Macromolecular tool box to elucidate CLAVATA3/Embryo Surrounding Region-Related-RLK binding, signaling and downstream effects

Madhumitha Narasimhan; Nina Jahnke; Felix Kallert; Elmehdi Bahafid; Franziska Böhmer; Laura Hartmann; Rüdiger Simon

doi:10.1093/jxb/erae206

Macromolecular tool box to elucidate CLAVATA3/Embryo Surrounding Region-Related-RLK binding, signaling and downstream effects

J Exp Bot. 2024 May 8:erae206. doi: 10.1093/jxb/erae206. Online ahead of print.

Authors

Madhumitha Narasimhan¹, Nina Jahnke², Felix Kallert², Elmehdi Bahafid¹, Franziska Böhmer², Laura Hartmann^{2

3}, Rüdiger Simon^{1

4}

Affiliations

¹ Institute for Developmental Genetics, Heinrich Heine University, Düsseldorf, North Rhine Westphalia, Germany.
² Institute of Organic Chemistry and Macromolecular Chemistry, Heinrich Heine University, Düsseldorf, North Rhine Westphalia, Germany.
³ Institute of Macromolecular Chemistry, University Freiburg, Freiburg im Breisgau, Baden-Wuerttemberg, Germany.
⁴ Institute for Developmental Genetics and Cluster of Excellence in Plant Sciences, Heinrich Heine University, Düsseldorf, North Rhine Westphalia, Germany.

PMID: 38717932
DOI: 10.1093/jxb/erae206

Abstract

Plant peptides communicate by binding to a large family of receptor-like kinases (RLKs) and they share a conserved binding mechanism, which may account for their promiscuous interaction with several RLKs. In order to understand the in vivo binding specificity of CLAVATA3/Embryo Surrounding Region-Related peptide family, we have developed a novel set of CLAVATA 3 (CLV3) based peptide tools. After carefully evaluating the CLE peptide binding characteristics, using solid phase synthesis process, we have modified the CLV3 peptide and attached a fluorophore and a photoactivable side group. We observed that the labeled CLV3 shows binding specificity within CLAVATA1 clade of RLKs while avoiding the distantly-related PEP RECEPTOR clade, thus resolving the contradictory results obtained previously by many in vitro methods. Furthermore, we observed that the RLK-bound CLV3 undergoes clathrin-mediated endocytosis and gets trafficked to vacuole via ARA7-labeled endosomes. Additionally, modifying CLV3 for light-controlled activation enabled spatial and temporal control over CLE signalling. Hence, our CLV3 macromolecular toolbox can be used to study rapid cell specific down-stream effects. Given the conserved binding properties, in the future our toolbox can also be used as a template to modify other CLE peptides.

Keywords: CLAVATA signaling; CLV3 peptide; chemical probe; endocytosis; fluorophore labeling; photoactivation; receptor specificity; solid phase peptide synthesis; sub-cellular trafficking.