The plant hormones brassinosteroids (BRs) modulate plant growth and development. Cysteine (Cys) residues located in the extracellular domain of a protein are of importance for protein structure by forming disulfide bonds. To date, the systematic study of the functional significance of Cys residues in BR‐insensitive1(BRI1)is still lacking.

We used the brassinolide (BL)‐induced exogenous bri1‐EMS‐Suppressor 1 (BES1) dephosphorylation in Arabidopsis thaliana protoplasts as a readout, took advantage of dramatic decrease of BRI1 protein levels during protoplast isolation, and of the strong phosphorylation of BES1 by BR insensitive 2 (BIN2) in protoplasts, developed a protoplast transient system to identify critical Cys sites in BRI1.

Using this system, we identified a set of critical Cys sites in BRI1, as substitution of these Cys residues with Ala residues greatly compromised the function of BRI1. Moreover, we identified two negative regulators of BR signaling, pattern‐triggered immunity compromised RLCK1 (PCRK1) and PCRK2 that were previously known to positively regulate innate immunity signaling.