Abstract The brassinosteroid (BR) hormone and its plasma membrane receptor BR INSENSITIVE1 (BRI1) is one of the best-studied receptor-ligand pairs for understanding the interplay between receptor endocytosis and signaling in plants. BR signaling is mainly determined by the plasma membrane pool of BRI1, whereas BRI1 endocytosis ensures signal attenuation. Since BRs are ubiquitously distributed in the plant, the tools available to study BRI1 function without interference from endogenous BRs are limited. Here, we designed a BR-binding-deficient mutant based on protein sequence-structure analysis and homology modeling of BRI1 and its close homologues. This new tool allowed us to re-examine the BRI1 endocytosis and signal attenuation model. We show that despite decreased phosphorylation and ubiquitination, the BR-binding-deficient BRI1 was internalized similar to the wild type form. These results reinforce the hypothesis that BRI1 is internalized via parallel endocytic routes and machineries. In addition, BR-binding-deficient mutant provides opportunities to study non-canonical ligand-independent BRI1 functions.

Phosphorylation and ubiquitination independent endocytosis of BRI1