A DNA Methylation Reader-Chaperone Regulator-Transcription Factor Complex Activates OsHKT1;5 Expression

Soil salinity is a major environmental constraint for rice（Oryza sativa L.）growth and production.Irrational irrigation of the land throughout the year has increased the salinization of the soil,which has adversely affected the current development of agricultural production of China.To respond to high sodium（Na⁺）concentrations,plants harbor multiple Na⁺transport systems.Rice HIGH-AFFINITY POTASSIUM（K⁺）TRANSPORTER 1;5（OsHKT1;5）,a Na⁺-selective transporter,maintains K⁺/Na⁺homeostasis in shoots of rice under salt stress.However,the mechanism of regulating OsHKT1;5 expression remains unknown.In this study,using forward genetic screening of a genome-scale mutagenesis library of Rice（Nipponbare）CRISPR/Cas9 mutant pool,we identified a mutant line sensitive to salt stress.In this mutant,due to the frameshift mutation in the gene coding region of a chaperone regulator named OsBAG4,the stop codon was generated in advance,which caused it to become a loss-of-function mutant of OsBAG4.Subsequently,we used multidisciplinary techniques and methods such as genetics,biochemistry,molecular biology,bioinformatics,and cell biology to investigate the functions and molecular mechanisms of OsBAG4 in the salt stress,and elucidate the epigenetic regulation mechanism of regulating OsHKT1;5 expression.The main experimental results indicates that:（1）The survival rates of osbag4 were lower than that of Nipponbare（NIP）after recovery from salt stress.The ROS content of osbag4 was higher than that of NIP under salt stress,and osbag4 mutants showed significantly reduced K⁺and elevated Na⁺levels in shoots under salt stress conditions;（2）Genome-wide RNA-sequencing（RNA-seq）analysis indicated that OsBAG4 altered the expression level of genes,and further real-time quantitative polymerase chain reaction（RT-q PCR）revealed that the root of osbag4-1 shows significantly reduced OsHKT1;5 expression,while the expression level of OsHKT1;5 is slightly reduced in the shoot of osbag4-1;（3）Using comparative interactomics,it is found that OsBAG4interacts with OsMYB106,and further investigation found that OsMYB106 binds to the consensus MYBE（MYB-binding cis-element）in the OsHKT1;5 promoter,while OsBAG4 increases the affinity of OsMYB106 for the target promoter;（4）Our analysis also revealed that OsSUVH7,which was identified in the IP-MS experiment,recognizes m CHH and m CHG using Fluorescence polarization assay,and we further detected direct binding of OsSUVH7-FLAG to the methylated MITE（miniature inverted-repeat transposable element）located in OsHKT1;5 promoter using chromatin immunoprecipitation assay（Ch IP）followed by bisulfite sequencing PCR（BSP）.ossuvh7 mutants exhibited lower survival rate than NIP under salt stress and expression of OsHKT1;5 was dramatically reduced in ossuvh7 mutants;（5）Elimination of the MITE(OsHKT1;5-MITE^KO)decreased OsHKT1;5 expression and increased salt sensitivity.Together,OsMYB106 and OsSUVH7 bound to the MYBE and MITE which located upstream of the MYBE in the OsHKT1;5 promoter,respectively.OsBAG4functioned as a bridge between OsSUVH7 and OsMYB106 to facilitate OsMYB106binding to the consensus MYBE in the OsHKT1;5 promoter,thereby activating the OsHKT1;5 expression to response to salt stress.Our findings revealed a transcriptional complex,consisting of a DNA methylation reader,a chaperone regulator,and a transcription factor,that collaboratively activate OsHKT1;5 expression and improve the salt tolerance of rice.It reveals the new function and molecular mechanism of the transposon evolving into transcriptional regulatory enhancer during the evolution process,which provides a theoretical basis and molecular manipulation target for the creation of new varieties of salt-tolerant rice.