependent manner suggesting that no eukar- of the promoter region. yotic host factor is needed for DNA binding and speci- ficity [7,8,9,17,18,21]. It is suggestive that the TAL technology versus zinc finger technology hypervariable residues, which determine the specificity The programmable DNA-binding specificity of TALs of each repeat directly interact with the DNA bases. enables biotechnological applications comparably to the zinc finger (ZF) technology [31]. One ZF domain binds to Generally, repeat-containing proteins carry tandem arrays 3–4 DNA bp, and the specificity of ZF domains can be of a short structural motif and the repetitive structure modified. A tandem array of 3–6 ZFs corresponds to target typically forms an extended interaction interface to bind sites of 9–18 bp which is sufficient to target unique sites in diverse ligands [28]. The repetitive units can be arranged complex genomes. The ZF technology has been used to similarly to a coil spring in a so-called solenoid structure generate artificial transcription factors, repressors, methy- [29]. The vast majority of solenoids in nature are right- lases, recombinases, and ZF nucleases (ZFNs) [31]. The handed (i.e. a right-handed coil). In contrast, the Zurdo binding specificity of a ZF array is not completely pre- (‘left-handed’ in Spanish) domain has recently been dictable, because specificities of neighboring ZFs inter- described as a left-handed alpha-helical solenoid that binds depend which results in highly laborious screening of to DNA [30]. Often, solenoid units are stacked slightly libraries to identify suitable candidates [32,33]. In con- shifted to each other resulting in a twisted right-handed or trast, TALs have an obvious advantage, because the left-handed continuous superhelix [29]. Based on predic- TAL–DNA-binding specificity is unambiguously pre- tions [23,24] and to accomodate the DNA, TAL repeats dictable and TAL repeat specificity is obviously neigh- likely twist in a right-handed superhelix around the right- bor-independent [9,10]. handed DNA helix. A manual fit of possible TAL–DNA complexes shows that right-handed or left-handed TAL TAL nucleases promote genome editing repeat solenoids might wrap around the DNA in a sun- Zinc finger nucleases (ZFNs) are fusion proteins of tan- flower-like shape (Figure 2). dem ZF domains and a nonspecific endonuclease, typi- cally the C-terminal nuclease domain of the type IIs TAL effectors with 17.5 repeats, for example, AvrBs3, restriction enzyme FokI [34–37]. FokI enzymatic activity interact with a target DNA sequence (one repeat per bp depends on dimerization after DNA binding and, accord- plus flanking sequences [9]) that corresponds to nearly ingly, two ZFNs are designed to bind to adjacent DNA two helical turns. The predicted right-handed superhelical sequences to allow FokI dimerization and DNA cleavage TAL repeat structure can be expected to be flexible to wrap within the spacer region between both binding sites ([36]; around the DNA double helix (Figure 2). The modeled Figure 3). DNA double-strand breaks are generally structure of the TAL repeat region [23] is too large repaired by one of two alternative mechanisms, nonho- (approximately twice the size) to accomplish interaction mologous end-joining (NHEJ) or homologous DNA of one repeat to one bp as required from its DNA specificity recombination (HDR) [36]. The first mechanism is typi- [9,10]. Interestingly, the TAL repeats undergo confor- cally accompanied by mutagenic deletions or insertions mational changes upon DNA interaction to a more compact whereas the latter can also mediate recombination be- structure and the helical content of the repeats is decreased tween endogenous sequences and exogenously supplied upon DNA binding [23]. This suggests that TAL–DNA DNA fragments. Targeted cuts enable targeted DNA complex formation is a dynamic process that involves integration, gene replacement or gene knockout in com- repeat and/or DNA conformational changes. plex genomes [34–37]. Accordingly, the ZFN technology has been used for genome modification of yeast, plants, Biotechnology aspects mammals, and humans [36]. TALs as transcriptional remote controls TALs can be tailored to target specific DNA sequences In addition to ZFNs, chimera between TALs and and, accordingly, specifically induce target reporter genes the FokI nuclease have recently been constructed and www.sciencedirect.com Current Opinion in Microbiology 2011, 14:47–53 50 Host-microbe interactions: bacteria

Figure 2

(a) side view top view N C

C

N 25.8 Å 12.6 Å 1 2 20 Å

10 3 (b)

36° tw is 34 Å C t 9 4 left-handed

r ig

TAL-repeat solenoid TAL-repeat h 8 t -h 5 a nd N ed su per helix 7 6 top view side view

