Molecular Mechanism Of Plasma Membrane-Localized Molecular Chaperone OsDNAJ15 Facilitating OsHKT1;5 Expression

Land salinization affects agricultural productivity,excessive sodium(Na~+)accumulation harms rice growth and development.An increase in crop salt stress tolerance would enable sustainable agriculture on marginal lands and improve crop yields.As sodium transporters impact sodium distribution in different tissues and organs to bestow salinity tolerance,significant attention has been paid to the function of HIGH-AFFINITY POTASSIUM(K~+)TRANSPORTER(HKT).HIGH-AFFINITY POTASSIUM(K~+)TRANSPORTER 1;5(OsHKT1;5)-dependent sodium(Na~+)transport is a key salt tolerance mechanism during rice growth and development.However,the molecular regulatory network regulating OsHKT1;5expression has yet to be improved.Using a previously generated high-throughput activation tagging-based T-DNA insertion mutant pool,we isolated a mutant exhibiting salt stress-sensitive phenotypes,caused by a reduction in OsHKT1;5 transcripts.The salt stress-sensitive phenotype of this mutant results from the loss of function of OsDNAJ15,which encodes plasma membrane-localized heat shock protein 40(Hsp40).In this study,the molecular mechanism of OsDNAJ15 involved in salt stress response was explored through the techniques and means of molecular biology,biochemistry,cell biology,genetics and other disciplines.The main findings are as follows:(1)Through salt stress phenotype,osdnaj15 mutant showed decreased tolerance to salt stress,the survival rates were about 13%lower than that of the wild-type Kitaake,while OsDNAJ15OE lines displayed salt stress-tolerant phenotypes compared with wild-type Kitaake and the survival rates improved by about 15%.(2)Byb-glucuronidase(GUS)staining and subcellular localization,the GUS signals were found in the leaf,panicle,glume,root tissues,stem and leaf sheath,relatively higher expressed in root tissues,stem and leaf sheath,and OsDNAJ15 is localized in the plasma membrane as a peripheral membrane protein.(3)The field phenotypes of osdnaj15 loss-of-function mutants showed decreased plant height,increased leaf angle,decreased grain number,shorter panicle length and decreased branch number,which were related to yield.(4)Co-IP,pull-down and luciferase reporting system were used to verify that OsDNAJ15 interacts with OsBAG4 in vitro and in vivo,and in the complex of OsBAG4,OsSUVH7 and OsMYB106,OsDNAJ15 only directly interacts with OsBAG4.(5)The expression level of OsHKT1;5 was detected by RT-qPCR,and the results suggested that OsHKT1;5 was a stress response gene of OsDNAJ15.Through genetic verification,we proved OsHKT1;5 is located in the genetic downstream of OsDNAJ15,and the deficiency of OsDNAJ15 affects the Na~+/K~+ion balance in shoots under salt stress.(6)By cell biology experiment and electrophoretic mobility shift assay(EMSA),salt stress facilitates the nuclear relocation of OsDNAJ15,and OsDNAJ15 enhances OsBAG4 activity to facilitate the DNA-binding activity of OsMYB106 to positively regulate the expression of OsHKT1;5.The results of this study reveal a novel function of OsDNAJ15,which is a member of the Hsp40 family,and OsBAG4 which is a molecular chaperone regulator,in synergistic regulation of transcription under salt stress.A model of OsHKT1;5 activation in rice under salt stress was proposed.Salt stress rapidly facilitates the relocation of OsDNAJ15 to the nucleus,thereby causing the interaction between OsDNAJ15 and OsBAG4 in the nucleus,which further facilitates OsSUVH7-OsBAG4-OsMYB106 transcriptional complex formation for OsHKT1;5 activation.From a new perspective,this study speculated whether plasma membrane-localized OsDNAJ15 directly regulates the activity of sodium ion transporters to expand the regulatory network of salt stress response,provides a new theoretical basis for the creation of new salt-tolerant rice varieties and molecular design breeding.