Protein Interaction And Transcriptome Analysis Of The Indica-japonica Hybrid Sterility System S5

Rice,a major food crop and model plant for genomic study of monocotyledon species,also provides a model system for studying reproductive isolation.The Asian cultivated rice is composed of two subspecies,indica and japonica.Their hybrids are usually highly sterile,which is a barrier for utilization of the strong vigor in indica-japonica hybrids.S5 is a major locus controlling indica-japonica hybrid embryo-sac sterility,which is composed of three closely-linked genes,ORF3,ORF4,and ORF5.Typical japonica and indica varieties carry ORF3-ORF4+ORF5-and ORF3+ORF4-ORF5+,respectively.Cell wall protein ORF5+,together with transmembrane protein ORF4+,kills the gametes by inducing ER stress that causes premature PCD.ER-resident ORF3+ protects the gametes from being killed thus restores the hybrids fertility by preventing/resolving ER stress.In this study,we further studied the mechanisms underlying S5 locus by molecular cytology and transcriptome.We constructed a yeast cDNA library of panicles from Balilla and then screened the library using the full-length and truncated ORF3,ORF4 and ORF5 proteins as baits.Two candidate genes were identified for ORF5-,but were further verified to be false positive.According to our presumption about S5,we investigated the interaction between ORF4+ and membrane-associated receptor proteins such as S1L4,LYP4 and LYP6,but they did not interact with each other.We studied the protein property of ORF5+ and ORF4+ by constructing stable BY2 suspension cell lines transformed with the GFP-tagged ORF5+ or ORF4+.To investigate whether ORF5+ releases oligosaccharides signals by damaging cell wall,we treated the ORF4+-GFP cell line with three types of oligosaccharides,and found they could not induce cell death.To figure out whether ORF5+ cleaves ORF4+,we co-expressed ORF5+ with fluorescent protein-tagged ORF4+ in N-terminal or C-terminal transiently in tobacco leaves and found the localization of ORF4+ was not changed.In both BY-2 cell line and transient expression in tobacco leaves,ORF5+ was not localized to the cell wall,indicating that the tag may affect the localization of ORF5+.However,we found some PR genes were down-regulated in Arabidopsis transformed with GFP-tagged ORF5.ORF5+ encodes an aspartic protease.The transgenic Balilla ORF5+ is sterile.We mutated the two aspartic protease activity sites D132 and D337 separately and simultaneously,and transformed them into Balilla.All transgenic plants were fertile,suggesting that the aspartic protease activity was essential for ORF5+ taking function.ORF4+ encodes a single transmembrane protein with N-terminal outside and C-terminal inside.ORF4+-GFP overlapped partially with the endocytosis marker suggesting that it may be involved in endocytosis.We studied the predicted endocytosis-related sites of ORF4+ by mutation,and found they were not related with the endocytosis of ORF4+.BalillaORF4+OX had reduced plant height and tiller numbers and exhibited increased resistance to leaf blight strain PXO341.Its leaves showed lesion mimic and had callose deposited.However,BalillaORF4-OX showed none of these phenotypes.These indicated the functional difference between ORF4+ and ORF4-.In addition,we created a rice plants with ORF3-/ORF4-/ORF5-at S5 locus in Balilla background by CRISPR.Based on the embryo-sac development processes of Balilla,sterile Balilla ORF5+and fertility-restored BalillaORF3+ORF5+,we divided the ovule abortion processes into three stages(MMC,MEI and AME)and analyzed the transcriptomic data from the three stages.Comparison of BalillaORF5+ and Balilla showed that ORF5+ induced large-scale up-regulation of cell wall remodeling genes such as EXP,XTH,GH9,PAE,PME,PGase and AGP at MMC,which resulted in massive biotic and abiotic stresses and OsbZIP50-mediated ER stress response possibly by damaging cell wall integrity.At MEI,the unresolved stresses activated TIP2,TDR,OsAP25 and OsAP37 mediated PCD pathway.In addition,aborted ovule cells accumulated cellulose and callose to strengthen cell wall by up-regulating secondary cell wall biosynthetic genes such as OsSWN1,OsMYB46,OsCesA4,OsCesA7 and OsCesA9 and modulating callose metabolism,respectively.At AME,aborted ovule was stuffed with cellulose and callose.Comparison of BalillaORF3+ORF5+ and Balilla showed that ORF5+ still induced the up-regulation of cell wall remodeling genes and stress responses at MMC,which was not as strong as in BalillaORF5+ and suppressed at MEI,thus the ovule developed normally.As ORF3+ is a chaperone,it was speculated that ORF3+ prevented the stress signals by modifying the interacting protein or downstream signal molecules of ORF4+.Comparison of BalillaORF3+ORF5+ with BalillaORF5+ showed that ORF3+ elevated the expression of defense-related genes greatly,such as OsMTs,OsLEAs and OsPR10 a.In this study,we further studied the mechanisms of S5 locus.Our results revealed the essentiality of ASP activity for ORF5+ and the correlation of ORF4+ with disease resistance.By analyzing the transcriptomes of Balilla,Balilla ORF5+ and BalillaORF3+ORF5+,we speculated that ORF5+ impaired the cell wall integrity,which was sensed by the transmembrane protein ORF4.ORF4+ then transmitted the signal into cells and induced biotic and abiotic stresses and ER stress and PCD.When ORF3+ existed,it alleviated ER stress and promoted the resistance to stress,which suppressed the stress induced by ORF5+ and saved the gametes.This study helped to better understand the mechanisms of S5,which provides theoretical basis for utilizing S5 in breeding.