36° tw is t

C

right-handed r ig h t- TAL-repeat solenoid TAL-repeat h N an de d sup erhelix

(c)

N N N N N N N

C C C C C C C C N R. solanacearum 30 aa 33 aa 34 aa 35 aa 39 aa 40 aa 42 aa 35 aa

Current Opinion in Microbiology

Possible TAL effector repeat arrangements. (a) NMR structure of 1.5 repeats of the TAL effector PthA2 (2KQ5) forming three a-helices [23]. (b) Cartoon of three consecutive repeats forming a predicted right-handed superhelix of repeats twisted 368 to fit the right-handed DNA helix. Manual fit of 10 individual repeats onto DNA (black). To connect individual helices, the long alpha helices would have to be kinked. Top and bottom models are based on left-handed and right-handed solenoids (repeat twist), respectively. Repeats of the right-handed solenoids are shown tilted. a-Helical

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Table 1

Number of TAL effectors per strain.

Host TALsa Referenceb Xanthomonas spp. X. campestris pv. musacearum Banana 0 ACHT01000000 X. campestris pv. vasculorum Sugarcane 0 ACHS01000000 X. campestris pv. vesicatoria Pepper and tomato 0–1[42]; AM039952 X. gardneri Pepper and tomato 0–1[43] X. campestris pv. campestris Brassicaceae 0–1[42]; AM920689; CP000050; AE008922 X. axonopodis pv. glycines Soybean 1 [44]; AY780631 X. axonopodis pv. manihotis Cassava 1c [45] X. campestris pv. armoraciae Brassicaceae 0–3[46]; www.cmr.jvci.org X. axonopodis pv. citri Citrus 1–4[47–49]; AE008923 X. fuscans subsp. aurantifolii Citrus 2–4 ACPX00000000; ACPY00000000 X. translucens pvs. Cereals and grasses 0–6[50] X. campestris pv. malvacearum Cotton 6–10 [51,52] X. oryzae pv. oryzae Rice 7–16 [50]; CP000967; AP008229; AE013598 X. oryzae pv. oryzicola Rice 12–28 [50]; AAQN01000001 Ralstonia spp. Ralstonia solanacearum Broad host range 0–1[11]; AL646052

a The number of TAL effectors per strain was determined by Southern blot or genomic sequence. Southern blots did not reveal pseudogenes and might not have identified all alleles. Further information on Xanthomonas genomes and TALs can be found at www.xanthomonas.org and www.cmr.jcvi.org. b GenBank accession numbers are given as reference for sequenced genomes and plasmids. c The probe for Southern analysis yielded several bands, but contained TAL and TAL-flanking sequences which precludes allele number determination.

Figure 3

(a) FokI TAL TAL nuclease

tailored DNA-binding specificity

(b) FokI dimer TAL nuclease “A” TAL nuclease “B”

target sequence A target sequence B DNA 15-31 bp spacer

DNA Δ DNA targeted deletions targeted insertions

Current Opinion in Microbiology

TAL nucleases (TALNs) promote genome editing. (a) TALNs are fusions between TAL effectors and the FokI endonuclease domain. A tailored TAL repeat domain controls DNA-binding specificity. (b) Two TALNs bind neighboring DNA boxes and FokI dimerization induces DNA cleavage in the spacer region between the boxes. DNA double-strand breaks can promote nonhomologous end-joining (NHEJ) or homologous DNA recombination (HDR) enabling targeted genome modifications like deletions or insertions. termed TAL nucleases (TALNs or TALENs; Figure 3; boxes promoted DNA cleavage, followed by targeted [38,39]). Different TALs were combined with a FokI HDR in yeast with comparable efficiency as ZFNs nuclease domain at the N-terminus or C-terminus. [38,39]. Similarly, TALNs with artificial arrangements Expression of TALN pairs that bind to adjacent DNA of repeats were designed to target a given DNA sequence

(Figure 2 Legend Continued) solenoids are most often right-handed in nature. (c) Cartoon of repeats with different lengths (aa, amino acids) occuring in nature. The predominant 34 aa-repeat is boxed. (a)–(c) Yellow: first helix; orange: second helix; green: hypervariable residues; red: duplications, insertions (solid) and deletions (dashed) in comparison to typical Xanthomonas 34 aa-TAL repeats; minor (1 aa) changes are indicated by a red arrow; purple: aa sequence differs from Xanthomonas repeats. www.sciencedirect.com Current Opinion in Microbiology 2011, 14:47–53 52 Host-microbe interactions: bacteria

