Brassinosteroids (BRs) are the sixth class of plant hormones that involved in numerous plant development processes such as leaf expansion,shoot elongation and pollen tube formation.Currently,many crucial details of BR signaling pathway remains unknown.In the downstream signaling pathway,the signal transmits from cytoplasm to nucleus,and this part is realized by three proteins:brassinosteroid insensitive 2 (BIN2),BRI1 EMS suppressor1 (BES1) and general regulatory factor 8 (GRF8,member of 14-3-3 proteins).BRs have been widely studied via traditional cell biology techniques.There are advantages in identifying components and functions of participants in the signaling pathway.However,these bulk methods cant report the transduction process in situ or resolve details at a rate matching the true signaling time-scale.Here we use single molecular assay based on total internal reflection fluorescence (TIRF)Microscopy to observe the interaction of the three proteins.The result shows that BIN2 can phosphorylate BES1 on the order of seconds.And the dimeric GRF8 can only bind with BES1 which has been phosphorylated.For the process of BIN2 binding to its substrate BES1,it is an oxygen-dependent reaction.It implies BIN2sinvolvement in plantsdefense mechanism against environmental stress.This is the first time to demonstrate plantsadversity-resistant using single molecular assay.