Specificity of MYB interactions relies on motifs in ordered and disordered contexts

Abstract

Physical interactions between members of the MYB and bHLH transcription factor (TF) families regulate many important biological processes in plants. Not all reported MYB–bHLH interactions can be explained by the known binding sites in the R3 repeat of the MYB DNA-binding domain. Noteworthy, most of the sequence diversity of MYB TFs lies in their non-MYB regions, which contain orphan small subgroup-defining motifs not yet linked to molecular functions. Here, we identified the motif mediating interaction between MYB TFs from subgroup 12 and their bHLH partners. Unlike other known MYB–bHLH interactions, the motif locates to the centre of the predicted disordered non-MYB region. We characterised the core motif, which enabled accurate prediction of previously unknown bHLH-interacting MYB TFs in Arabidopsis thaliana, and we confirmed its functional importance in planta. Our results indicate a correlation between the MYB–bHLH interaction affinity and the phenotypic output controlled by the TF complex. The identification of an interaction motif outside R3 indicates that MYB–bHLH interactions must have arisen multiple times, independently and suggests many more motifs of functional relevance to be harvested from subgroup-specific studies.

Evolution of A bHLH Interaction Motif

“…Interactions between proteins from the two most expanded plant TF families, the MYB (v-myb avian myeloblastosis viral oncogene homolog) family and the bHLH (basic helix-loop-helix) family, are common, and together they regulate a large variety of biological processes [ 2 , 3 ]. Recently, we determined the binding site responsible for interactions between eight Arabidopsis thaliana MYB TFs and their bHLH partners, MYC2, MYC3, and MYC4 [ 4 ]. The binding site constitutes a short linear motif (SLiM) and coincides with a sequence motif defining a subgroup of six MYB TFs (MYB28, MYB29, MYB76, MYB34, MYB51, and MYB122) that require MYC-interaction for activity [ 4 , 5 ].…”

“…Recently, we determined the binding site responsible for interactions between eight Arabidopsis thaliana MYB TFs and their bHLH partners, MYC2, MYC3, and MYC4 [ 4 ]. The binding site constitutes a short linear motif (SLiM) and coincides with a sequence motif defining a subgroup of six MYB TFs (MYB28, MYB29, MYB76, MYB34, MYB51, and MYB122) that require MYC-interaction for activity [ 4 , 5 ]. All members of this subgroup are involved in regulating glucosinolate (GLS) biosynthesis [ 6 , 7 ], and have homologs throughout the Brassicaceae [ 8 ].…”

“…The MIM consists of the six residues [L/F]LN[K/R][V/L]A, with the core motif (residue positions that are absolutely required) being xLNxxA. Mutations in these core residues abolish the interaction [ 4 ].…”

“…For example, SLiMs within IDRs facilitate interactions with SLiM-binding pockets in globular domains [ 19 ]. The MIM is a SLiM located outside the DBD [ 4 ], and the short length combined with its presence in a long disordered context increases the chance of ex nihilo evolution [ 20 ], i.e., the likelihood that proteins from other families could evolve an analogous interaction motif.…”

“…In contrast to the evolutionarily young GLSs, the molecular components required for JA biosynthesis, signaling, and perception, including homologs of MYC2, are much more widespread in the plant lineage, being omnipresent in land plants [ 36 , 37 ]. Although the eight known MIM-containing TFs from A. thaliana are all R2R3 MYB TFs, and the MYC-interaction is essential for activating GLS biosynthesis [ 4 ], the direct role of the MIM is to link TF activity to JA signaling through MYC-interaction. We therefore hypothesized that MIMs could be present in proteins from other families, and in TFs that are not involved in GLS regulation but instead regulate other JA-responsive genes.…”