 [38]. Surprisingly, some artificial TALNs showed no First report (with Ref. [8 ]) that TAL effectors bind to a specific DNA sequence in promoters of target plant genes. AvrBs3 induces hypertro- activity, indicating that some unknown parameters still phy via expression of UPA20, a cell-size regulator from pepper.  govern efficiency of TALNs [38 ]. The ZFN technology 8. Ro¨ mer P, Hahn S, Jordan T, Strauß T, Bonas U, Lahaye T: Plant- has been hampered by cytotoxicity possibly due to off-  pathogen recognition mediated by promoter activation of the pepper Bs3 resistance gene. Science 2007, target cleavage at multiple genomic sites [40,41]. In 318:645-648. contrast, TALs are highly sensitive to changes in their First report (with Ref. [7]) that TAL effectors bind to a specific DNA target DNA boxes and three to four mutations typically sequence in promoters of target induced plant genes. AvrBs3 induces  expression of the pepper resistance gene Bs3 leading to cell death. Bs3 block recognition [9 ,19] which indicates that off-targets encodes a putative flavin-monooxygenase. might be limited. 9. Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S,  Lahaye T, Nickstadt A, Bonas U: Breaking the code of DNA Conclusions binding specificity of TAL-type III effectors. Science 2009, 326:1509-1512. Rarely has a protein in nature been identified whose Experimental evidence that solved the code of DNA recognition speci- specificity can be readily predicted from its amino acid ficity of TAL effectors. First artificial TALs were generated and shown to exhibit the predicted specificity. See also Ref. [10]. sequence and furthermore, easily modified. The simple structure-function encryption of the TAL–DNA-binding 10. Moscou MJ, Bogdanove AJ: A simple cipher governs DNA  recognition by TAL effectors. Science 2009, 326:1501. specificity is both elegant and intriguing. It immediately Bioinformatic analysis that supported a model for DNA recognition suggests use of TAL effectors as programmable DNA- specificity of TAL effectors. See also Ref. [9]. targeting devices. First results showed that TAL 11. Heuer H, Yin Y-N, Xue Q-Y, Smalla K, Guo J-H: Repeat domain diversity of avrBs3-like genes in Ralstonia solanacearum nucleases have specific activity and certainly other appli- strains and association with host preferences in the field. Appl cations will be explored, soon. TAL proteins have the Environ Microbiol 2007, 73:4379-4384. potential to rival the existing ZF technology and establish 12. Salanoubat M, Genin S, Artiguenave F, Gouzy J, Mangenot S, a game-changing variety of novel DNA sequence-specific Arlat M, Billault A, Brottier P, Camus JC, Cattolico L et al.: Genome sequence of the plant pathogen Ralstonia solanacearum. applications (specific gene activators, repressors, DNA- Nature 2002, 415:497-502. probes, gene replacement, and mutations) and hopefully 13. Chu Z, Yuan M, Yao J, Ge X, Yuan B, Xu C, Li X, Fu B, Li Z, therapeutics. Bennetzen JL et al.: Promoter mutations of an essential gene for pollen development result in disease resistance in rice. Note added in proof Genes Dev 2006, 20:1250-1255. Recently, TAL nucleases were successfully employed to 14. Gu K, Yang B, Tian D, Wu L, Wang D, Sreekala C, Yang F, Chu Z,  Wang G-L, White FF et al.: R gene expression induced by a edit human chromosomal targets and designed TALs type-III effector triggers disease resistance in rice. Nature induced expression of endogenous human genes [53]. 2005, 435:1122. First report that plant resistance genes against TAL effectors can be transcriptionally induced by TAL effectors. The rice resistance gene Xa27 Acknowledgements and the cognate TAL AvrXa27 were cloned. We thank D. Bu¨ ttner, R. Koebnik, and U. Bonas for discussions and helpful suggestions on the manuscript. Research in our laboratory is supported by a 15. Yang B, Sugio A, White FF: Os8N3 is a host disease- susceptibility gene for bacterial blight of rice. Proc Natl Acad grant from the Deutsche Forschungsgemeinschaft (SPP 1212) to J.B.  Sci U S A 2006, 103:10503-10508. Report of the TAL-induced susceptibility gene Os8N3 from rice that References and recommended reading supports bacterial proliferation. Os8N3 is similar to the nodulin MtN3 Papers of particular interest, published within the period of review, from Medicago truncatula which is induced during symbiosis. have been highlighted as: 16. 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