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

Abstract:Cytoplasmic male sterility?CMS?and fertility restoration?Rf?are genetic bases for heterosis utilization in crops.Current production of three-line hybrid rice is mainly dependent on wild-abortive CMS?CMS-WA?system.The CMS transcript of WA352 in CMS-WA is decreased by a P-type PPR?pentatricopeptide repeat?encoded by RF4 restorer.However,the molecular mechanism underlying binding and degradation of WA352 mRNA involved in RF4-mediated restoration remain largely unknown.In this study,I identified a Rf4-interacting protein,an endonuclease OsMUS81,by pull-down assay and mass spectrometry?MS?analysis.The interaction between OsMUS81 and RF4 was further verified by yeast two-hybrid,pull-down and bimolecular fluorescence complementation assays.The biological function of OsMUS81 in CMS-WA system was studied by CRISPR/Cas9 editing system.My main results are as follow:1)The total proteins of ZSRf4 were applied in a pull-down assay using RF4 as bait in vitro.An endonuclease OsMUS81,which contains an ERCC4 domain,was identified by MS analysis.2)The interaction between OsMUS81 and RF4 was further verified by Y2H,Pull-down assay and BiFC?3)Protoplasts were prepared from ZSRf4?rf3rf3Rf4Rf4?and ZS97A?rf3rf3rf4rf4?with different genetic background.Subcellular localization of OsMUS81 was performed with resultant protoplasts.The results showed that OsMUS81 localized in the nucleus.4)The OsMUS81 was knocked out by CRISPR/Cas9 gene editing in rice ZSRf4.The pollen fertility of resultant osmus81 T₀ mutants decreased and the transcriptional level of WA352 was significantly increased in the osmus81 knockout plants compared to ZSRf4.Taken together,my results indicated that OsMUS81 is required for pollen development and involved in RF4-mediated degration of WA352 mRNA for the fertility restoration.