Spatial-Temporal Dynamic Reorganization Of BSK1 Regulates Signal Activation For Growth And Immunity

In plants,growth and immunity are two opposing processes competing for cellular resources and proper resource allocation is crucial for plant survival.In fact,constitutive activation of defense responses causes serious problem in plant growth.Thus,fundamental questions are how plants balance the two opposing processes depending on the unexpected pathogen attacks with varying degrees of severity without jeopardizing the growth.Although BSK1 has been shown to play a central role in the two opposing processes and may function as a toggle switch for the activation of either growth or immunity depending on the intrinsic and environmental conditions,how it occurs remains elusive.We used a set of single-molecule techniques and biochemical assays to investigate the spatiotemporal dynamics of plasma membrane(PM)proteins in their native environment at the nano-scale.Using these methods,we were able to dissect the dynamics and the nature of complex assembly of BSK1 in a ligand-specific manner at the nano level and provide insight into the action mechanisms of BSK1 in plant growth or immunity responses depending on stimulatory ligands in a great detail.The main results are as follows:(1)BR perception could inhibit several flg22-triggered PTI outputs,which was a unidirectional crosstalk between BRI1-regulated growth and FLS2-mediated immunity responses.Brassinosteroid signal kinase 1(BSK1),which associates with FLS2 or BRI1 respecrtively,was an attractive candidate to regulate the tradeoff between flg22-induced PTI responses and BR-regulated growth signaling pathways.(2)Using laser scanning confocal microscope(LSCM)and variable-angle total internal reflection fluorescence microscope(VA-TIRFM),it was found that BSK1 in both resting and activated states localized to the PM in a non-random distribution pattern.(3)BSK1 underwent limited endocytosis with or without ligand stimulation in contrast to BSK1-associating FLS2 or BRI1.However,the density of BSK1 significantly decreased with different ligand treatments.The stoichiometry of BSK1 at the PM with or without ligand stimulation showed that BSK1 existed mainly as monomers in the steady state,but underwent a higher degree of oligomerization upon stimulation by ligands.(4)Ligand stimulation induced dissociation of BSK1 from preassembled RLKs/BSK1 complexes by using FLIM-FRET.Importantly,BR could inhibit flg22-induced BSK1 dissociation form BSL1/FLS2 complex.(5)At steady state,BSK1 localizated in At Flot1-labbed membrane rafts.Interestingly,e BL-activated BSK1 largely remained in the membrane rafts whereas flg22-activated BSK1 translocated from membrane rafts to non-raft regions,which provides the mechanistic insight of how BSK1 can differentially activate the growth or immunity upon stimulation with e BL or flg22.These results provide an insight into the action mechanism by which e BL and flg22 compete to promote growth stimulation and induce immune responses,respectively,via BSK1.Differential localization of BSK1 to either membrane rafts or non-raft regions can lead to differential activation of the downstream components that are also differentially localized to membrane rafts or non-raft regions.Importantly,the growth stimulating e BL inhibits the flg22-triggerred immune responses at the level of BSK1 dissociation from the FLS2/BSK1 signaling units and BSK1 translocation.Taken together,our findings provide a multiple lines of evidence that BSK1 plays a pivotal role in fine-tuning the growth-defense tradeoff between BRI1-and FLS2-mediated signaling via a unique mechanism.