Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Feb 28;50(4):2387-2400.
doi: 10.1093/nar/gkac098.

Flexible TALEs for an expanded use in gene activation, virulence and scaffold engineering

Sebastian Becker  1 , Stefanie Mücke  1 , Jan Grau  2 , Jens Boch  1
Affiliations

Flexible TALEs for an expanded use in gene activation, virulence and scaffold engineering

Sebastian Becker et al. Nucleic Acids Res. .

Abstract

Transcription activator-like effectors (TALEs) are bacterial proteins with a programmable DNA-binding domain, which turned them into exceptional tools for biotechnology. TALEs contain a central array of consecutive 34 amino acid long repeats to bind DNA in a simple one-repeat-to-one-nucleotide manner. However, a few naturally occurring aberrant repeat variants break this strict binding mechanism, allowing for the recognition of an additional sequence with a -1 nucleotide frameshift. The limits and implications of this extended TALE binding mode are largely unexplored. Here, we analyse the complete diversity of natural and artificially engineered aberrant repeats for their impact on the DNA binding of TALEs. Surprisingly, TALEs with several aberrant repeats can loop out multiple repeats simultaneously without losing DNA-binding capacity. We also characterized members of the only natural TALE class harbouring two aberrant repeats and confirmed that their target is the major virulence factor OsSWEET13 from rice. In an aberrant TALE repeat, the position and nature of the amino acid sequence strongly influence its function. We explored the tolerance of TALE repeats towards alterations further and demonstrate that inserts as large as GFP can be tolerated without disrupting DNA binding. This illustrates the extraordinary DNA-binding capacity of TALEs and opens new uses in biotechnology.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Aberrant repeat variants in Xanthomonas TALEs and their impact on DNA binding. (A) Amino acid alignment of natural repeat length polymorphisms. A standard 34 amino acid (aa) repeat is shown in bold with helix-forming residues underlined. Duplicated or deleted regions are shaded in grey. Xanthomonas oryzae pv. oryzae, Xoo; X. oryzae pv. oryzicola, Xoc; X. axonopodis pv. citri, Xac; X. theicola, Xt. (B) RVD composition of constructed TALEs. Position 7 (boxed) contains a standard 34 aa repeat (top row) or an aberrant repeat (XX aa) with 42, 40, 37, 36, 35, 30 or 28 aa. The TALE box (optimal, op, or with deletion at position 7, −1 p7) is fused to a minimal promoter and a β-glucuronidase (GUS) reporter gene. (C) TALE with type III secretion signal (T3SS), four noncanonical repeats (NCR), TFIIAγ binding site (TFB), nuclear localization signals (NLS) and acidic activation domain (AAD). The x indicates the aberrant repeat. (D) DNA specificities of selected RVDs. (E) In planta GUS assay for aberrant repeat-containing TALEs on boxes shown in panel (B). Error bars represent standard deviation (n = 3).
Figure 2.
Figure 2.
TALEs with multiple aberrant repeats in tandem loop out multiple repeats. (A) TALE set-up. Altered repeats are indicated by x. (B) RVDs of constructed TALEs containing zero (control) or one to six aberrant 40 aa repeats (boxed RVDs). TALE boxes were optimal (op) or frameshift variants with one to six nucleotides deleted (−1 to −6; dashed squares). (C) GUS assay of TALEs on boxes shown in panel (B). Error bars represent standard deviation (n = 3). Colours refer to the boxes in panel (B). (D) Possible binding behaviour of the TALE with four aberrant repeats (longer rectangles). Activity was observed only if combined with boxes that had three or four nucleotides deleted (−3 or −4), suggesting a simultaneous looping out of multiple aberrant repeats (tilted rectangles).
Figure 3.
Figure 3.
Two distant aberrant repeats can function independently. (A) TALE set-up. Altered repeats are indicated by x. (B) RVDs of constructed TALEs. Aberrant repeats are boxed. TALE boxes were optimal (op) or frameshift variants with one to six nucleotides deleted (−1 to −6; dashed squares). (C) GUS assay of TALEs on boxes shown in panel (B). Error bars represent standard deviation (n = 3). Colours refer to the boxes in panel (B). (D) Possible binding behaviour of the TALE with aberrant 40 aa repeats at positions 7 and 10 (longer rectangles). Binding likely occurs by looping out none (op), one (−1) or both (−2) aberrant repeats or by looping out both aberrant repeats together with several normal repeats in between (−3 and −4).
