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 email@example.com.Show MeSH
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Mentions: The tryptophan (W232) that coordinates TALE contact to the base at position N0 is not conserved in TalC from the rice pathogen X. oryzae pv. oryzae (15). Instead, TalC contains a cysteine residue, which when introduced into AvrBs3 led to low activity [AvrBs3(W232C); Figure 2B]. Notably, the natural target box of TalC starts with C0 (15). TalC harbors additional substitutions and a deletion of 23 aa in the NTR (Supplementary Figure S4A). To test whether the NTR of TalC confers a preference for an EBE with C0 we compared the activities of AvrBs3, AvrBs3(W232C) and chimeras between TalC and AvrBs3 (Figure 3A). As targets we used the same four different EBEAvrBs3-reporters as in Figure 2. Surprisingly, the swap of the NTRs resulted in a non-functional AvrBs3 protein. Sequence comparison (Supplementary Figure S4A) revealed an amino acid difference in TalC repeat 0, which according to 3D data (9) is located in an α-helical region that is tightly packed together with the neighboring helix of the canonical repeat 1. We therefore tested chimeras between TalC-NTR and AvrBs3 in which we shortened the fragment contributed by TalC. As shown in Figure 3A, AvrBs3 activity improved when the protein contained only the very NTR of TalC including repeat −2 [AvrBs3-N2(TalC)]. However, AvrBs3 containing only repeat −1 and repeat 0 from TalC displayed very low activity [AvrBs3-N3(TalC)]. Only the exchange of repeat 0 is tolerated (AvrBs3-N5(TalC) but led to reduced AvrBs3 activity (Figure 3A). This confirms our hypothesis that amino acid differences in the helices of repeat 0 and repeat 1 affect protein activity. Furthermore, the results underpin the necessity of W232 for AvrBs3 function.
Affiliation: Department of Genetics, Martin Luther University, Weinbergweg 10, 06120 Halle (Saale), Germany firstname.lastname@example.org.