Elucidating the evolutionary conserved DNA-binding specificities of WRKY transcription factors by molecular dynamics and in vitro binding assays

Abstract

WRKY transcription factors constitute a large protein family in plants that is involved in the regulation of developmental processes and responses to biotic or abiotic stimuli. The question arises how stimulus-specific responses are mediated given that the highly conserved WRKY DNA-binding domain (DBD) exclusively recognizes the ‘TTGACY’ W-box consensus. We speculated that the W-box consensus might be more degenerate and yet undetected differences in the W-box consensus of WRKYs of different evolutionary descent exist. The phylogenetic analysis of WRKY DBDs suggests that they evolved from an ancestral group IIc-like WRKY early in the eukaryote lineage. A direct descent of group IIc WRKYs supports a monophyletic origin of all other group II and III WRKYs from group I by loss of an N-terminal DBD. Group I WRKYs are of paraphyletic descent and evolved multiple times independently. By homology modeling, molecular dynamics simulations and in vitro DNA–protein interaction-enzyme-linked immunosorbent assay with AtWRKY50 (IIc), AtWRKY33 (I) and AtWRKY11 (IId) DBDs, we revealed differences in DNA-binding specificities. Our data imply that other components are essentially required besides the W-box-specific binding to DNA to facilitate a stimulus-specific WRKY function.

ArabidopsisTTG2 RegulatesTRYExpression through Enhancement of Activator Complex-Triggered Activation

“…These bidirectional interaction possibilities could in turn cause the multi-TTG2/DNA aggregates in EMSA studies when using TTG2 instead of the WRKY18 DNA binding domain alone. TTG2 itself contains two WRKY domains and belongs to group1 of the WRKY family (Brand et al, 2013). Studies with the ELISA-DPI technique showed that generally both domains of WRKY33, another group 1 member, could bind DNA.…”

“…Studies with the ELISA-DPI technique showed that generally both domains of WRKY33, another group 1 member, could bind DNA. Nevertheless the binding of the cDBD (C-terminal domain) is stronger than of the nDBD (Brand et al, 2013). Whether both WRKY domains of TTG2 can bind to the two W-boxes remains to be investigated.…”

The Enzyme-Like Domain of Arabidopsis Nuclear β-Amylases Is Critical for DNA Sequence Recognition and Transcriptional Activation

“…We tested in vitro DNA binding of the chimeric proteins in a DNAprotein interaction (DPI)-ELISA assay (Brand et al, 2010(Brand et al, , 2013 and in vivo transactivation activity in leaf protoplasts. Intriguingly, fusion of the BAM domain of BAM8 to the N-terminal part of BAM7 (BAM78) resulted in a protein that bound to the BBRE motif in vitro and activated reporter gene expression in protoplasts.…”

“…DNA binding properties of BZR1-BAM wild-type and mutated proteins were determined using the DPI-ELISA method as described before (Brand et al, 2010(Brand et al, , 2013. The amount of total protein input was normalized to the level of heterologous expressed protein (Supplemental Figure 9).…”

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“…We speculated that the W-box consensus might be more degenerate and yet undetected differences in the W-box consensus of WRKYs of different evolutionary descent exist. Simulation analysis was performed to explore differences in DNA-binding specificities 38 .…”