Screening and identification of a non-peptide antagonist for the peptide hormone receptor in Arabidopsis

Hidefumi Shinohara,Yoshikatsu Matsubayashi,Naoko Yasue,Yasumitsu Kondoh,Minoru Yoshida,Tetsuo Onuki

doi:10.1038/s42003-019-0307-8

Abstract

Intercellular signaling mediated by peptide hormones and membrane-localized receptor kinases plays crucial roles in plant developmental processes. Because of their diverse functions, agonistic or antagonistic modulation of peptide signaling holds enormous promise for agricultural applications. Here we established a high-throughput screening system using a bead-immobilized receptor kinase and fluorescent-labeled peptide ligand to identify small molecules that bind peptide hormone receptors in competition with natural ligands. We used the Arabidopsis CLE9-BAM1 ligand-receptor pair to screen a library of ≈30,000 chemicals and identified NPD12704 as an antagonist for BAM1. NPD12704 also inhibited CLV3 binding to BAM1 but only minimally interfered with CLV3 binding to CLV1, the closest homolog of BAM1, demonstrating preferential receptor specificity. Treatment of clv1-101 mutant seedlings with NPD12704 enhanced the enlarged shoot apical meristem phenotype. Our results provide a technological framework enabling high-throughput identification of small non-peptide chemicals that specifically control receptor kinase–mediated peptide hormone signaling in plants.

Highlights

Intercellular signaling mediated by peptide hormones and membrane-localized receptor kinases plays crucial roles in plant developmental processes
We used Arabidopsis receptor kinase BAM111 as a model, primarily because this receptor kinase plays a pivotal role in regulating shoot apical meristem (SAM) size redundantly with the closely related receptor kinase CLV112 by recognizing the peptide ligand CLV313–16
When BAM1-Sepharose microbeads were incubated with Alexa488-CLE9 at 100 nM, strong green fluorescence of Alexa488-CLE9 was detected on the outer surface of the microbeads under confocal laser scanning microscopy (Fig. 1c)

Summary

Introduction

Intercellular signaling mediated by peptide hormones and membrane-localized receptor kinases plays crucial roles in plant developmental processes Because of their diverse functions, agonistic or antagonistic modulation of peptide signaling holds enormous promise for agricultural applications. Extracellular signaling mediated by small peptide hormones and membrane-spanning receptor kinases plays crucial roles in numerous developmental processes in plants, including vegetative growth, stem cell regulation, vascular differentiation, nitrogen acquisition, pollen tube guidance, tissue abscission, symbiosis regulation, stomata differentiation, and diffusion barrier formation[1–4]. BAM1 interacts with several CLV3 homologs with high affinity, including CLE9 peptide, which enabled us to synthesize a high-affinity fluorescent-labeled ligand by introducing a fluorescent group into evolutionarily unconserved residues[17] Using this system, we screened a library of ~30,000 chemicals and identified one compound that acts as an antagonist for BAM1

Methods

Results

Conclusion