In vitro evaluation of protein–protein interactions in the rice KAI2 ligand signaling complex

Abstract KARRIKIN INSENSITIVE 2 (KAI2)/DWARF14-LIKE (D14L) plays key roles in land plant development, environmental responses, and the establishment of arbuscular mycorrhizal symbiosis, likely acting as the receptor for unidentified signaling molecules termed KAI2 ligands (KLs). KL perception by KAI2/D14L promotes DWARF3 (D3)/MORE AXILLARY GROWTH2 (MAX2) F-box protein-mediated ubiquitination of SUPPRESSOR OF MAX2 1 (SMAX1) proteins, thereby transducing the KL signals. Although genetic and in vivo assays have demonstrated the functions of these components, the biochemical details of their interactions remain elusive. Here we investigated physical interactions between rice D14L, D3, and OsSMAX1 in vitro using desmethyl germinone (dMGer), a recently developed KL analog. dMGer elicited KL responses in rice with higher activity and pathway specificity than a widely used KL analog (−)-GR24. dMGer, but not (−)-GR24, directly bound to D14L and promoted the interaction between D14L and D3 in vitro . The interaction between D14L and OsSMAX1 was also enhanced by dMGer. Furthermore, we identified the domain of OsSMAX1 that distinguishes it from its paralog DWARF53 (D53), which is associated with the strigolactone signaling complex. These findings propose a model of the interactions among KL signaling components and highlight the role of the ligand in the signaling complex.