Brassinosteroids, the steroid hormones of plants, control physiological and developmental processes through its signaling pathway. The major brassinosteroid signaling network components, from the receptor to transcription factors, have been identified in the past two decades. The development of biotechnologies has driven the identification of novel brassinosteroid signaling components, even revealing several crosstalks between brassinosteroid and other plant signaling pathways. Herein, we would like to summarize the identification and improvement of several representative brassinosteroid signaling components through the development of new technologies, including brassinosteroid-insensitive 1 (BRI1), BRI1-associated kinase 1 (BAK1), BR-insensitive 2 (BIN2), BRI1 kinase inhibitor 1 (BKI1), BRI1-suppressor 1 (BSU1), BR signaling kinases (BSKs), BRI1 ethyl methanesulfonate suppressor 1 (BES1), and brassinazole resistant 1 (BZR1). Furthermore, improvement of BR signaling knowledge, such as the function of BKI1, BES1 and its homologous through clustered regularly interspaced short palindromic repeats (CRISPR), the regulation of BIN2 through single-molecule methods, and the new in vivo interactors of BIN2 identified by proximity labeling are described. Among these technologies, recent advanced methods proximity labeling and single-molecule methods will be reviewed in detail to provide insights to brassinosteroid and other phytohormone signaling pathway studies.
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