Abstract
TAL effectors are re-targetable transcription factors used for tailored gene regulation and, as TAL effector-nuclease fusions (TALENs), for genome engineering. Their hallmark feature is a customizable central string of polymorphic amino acid repeats that interact one-to-one with individual DNA bases to specify the target. Sequences targeted by TAL effector repeats in nature are nearly all directly preceded by a thymine (T) that is required for maximal activity, and target sites for custom TAL effector constructs have typically been selected with this constraint. Multiple crystal structures suggest that this requirement for T at base 0 is encoded by a tryptophan residue (W232) in a cryptic repeat N-terminal to the central repeats that exhibits energetically favorable van der Waals contacts with the T. We generated variants based on TAL effector PthXo1 with all single amino acid substitutions for W232. In a transcriptional activation assay, many substitutions altered or relaxed the specificity for T and a few were as active as wild type. Some showed higher activity. However, when replicated in a different TAL effector, the effects of the substitutions differed. Further, the effects differed when tested in the context of a TALEN in a DNA cleavage assay, and in a TAL effector-DNA binding assay. Substitution of the N-terminal region of the PthXo1 construct with that of one of the TAL effector-like proteins of Ralstonia solanacearum, which have arginine in place of the tryptophan, resulted in specificity for guanine as the 5’ base but low activity, and several substitutions for the arginine, including tryptophan, destroyed activity altogether. Thus, the effects on specificity and activity generated by substitutions at the W232 (or equivalent) position are complex and context dependent. Generating TAL effector scaffolds with high activity that robustly accommodate sites without a T at position 0 may require larger scale re-engineering.
Highlights
Transcription activator-like (TAL) effectors are a class of DNA binding transcription factors injected into host cells by members of the plant pathogenic bacterial genusXanthomonas for targeted activation of specific host genes during infection
To test whether W232 accounts for TAL effector specificity for T at the 0th position of the binding site, we generated a full length TAL effector construct encoding the repeat region of X. oryzae TAL effector PthXo1 (Figure 2B) with the wild-type W232 as well as variants with all 19 possible single amino acid substitutions at this position
When the latter substitutions were tested in a second effector, TAL868, the effects were different, revealing that the effects of substitutions on activity and 0th position specificity depend on TAL effector context, the composition of the central repeat region (CRR)
Summary
Xanthomonas for targeted activation of specific host genes during infection. They are important tools for biological engineering and genome editing because they can be readily engineered to bind to almost any DNA sequence of interest. TAL effector binding site specificity is determined by a central repeat region (CRR) composed of a variable number of tandem repeats each typically 33-34 amino acids in length. Discovery of the TAL effector-DNA binding code has enabled the identification of candidate EBEs for specific TAL effectors, which facilitates the identification of TAL effector-targeted plant genes that play important roles in diseases caused by Xanthomonas spp. The repeatencoded targeting mechanism has proven to be modular, with no qualitative neighbor or context effects on specificity, making it possible to generate custom TAL effectors with desired specificities by assembling a CRR with an RVD sequence that corresponds to the intended target site (e.g. 6)
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