Functional analysis of molecular interactions in synthetic auxin response circuits

Abstract

Auxin-regulated transcription pivots on the interaction between the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) repressor proteins and the AUXIN RESPONSE FACTOR (ARF) transcription factors. Recent structural analyses of ARFs and Aux/IAAs have raised questions about the functional complexes driving auxin transcriptional responses. To parse the nature and significance of ARF-DNA and ARF-Aux/IAA interactions, we analyzed structure-guided variants of synthetic auxin response circuits in the budding yeast Saccharomyces cerevisiae. Our analysis revealed that promoter architecture could specify ARF activity and that ARF19 required dimerization at two distinct domains for full transcriptional activation. In addition, monomeric Aux/IAAs were able to repress ARF activity in both yeast and plants. This systematic, quantitative structure-function analysis identified a minimal complex-comprising a single Aux/IAA repressing a pair of dimerized ARFs-sufficient for auxin-induced transcription.auxin signaling | synthetic circuits | transcription | ARF | PB1

Structure of the Arabidopsis TOPLESS corepressor provides insight into the evolution of transcriptional repression

“…6). Expression of AtTPL202 lacking the two WD40 domains repressed DR5 activity similarly consistent with previous assays in yeast (30,31). Conversely, the AtTPL202 mutant forms affecting the EAR peptide binding G3 groove (TPL202m/F74Q) and the dimeric mutant TPL202m/ K102S-T116A-Q117S-E122S lost their repression activity.…”

Accelerating structure‐function mapping using the ViVa webtool to mine natural variation

“…However, it is unclear whether ARF multimerization occurs or plays a significant role in vivo. Interfering with ARF dimerization in either the DNA‐binding proximal dimerization domain or the PB1 domain decreases the ability of class A ARFs to activate transcription in a heterologous yeast system (Pierre‐Jerome, Moss, Lanctot, Hageman, & Nemhauser, ).…”

Regulation of auxin transcriptional responses

“…However, expression of nonoligomerizing IAA17 and IAA19 in protoplasts had an intermediate effect on repressive activity of these Aux/IAAs; expression of nonoligomerizing IAA1a in Physcomitrella had an intermediate effect on IAA1a repressive activity . Further, expression of a stabilized nonoligomerizing IAA14 variant efficiently repressed auxin responses, consistent with IAA14 oligomerization being unnecessary for ARF repression. In addition, Aux/IAA multimerization may be required for efficient recruitment of TPL, as structural studies have shown that binding affinity of the TPL/TPR co‐repressor increases in the presence of oligomerized EAR‐motif containing repressors .…”

“…Mutation of either the conserved lysine or OPCA residues is sufficient to disrupt PB1 domain interactions, however additional, less conserved residues within each of the domain interaction faces also contribute to PB1 domain binding affinity . Further, recapitulation of the auxin signaling pathway in a synthetic yeast system revealed potential face preference in PB1 domain interactions between ARF and Aux/IAA pairs . Additional work is necessary to determine if sequence variation in individual PB1 domain faces regulates ARF‐Aux/IAA interaction specificity and whether these pair preferences play a functional role in auxin signaling.…”

“…In addition to regulating auxin response through interactions with Aux/IAA repressors, ARF PB1 domain interactions enigmatically play a critical role in DNA binding. Deletion of the ARF PB1 domain results in reduced dimerization of ARF protein DBDs and consequent ability to bind DNA, suggesting that while the DBD is sufficient for ARF dimerization in vivo, interactions through the PB1 domain may act to stabilize DBD dimers …”