Brassinosteroids (BRs) are steroid hormones that play essential roles in plant growth and development. In this study, we identified a new dwarf mutant in Brassica napus. By map-based cloning, BnaC04.BIL1 (BnaC04g41660D) gene, a BIN2-like1 (BIL1) encoding a GLYCOGEN SYNTHASE KINASE 3 (GSK3-like) protein kinase, was isolated. To date, how BIL1 involves in BR signal transduction remains uncovered. Genetic transformation experiments confirmed that the BnaC04.BIL1 is responsible for the plant dwarf

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... henotype in the Bndwarf2 mutants. Overexpression of BnaC04.BIL1 not only reduced plant height, but also resulted in compact plant architecture. Using CRISPR/Cas9, two sgRNAs were designed to target BnaC04.BIL1 gene. The gene editing experiments generated mutations of BnaC04.BIL1 sequence, which were stably transmitted to successive generations, and lead to restoration of plant height and plant architecture. The molecular mechanism of Bndwarf2 dwarfing was further verified by Y2H and BiFC assays. Results shown that a Thr187Ser amino acid substitution residing in the conserved region promotes the interaction between BnaC04.BIL1Mut with BnaBZR1, thus enhances the negative regulation of plant growth. The genetic and molecular evidence clarifies first the BnaC04.BIL1 can sharply change plant architecture in natural plant accessions in allotetraploid, and provides new insights into the molecular mechanisms of BR signaling.