Structural And Functional Study Of WA352 In Rice Wild Abortive Cytoplasmic Male Sterility

Cytoplasmic male sterility(CMS)and its restoration are the genetic basis of rice heterosis.Since the beginning of the 1970 s,CMS-based three-line hybrid breeding in China has played a decisive role in the improvement of rice yield.The most widely used CMS system is the wild abortive type cytoplasmic male sterility(CMS-WA),of which sterile gene is a mitochondrial gene WA352 composed of three mitochondrial presumed gene fragments orf284,Orf224 and orf288,and an unknown source of DNA fragments.The mechanism of wild abortive type cytoplasmic male sterility is caused by the interaction of the sterile protein WA352 with nuclear gene encoded protein Cox11(Cytochrome c Oxidase Assembly Factor).WA352 inhibits the degradation of ROS by binding to Cox11,resulting in the accumulation of ROS in the cells,which promotes the release of cytochrome c,causing PCD of the pollen tapetum,eventually leading to pollen abortion.However,the molecular mechanism of interaction between WA352 and Cox11 is still unclear,which requires further interpretation of the structural biology results.This study comprehensively uses the methods of structural biology and bioinformatics to explain the structural basis of cytoplasmic male sterile protein WA352.We made a different truncation of WA352,a total of 80 clones,screened 20000 conditions,and finally found the WA352 protein crystals in three conditions,and obtained the native data.The phase information of the WA352 protein was obtained by the method of selenomethionine,and the high-resolution crystal structure of the C-terminal functional domain of WA352 was analyzed.By comparing the Dali search results of the WA352 structure,it was found that the structure closest to WA352 was leucyl-t RNA synthetase.This study also concerns the interaction mechanism of WA352/Cox11.We tried to co-express the C-terminal functional domain of WA352 with Cox11 in different truncation and different expression vector,but we didn’t obtain the complex protein.However,the yeast two-hybrid experiment confirmed that the C-terminal functional domain of WA352 could interact with Cox11.The sequence alignment showed that the WA352 and Cox 11 protein sequences were conserved in plants,revealing the conserved biological significance of WA352 and Cox11 interaction mechanism.The homology structure of Cox11 has been resolved yet,we hope to explain the molecular interaction mechanism by the molecular simulation of WA352 and Cox11.The results show that the WA352 may interact with Cox11 through its two positive charge regions of the surface.