Figure 4.
Figure 4.
TalBK2 binds within the OsSWEET13 promoter. (A) RVDs of artBK2. Aberrant repeats are boxed (both 36 aa v1). TALE boxes were optimal (op) or frameshift variants with various deletions at positions 9–12 (−1 p9, −1 p12, −2 p9/12 and −4 p9–12). (B) GUS assay of constructs in panel (A). Error bars represent standard deviation (n = 3). (C) RVD composition of artAM2, artBK2 and a TALE designed to address all four shown OsSWEET13 promoter variants (flexTALE). Aberrant repeats are boxed (36 aa v1). The 1000-bp fragments of the OsSWEET13 promoter from different rice cultivars (Zhenshan, Zhen; Nipponbare, Nip; Sadu Cho, Sadu; and IR24) were amplified and fused in front of the GUS reporter gene. Only the TALE target region is shown. Differences between cultivars are indicated by dashed boxes. (D) GUS assay of constructs shown in panel (C). Error bars represent standard deviation (n = 3). (E) Representative examples from X. oryzae TALE classes targeting OsSWEET genes. (F) Schematic of constructed TALEs. N- and C-terminal regions are derived from Hax3.
Figure 5.
Figure 5.
Tolerance of a TALE repeat for insertions. (A) Constructed repeat variants. A 7 aa serine–glycine linker (SSGGGGS) was placed at different positions (#) throughout a TALE repeat. RVDs, boxed; inserted amino acids, grey. Functional motifs are coloured according to panel (D). (B) TALE set-up. The altered repeat is indicated (x). (C) RVDs of constructed TALEs with the repeat at position 8 containing the different linker insertions. TALEs were tested on an optimal box (op) and a −1 frameshift derivative (−1 p8). (D) Functional motifs found within a TALE repeat according to Deng et al. (66). (E, F) Tolerance of a TALE to the insertion of 7 aa serine–glycine linkers in a single repeat based on the data in panel (G). Colour code: activity at optimal box (no looping out), green; activity at frameshift sequences (looping out), orange; flexible binding, light green; loss of TALE activity, red; no data, black. Number of lines below the amino acids correspond to relative activity in panel (G). RVDs, boxed. (G) GUS assay of TALEs with repeat variants on boxes shown in panel (C). Error bars represent standard deviation (n = 3). (H) Expression of the TALEs in planta was confirmed after immunoblotting using an anti-GFP antibody. Asterisks indicate expected sizes.
Figure 6.
Figure 6.
Insertion of foreign domains into TALE repeats. (A) RVDs of constructed TALEs. GFP (*) was inserted into the TALE repeat array at three different positions (TA–GFP–LE v1–v3) or at all three positions simultaneously (TA–3×GFP–LE). (B) GUS assay of TALEs on boxes shown in panel (A). Error bars represent standard deviation (n = 3). (C) Confocal microscopy images of GFP or GFP-tagged TALEs. Size bar is 100 μm. The set-up of the different constructs is visualized by cartoons.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Boch J., Bonas U.. Xanthomonas AvrBs3 family-type III effectors: discovery and function. Annu. Rev. Phytopathol. 2010; 48:419–436. - PubMed
    1. Perez-Quintero A.L., Szurek B.. A decade decoded: spies and hackers in the history of TAL effectors research. Annu. Rev. Phytopathol. 2019; 57:459–481. - PubMed
    1. Wilkins K., Booher N., Wang L., Bogdanove A.. TAL effectors and activation of predicted host targets distinguish Asian from African strains of the rice pathogen Xanthomonasoryzae pv. oryzicola while strict conservation suggests universal importance of five TAL effectors. Front. Plant Sci. 2015; 6:536. - PMC - PubMed
    1. Tran T.T., Pérez-Quintero A.L., Wonni I., Carpenter S.C.D., Yu Y., Wang L., Leach J.E., Verdier V., Cunnac S., Bogdanove A.J.et al. .. Functional analysis of African Xanthomonasoryzae pv. oryzae TALomes reveals a new susceptibility gene in bacterial leaf blight of rice. PLoS Pathog. 2018; 14:e1007092. - PMC - PubMed
    1. Mücke S., Reschke M., Erkes A., Schwietzer C.-A., Becker S., Streubel J., Morgan R.D., Wilson G.G., Grau J., Boch J.. Transcriptional reprogramming of rice cells by Xanthomonasoryzae TALEs. Front. Plant Sci. 2019; 10:162. - PMC - PubMed

Publication types

MeSH terms