Kinase domain diversification drives specificity in BRI1 and non-BRI1 RLKs in brassinosteroid signaling

Receptor-like kinases (RLKs) are one of the largest families of Eukaryotic protein kinases (EPKs) that evolved through repeated duplication and diversification events in plants. RLKs regulate diverse roles of plant growth and development. Brassinosteroid Insensitive 1 (BRI1) and its family members BRI1-Like 1 (BRL1/3), BRL2, Excess Microsporocytes 1 (EMS1), and Nematode-Induced LRR-RLK 1 (NILR1) that belong to the LRR-RLK family of RLKs, control distinct biological functions through a conserved brassinosteroid (BR) signaling pathway. We previously demonstrated that the kinase specificity between BRI1 and GASSHO1 (GSO1) is allosterically regulated by merely two subdomains, raising a question of how different RLKs control distinct biological functions through their conserved kinase domain (KD). Here, we engineered chimeric receptors by fusing the extracellular domain (ECD) of BRI1 with KD of the BRI1 family and with non-BRI1 family RLKs, including BAK1-Interacting Receptor-like Kinase 1 (BIR1), BIR2, TOAD2 (RPK2), Barely Any Meristem (BAM1), CLAVATA 1 (CLV1), SOBIR1, Elongation Factor (EF-Tu) Receptor (EFR), Glycan Perception 4 (IGP4), and Strubbelig-Receptor Family 8 (SRF8), and confirmed that only the BRI1 family achieved BR signal output but not the others. We then replaced the S1 and S2 subdomains of the chimeric receptors with the corresponding S1 and S2 subdomains of the BRI1 kinase and found that except GSO1^BRI1-S1S2, no other chimeric receptor could induce BR signaling in bri1-301 mutants. However, chimeric receptors RPK2^BRI1-S1(E)S2, EFR^BRI1-S1(E)S2, IGP4^BRI1-S1(E)S2, BAM1^BRI1-S1(E)S2, and SRF8^BRI1-S1(E)S2 with an extended S1 subdomain S1(E) of BRI1 not only rescued bri1-301, but also achieved molecular phenotypes. In conclusion, this study provides evidence that signaling specificity of the RLKs has evolved through evolution of the S1 and S2 subdomains.