Comprehensive identification of <i>Arabidopsis thaliana</i> MYB transcription factors interacting with R/B‐like BHLH proteins

TLDR

Protein–protein interaction specificity of Arabidopsis thaliana MYB transcription factors is governed by a conserved amino‑acid signature that underlies their binding to R/B‑like BHLH proteins and informs on their multifunctionality, specificity, and redundancy. This study aims to comprehensively catalogue MYB proteins that interact with R/B‑like BHLH partners within the TTG1‑dependent regulatory network. The signature resides on helices 1 and 2 of the R3 repeat, forming a surface‑exposed hydrophobic/charged pattern, and its functional relevance was assessed in vivo using a transient DFR promoter activation assay. The conserved motif accurately predicts new MYB/BHLH interactions, distinguishes closely related MYB proteins, is shared by 14 R2R3 and 6 1R MYBs, and single‑site mutations or loss of two key residues abolish binding and impair promoter activation.

Abstract

Summary In‐depth analysis of protein–protein interaction specificities of the MYB protein family of Arabidopsis thaliana revealed a conserved amino acid signature ([DE]Lx 2 [RK]x 3 Lx 6 Lx 3 R) as the structural basis for interaction between MYB and R/B‐like BHLH proteins. The motif has successfully been used to predict new MYB/BHLH interactions for A. thaliana proteins, it allows to discriminate between even closely related MYB proteins and it is conserved amongst higher plants. In A. thaliana , the motif is shared by fourteen R2R3 MYB proteins and six 1R MYB proteins. It is located on helices 1 and 2 of the R3 repeat and forms a characteristic surface‐exposed pattern of hydrophobic and charged residues. Single‐site mutation of any amino acid of the signature impairs the interaction. Two particular amino acids have been determined to account for most of the interaction stability. Functional specificity of MYB/BHLH complexes was investigated in vivo by a transient DFR promoter activation assay. Residues stabilizing the MYB/BHLH interaction were shown to be critical for promoter activation. By virtue of proved and predicted interaction specificities, this study provides a comprehensive survey of the MYB proteins that interact with R/B‐like BHLH proteins potentially involved in the TTG1‐dependent regulatory interaction network. The results are discussed with respect to multi‐functionality, specificity and redundancy of MYB and BHLH protein function.