Regulation of selected genome loci using de novo-engineered transcription activator-like effector (TALE)-type transcription factors

Abstract

Proteins that can be tailored to bind desired DNA sequences are key tools for molecular biology. Previous studies suggested that DNA-binding specificity of transcription activator-like effectors (TALEs) from the bacterial genus Xanthomonas is defined by repeat-variable diresidues (RVDs) of tandem-arranged 34/35-amino acid repeat units. We have studied chimeras of two TALEs differing in RVDs and non-RVDs and found that, in contrast to the critical contributions by RVDs, non-RVDs had no major effect on the DNA-binding specificity of the chimeras. This finding suggests that one needs only to modify the RVDs to generate designer TALEs (dTALEs) to activate transcription of user-defined target genes. We used the scaffold of the TALE AvrBs3 and changed its RVDs to match either the tomato Bs4, the Arabidopsis EGL3, or the Arabidopsis KNAT1 promoter. All three dTALEs transcriptionally activated the desired promoters in a sequence-specific manner as mutations within the targeted DNA sequences abolished promoter activation. This study is unique in showing that chromosomal loci can be targeted specifically by dTALEs. We also engineered two AvrBs3 derivatives with four additional repeat units activating specifically either the pepper Bs3 or UPA20 promoter. Because AvrBs3 activates both promoters, our data show that addition of repeat units facilitates TALE-specificity fine-tuning. Finally, we demonstrate that the RVD NK mediates specific interaction with G nucleotides that thus far could not be targeted specifically by any known RVD type. In summary, our data demonstrate that the TALE scaffold can be tailored to target user-defined DNA sequences in whole genomes.

Fig. 1.

De novo-engineered dTALEs specifically activate promoters with matching UPT boxes. (A) In planta functional analysis of different TALE-promoter combinations. The uidA reporter constructs under transcriptional control of the promoters indicated at left were codelivered via A. tumefaciens into N. benthamiana leaves with the 35S promoter-driven TALE genes indicated above leaf discs. GUS assays were carried out 40 hpi. Leaf discs were stained with 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid, cyclohexylammonium salt (X-Gluc) to visualize activity of the GUS reporter. Asterisks (*) indicate TALE genes with optimized codon usage and corresponding gene products. (B) The dTALE[Bs4S] transcriptionally activates the endogenous Bs4S gene in tomato. Semiquantitative RT-PCR was carried out on RNA from tomato leaf tissue 36 h after A. tumefaciens-mediated delivery of the depicted 35S promoter-driven TALE genes. Isolated RNA was used for cDNA synthesis and semiquantitative RT-PCR was performed with primers specific for the tomato Bs4S gene. The constitutively expressed gene elongation factor 1α (EF1α) served as a normalization control.

Fig. 2.

The dTALE[EGL3] and dTALE[KNAT1] transcriptionally activate the EGL3 and KNAT1 endogenes in A. thaliana, respectively. Semiquantitative RT-PCR was carried out on RNA from the Arabidopsis ecotype Columbia (Col-0) and corresponding lines containing 35S promoter-driven dTALE[EGL3] or dTALE[KNAT1] transgenes. RNA was isolated from leaves and used for cDNA synthesis. Semiquantitative RT-PCR was performed with EGL3 and KNAT1 specific primers. The constitutively expressed gene actin2 served as an internal normalization control.

Fig. 3.

AvrBs3 derivatives with four additional C-terminal repeat units discriminate between the pepper Bs3 and UPA20 promoter. The depicted 35S promoter-driven TALE genes were delivered via A. tumefaciens into the pepper genotypes ECW or ECW-30R. Semiquantitative reverse-transcription RT-PCR was carried out on RNA isolated from leaf material 36 h upon infection with primers specific for the Bs3 or UPA20 gene. The constitutively expressed gene EF1α served as an internal normalization control.