Arabidopsis CRY2 And TCP8 Form Blue-Light-Specific Phase Separation Condensates To Regulate Immune-Related Gene ICS1 Expression

Light is one of the most important environmental factors that plants depend on for survival and growth.Plants have evolved many light receptors to sense light signals,such as red-light receptor PHY,ultraviolet light receptor UVR8,blue light receptor CRY,etc.Among them,the blue light receptor cryptochrome 2(CRY2)is of great significance in plant.CRY2 functions as light receptor to receive light signals that regulates photoperiod flowering,hypocotyl elongation and circadian clock rhythm,etc.CRY2 can forms photobodies under blue light activation,while this phenomenon has not been revealed precisely.Currently,much attention has been drawn to the study of phase separation.Many studies indicate that biomacromolecules could form a non-membrane structure to enhance the local concentration of molecular and increase efficiency of biochemical reactions,controlling the reaction in time and space.The photobodies formed by CRY2 and its photobodies were observed and analyzed from the formation time,circularity and partition ratio.The IDR region of different proteins were predicted.The aim of this study is to identify whether CRY2and TCP8 could forms liquid-liquid phase separation condensates and briefly explores the function of these condensates.The specific results are as follows:1.Lasers from confocal microscope were used to activate CRY2 and its interacting protein to form photobodies.The observed pictures were analyzed by Image J.The result shows that CRY2 and TCP8 could form the photobodies with the fastest speed.The analysis of circularity and partition ratio reveals that photobodies of CRY2-TCP8 has the highest circularity and partition ratio.The CCE region of CRY2contains IDR region and TCP8 has relatively long IDR region in its N and C terminals.So the CRY2-TCP8 group was chosen to conduct phase separation identification.2.Protoplasts after 1,6-HEX treatment were observed using confocal microscope,which pointed the 1,6-HEX could inhibit the formation of photobodies of CRY2-TCP8 and CRY.TCP8 and CRY2 mutant CRY2^D387A could not form photobodies with TCP8.Meanwhile,CRY2 and TCP8 could forms circular photobodies,and two photobodies could fuse into a new photobides,whose shape would return to circular after fusing.The fluorescence recover after photo-bleaching assay shows that the fluorescence signal of CRY2-TCP8 photobodies could recover up to 65%of non-bleached photobodies while the signal of photobodies of CRY2-CRY2 could only recover to 20%.These results could proletary defines that photobodies formed by CRY2 and TCP8 was the liquid-liquid phase separation condensates.3.Pathogen infection assay shows the TCP8-OX/cry2 and cry2 owns same phenotype,reveals the function of TCP8 in plant immunity was relied on CRY2.After HEX treatment,the activation of expression of ICS1 by blue light was inhibited.4.TCP8 alone could not activate the expression of ICS1 in blue light or dark.After transfected CRY2^D387A and LWD2,TCP8 failed activate the expression of ICS1.When CRY2,LWD2,TCP8 were transfected into cells,the ICS1 expression level was increased under blue light treatment.CRY2-TCP8 condensates was co-phase separated with LWD,which shows CRY2 might recruit the LWD into the condensates.