The Crosstalk Between Unfolded Protein Response And Salicylic Acid Signaling Mediated By CPR5 In Arabidopsis

Growth and stress adaptation rely on the biosynthetic capacity of the Endoplasmic Reticulum(ER)for the production of secretory proteins.In specific development and stress situations requiring enhanced protein synthesis,the ER biosynthetic capacity can be overwhelmed leading to a potentially lethal condition,known as ER stress,which results from the accumulation of unfolded proteins in the ER.During ER stress,a sophisticated signaling pathway,known as the UPR,is actuated to modulate gene expression and restore the ER homeostasis through chaperone-assisted protein folding,translational attenuation and ER-associated protein degradation(ERAD).Though it is reported about the protective roles of UPR in abiotic stress such as heat,salt and drought,little is known about how UPR works in the pathogen defense and physiological growth.In addition,if the ER balance was successfully maintained,the UPR should be terminated.On contrary,if the intense and severe ER stress was unresolvable,the protective UPR needs to be inhibited and switched to the programmed cell death(PCD).It is conceivable that some negative regulators function in these two situations above.However,it remains unknown about the negative regulators in the plant UPR pathway.Through the Y2H assay,CPR5 was screened as an interactor of bZIP60 which was a critical transcriptional factor in one of UPR signaling branches in Arabidopsis.There are five transmembrane domains in the C terminus and a nucleus signal sequence in the N terminus of CPR5 in agreement with our observation of its localization in the tobacco leaves.We detected it localizing in the ER,golgi and nucleus under confocal microscopy,similar to bZIP28 which was also a crucial transcription factor in the other UPR signaling branch.Besides bZIP60,CPR5 could interact with bZIP28 in Y2H and AP-FRET assays.cpr5,a loss of function mutant,was insensitive to chronic ER stress and had enhanced expression of UPR biomarker genes such as bZIP60 and BiP3.We uncovered the epistatic relationship by analysis of cpr5 bzip28,cpr5 bzip60 and cpr5 bzip28 bzip60 phenotypes,CPR5 was in the upstream of bZIP28 and bZIP60.Above all,we concluded that CPR5 was a negative regulator in the plant UPR and directly repressed the functions of bZIP28 and bZIP60 by protein interactions.cpr5 showed retarded growth,necrotic cytoletons and defected trichomes.However,in the cpr5 bzip60,genetic deletion of bZIP60 partially recovered the cpr5 growth defects including the primary root length,the number of lateral roots and the shoot fresh weight.It was reported that bZIP60 mRNA was cleaved by the endoribonuclease domain of IRE1 and then translocated to the nucleus.The loss of functions of IRE1 in cpr5 irela and cpr5 irelb also partially rescued the cpr5 retarded growth.cpr5 ire1a ire1b showed the same root phenotype as ire1a ire1b.Totally,CPR5 could control the plant physiological growth through IRE1 regulation.It was demonstrated that cpr5 was resistant to pathogen infection because of the high endogenous SA content and constitutive expression of PR genes.Since exogenous SA was able to induce UPR signaling,we eliminated SA by crossing cpr5 with sid2,a SA synthesis deficient mutant and could still detect the enhanced UPR activation in cpr5 sid2.In this case,UPR activation in cpr5 was not resulted from the high SA content.Meanwhile,several other high SA accumulation mutants like sncl,bonl and sizl did not show the similar ER stress-related phenotypes to cpr5,indicating that CPR5 specifically played roles in the UPR.In order to inhibit the SA-NPR1-PR1 pathway,we produced the cpr5 sid2 and cpr5 nprl and found that their sensitivities to chronic ER stress were significantly different from cpr5,but the molecular levels of UPR genes were indistinguishable.We concluded that the canonical UPR and SA signaling regulated the plant development independently under the normal growth condition,but had the overlapping roles under the chronic ER stress.On the other hand,we tested the phenotype of pathogen infection in various genotypes as follows,WT,cpr5,bzip28 bzip60,cpr5 bzip28 bzip60,ire1a ire1b and cpr5 ire1a ire1b.Consistent with the published results,cpr5 was resistant to pathogen and ire1a ire1b was susceptible to pathogen.We found that bzip28 bzip60 was as susceptible as ire1a ire1b,however,cpr5 bzip28 bzip60 and cpr5 ire1a ire1b were less resistant than cpr5.Base on the results above,we predicted that UPR could also contribute to pathogen defense,probably through the actions of CPR5.In summary,we discovered CPR5 as the negative regulator in the plant UPR,providing evidence to support the functions of UPR in the plant physiological growth and pathogen response.