Molecular Mechanism For Rf4-mediated Restoration Of Wild-abortive Cytoplasmic Male Sterility In Rice(Oryza Sativa L.)

Cytoplasmic male sterility（CMS）and its restoration are genetic bases for heterosis utilization in crops.Current production of three-line hybrid rice is mainly dependent on wild-abort cytoplasmic male sterility（CMS-WA）system.The CMS transcript WA352 of CMS-WA is negatively controlled by a PPR（pentatricopeptide repeat）-type restorer RF4.However,molecular mechanism underlying the binding and degradation of WA352 mRNA in RF4-mediated restoration is unclear.In order to study the molecular mechanism of Rf4-mediated restoration,candidates of RF4-interacting factors were identified by yeast two-hybrid（Y2H）screening.The interaction between RF4 and candidates was verified by Pull-down assays.The biological function of the RF4-interacting factors in the restoration of CMS-WA was studied by CRISPR/Cas9 gene editing.The main results were as follows:1.Ten RF4-interacting candidate proteins were obtained in Y2H screening.Among them,three proteins in full length,TPP243,DUF143 and Di19 can interact with RF4.2.The interactions of RF4 with TPP243,DUF143 and Di19 were further confirmed by Pull-down assays.3.The Y2H and Pull-down assay showed that TPP243,DUF143 and Di19 interact with each other,and Di19 may form homodimers,indicating that TPP243,DUF143 and Di19may function in a restoration complex with RF4.4.qRT-PCR analysis showed that TPP243,DUF143 and Di19 were constitutively expressed in rice tissues.Subcellular localization of TPP243 was partially localized in mitochondria,DUF143 was localized in the nucleus,and Di19 was localized in mitochondria.5.TPP243,DUF143 and Di19 were knocked out by using CRISPR/Cas9 gene editing technique in rice Zhenshan R5（ZSR5,CMS cytoplasm,nucleus carrying rf3rf3Rf4Rf4）.The pollen of resultant T₀ mutants tpp243,duf143 and di19 showed decreased fertility,indicating that TPP243,DUF143 and Di19 were required for the restoration of male fertility in ZSR5.6.Next,the expression level of WA352 was analyzed in the tpp243,duf143 and di19 T₀knockout plants by qRT-PCR.The results showed that the transcriptional level of WA352was significantly increased in the knockout plants compared with ZSR5,suggesting that the TPP243,DUF143 and Di19 are required for the function of restorer gene Rf4 in degradation of WA352 to fertility restoration.In this study,RF4-interactiong factors such as TPP243,DUF143 and Di19 were identified by Y2H screening and validated by Pull-down assays.Moreover,the expression and biological function of TPP243,DUF143 and Di19 were studied by qRT-PCR and CRISPR/Cas9-based knockout.This study sheds some light on the molecular mechanism of RF4-mediated resotoration of CMS-WA and lays foundation for further study of restoration complex（es）.