A Super Strong Engineered Auxin–TIR1 Pair

Abstract

Auxin regulates diverse aspects of plant growth and development through induction of the interaction between TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX proteins (TIR1/AFBs) and AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) co-receptor proteins and the subsequent transcriptional regulation. The artificial control of endogenous auxin signaling should enable the precise delineation of auxin-mediated biological events as well as the agricultural application of auxin. To this end, we previously developed a synthetic auxin-receptor pair that consists of 5-(3-methoxyphenyl)-IAA (convexIAA, cvxIAA) and the engineered TIR1 whose phenylalanine at position 79 in the auxin-binding pocket is substituted to glycine (TIR1F79G) (concaveTIR1, ccvTIR1). This synthetic auxin-receptor pair works orthogonally to natural auxin signaling in transgenic plants harboring the engineered TIR1 by exogenous application of 5-(3-methoxyphenyl)-IAA, and has potential to be utilized as novel agricultural/horticultural tools. In the present study, we report an improved version of the synthetic cvxIAA-ccvTIR1 pair such that synthetic IAA can act at lower concentrations. Using a yeast two-hybrid system, we screened various 5-substituted IAAs and identified 5-adamantyl-IAA, named pico_cvxIAA, which mediates interaction of TIR1F79G and IAA3 proteins at a 1,000-fold lower concentration than the original version, 5-(3-methoxyphenyl)-IAA. Furthermore, we found that TIR1F79A interacts with IAA3 protein in the presence of picomolar concentrations of 5-adamantyl-IAA, 10,000-fold lower than our prototype version of the cvxIAA-ccvTIR1 pair. In addition, pull-down assays confirmed that 5-adamantyl-IAA mediates in vitro interaction of TIR1F79A and IAA7-DII peptides at lower concentrations. The improved synthetic IAA-TIR1 pair with high affinity would be beneficial for basic science as well as for practical use in agriculture/horticulture.

A super sensitive auxin-inducible degron system with an engineered auxin-TIR1 pair

“…We observed fewer growth defects in cells expressing OsTIR1 F74G , OsTIR1 F74C , or OsTIR1 F74S than in cells expressing OsTIR1 F74A in the presence of 5-Ad-IAA (Fig. 1c), which was consistent with a previous finding that AtTIR1 F79G and AtTIR1 F79S show lower affinities to auxin derivatives than AtTIR1 F79A 8 9 . Therefore, OsTIR1 F74A most efficiently degrades mScarlet-mAID-CENP-H in the presence of the 5-Ad-IAA in DT40 cells.…”

“…To overcome auxin cytotoxicity, we took advantage of an orthogonal auxin-TIR1 system engineered via “bump and hole” approach. Here, a high-affinity interaction occurs pairwise between an auxin derivative with an extra bump, 5-adamantyl-IAA (5-Ad-IAA, also known as pico-cvxIAA), and a modified Arabidopsis TIR1 protein with a complementary cavity in the auxin binding pocket achieved by an F79A amino acid (aa) substitution (AtTIR1 F79A ) 8 9 . First, we performed a yeast two-hybrid assay to determine the effective concentrations of 5-Ad-IAA for the interaction of TIR1 with the auxin-responsive protein Arabidopsis thaliana IAA17 (AtIAA17) that was used as the AID-tag (Supplementary Fig.…”

Harnessing synthetic chemistry to probe and hijack auxin signaling

“…Surprisingly, IAA with a nonaromatic substitution at the 5 th position, 5‐adamantyl‐IAA (Fig. ), triggers an interaction of ccvTIR1 and DII peptide at a concentration as low as a pico‐molar, 10 000‐fold lower than the prototype version of cvxIAA‐ccvTIR1 pair in vitro (Yamada et al ., ). Indeed, the new 5‐adamantly‐IAA, now named pico‐cvxIAA, could elicit severe root growth inhibition of Arabidopsis seedlings overexpressing the engineered ccvTIR1 (Yamada et al ., ).…”

“…), triggers an interaction of ccvTIR1 and DII peptide at a concentration as low as a pico‐molar, 10 000‐fold lower than the prototype version of cvxIAA‐ccvTIR1 pair in vitro (Yamada et al ., ). Indeed, the new 5‐adamantly‐IAA, now named pico‐cvxIAA, could elicit severe root growth inhibition of Arabidopsis seedlings overexpressing the engineered ccvTIR1 (Yamada et al ., ). It is difficult to predict the extreme potency of pico‐cvxIAA based on the structural modeling a priori , and its explanation would require future structural analysis of the pico‐cvxIAA‐ccvTIR1‐DII complex.…”

Plant Chemical Biology

“…To this end, the recent report of a synthetic auxin-receptor pair that works orthogonally to natural auxin signaling to evoke auxinmediated developmental and physiological responses is an impressive step forward . In this issue, Yamada et al (2018) further describe improved versions of synthetic auxin-receptor pairs that can now act at much lower concentrations-in some cases 10,000-fold lower than their prototype version. The improved synthetic auxin-receptor pair with such high affinity will not only benefit fundamental research but should also have applications in agriculture/horticulture.…”