Repeat 1 of TAL effectors affects target specificity for the base at position zero.
Bottom Line: AvrBs3, the founding member of the Xanthomonas transcription-activator-like effectors (TALEs), is translocated into the plant cell where it localizes to the nucleus and acts as transcription factor.T0 and W232 appear to be particularly important if the RVD of the first repeat is HD ('rep1 effect').Our findings provide novel insights into the mechanism of T0 recognition by TALE proteins and are important for TALE-based biotechnological applications.
Affiliation: Department of Genetics, Martin Luther University, Weinbergweg 10, 06120 Halle (Saale), Germany firstname.lastname@example.org.Show MeSH
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Mentions: Structural data revealed that a tryptophan residue located in the ‘RVD-loop’ of repeat −1 is the most proximal amino acid to T0 in the target DNA. The tryptophan is believed to interact with the base by van der Waals forces (10). Both the tryptophan residue and T0 are highly conserved in natural TALEs and target sequences, respectively (18). To investigate the importance of tryptophan at position 232 (W232) in AvrBs3 and to identify amino acids that broaden or change target specificity for N0 we generated avrBs3 mutant derivatives. The activity of AvrBs3 and derivatives was determined using the GUS reporter system containing the optimal EBEAvrBs3 (Figure 2A). Figure 2B shows that most amino acid substitutions in AvrBs3 led to drastically reduced activity. However, substitutions of W232 by the aromatic amino acids tyrosine (W232Y) and phenylalanine (W232F) retained the highest activity (∼70 and 50%) compared to the wild-type (WT) protein and, like WT AvrBs3, worked best with T0 (Figure 2B). Expression of all proteins was confirmed by immunoblot (Supplementary Figure S2). Together, these results confirm the crucial importance of W232 in AvrBs3. There were no AvrBs3 derivatives with single substitutions that significantly performed better with any nucleotide at position zero (N0) than the WT. Only AvrBs3(W232R) showed slightly increased activity in combination with G0.
Affiliation: Department of Genetics, Martin Luther University, Weinbergweg 10, 06120 Halle (Saale), Germany email@example.com.