Identification And Preliminary Study Of The Minimum Motif For BKI1 Interacting With BRI1

BackgroundBrassinosteroids (BRs), a class of steroid hormones, are ubiquitously distributed throughout the plant kingdom and play essential roles in regulating plant growth and development. After a decade of intensive studies, some key components of the BR signaling pathway have been identified, and a big picture of the entire BR signaling pathway has come into view.BRs are first perceived by BRI1, a protein kinase localized on the plasma membrane, and then, BKI1, a negative regulator of BRI1, dissociates to release its inhibition, which leads to a BR signaling cascade. Up to now, we know few about the regulation mechanism between BRI1 and its interaction components, and as the negative regulator of BRI1, how BKI1 interacts with BRI1 is unclear either.AimExplore the underlying mechanisms of BKI1-BRI1 interaction and how BKI1 executes its negative regulatory effects. Do further research about BKI1 upon its structure prediction.Materials and Methods:Materials:Kinds of transgenic plants, including BKI1-RNAi,△304-325-BKI1-YFP, 304-325-BKI1-YFP, and a series of truncated BKI1-pEXP-AD502 vectors.Methods:1. To test the expression level of BKI1 by qRT-PCR;2. To analyze the outputs of BR signal through the hypocotyl elongation experiment;3. To study on BKI1 through some bioinformatics methods, such as Blast, Multi-alignment and 2D structure prediction;4. Identified the minimum and indispensable region for BKI1 interacting with BRI1 via yeast two-hybrid assay.5. Transient expression technology was used to observe the Subcellular-localization of BKIl,304-337, A304-325-BKI1 in the pavement cell of TobaccoResultsIn this research, the phenotype, BKI1 expression level and hypocotyl length were first studied in BKI1-RNAi transgenic line, in order to validate that the reduction of endogenous BKI1 can enhance BR signal. A 22 amino-acid motif, one of the two conserved regions in the C-terminus of BKI1, is the onlyα-helix, based on later structure prediction and homology analysis. Subsequent yeast two hybrid experiment proved that this motif is the minimum and indispensable interaction region, and we finally observed that, without the 22-amino acid region, the fluorescent signal of BKI1 emerges in nucleus, which is different from the normal localization.ConclusionsAs a conserved region in the C-terminus of BKI1,304-325 is also the only a-helix. This motif is the minimum and indispensable interaction region for BKI1 and BRI1, and it plays a role in maintaining the structure of BKI 1.