Functional Analysis Of A Novel PPR Protein PPR-DUF In Arabidopsis Thaliana

During the evolution of plants,most mitochondrial genes are lost or transferred to the nucleus or chloroplast genome,resulting in the current mitochondrial genome with only 5%of the genes from their ancestor.The transcriptional products of these genes need to undergo a series of post-transcriptional processing to mature,and these processes require a large number of nuclear gene-coded proteins to participate,including PPR(Pentatricopeptide Repeat)protein,RNA helicase,CRM(Chloroplast RNA splicing and ribosome maturation)protein,MORF(Multiple Organellar RNA editing Factors)/RIP(RNA editing Interacting Proteins)protein,mTERF(Mitochondrial transcription termination factor)protein ect.PPR protein is one of the largest protein families in higher plants and consists of a series of tandem repeat motifs(PPR motif).According to the motif structure and composition of each member,it is divided into P type and PLS type.PPR protein is usually located in mitochondria or chloroplasts,or dually located in both mitochondria and chloroplasts,and some are located in the nucleus.PPR protein is widely involved in the post-transcriptional processing of organelle genes,such as RNA splicing,C-to-U editing,3’/5’ end maturation and translation initiation,etc.This paper analyzes the structure of Arabidopsis PPR protein,a special PPR protein is found,its C-terminus contains an unknown functional domain,so the protein is named PPR-DUF(Domain of Unknown Function).PPR-DUF is localized in mitochondria,chloroplasts and nucleus.A T-DNA insertion mutant ppr-duf is obtained from the mutant library.In the selfed progeny of PPR-DUF/-,obtained 25%aborted embryos,indicating that PPR-DUF plays a very important function in embryogenesis.To study the function of the embryonic lethal gene PPR-DUF,the embryo-specific promoter p2S1 is used to drive the expression of PPR-DUF in the ppr-duf mutant.The ppr-duf mutant with embryonic complementation can germinate.but only two cotyledons can develop.In the ppr-duf mutant with embryonic complementation,the expression level of PPR-DUF is lower than 1/10 compared with wild-type.Therefore,the complementing mutant is used in follow-up research.The study found that of the 596 editing sites in Arabidopsis mitochondria,the editing efficiency of 208 sites was significantly reduced in complementing mutant.Most of the editing defect sites can lead to changes in encoded amino acids.Mitochondrial editing defects often lead to slow plant growth and even embryo-lethal.Therefore,editing defects in ppr-duf are one of the main causes of embryo-lethal.PPR-DUF and DYW2,an atypical PPR-DYW protein,share 45 edited sites.DYW2 participates in the mitochondria C-to-U editing at 353 sites,and maybe play roles in providing deaminase during the editing process.We guess that PPR-DUF may participate in editing by connecting to DYW2.Indeed,the yeast two-hybrid results show that PPR-DUF interacts with DYW2.DYW2 belongs to a special kind of atypical PPR-DYW protein.In addition to DYW2,there are four members of this class of proteins,including MEF8,MEF8S,DYW3 and DYW4,Yeast two-hybrid results show that PPR-DUF interacts with MEF8,MEF8S and DYW4,respectively.MEF8 and MEF8S are homologous proteins with redundant functions.The functions of MEF8 and MEF8S on RNA editing are revealed by studying the respective single mutants.but the mef8/mef8s double mutant embryos are lethal,it means that only the deletion of these two genes at the same time can truly reveal their functions.The functions of DYW3 and DYW4 are not characterized.In order to study the functions of MEF8 and MEF8S,we try to knock out MEF8 with CRISPR-Cas9 technology in mef8s mutant,and use p2Sl::MEF8S for embryo complementation.But we do not get such complementary plants.Fortunately,we get a weak phenotypic double mutant.In this mutant,the 5’ end of the MEF8 is deleted by 192 bp,resulting in a deletion of 64 amino acids at the N-terminal of the encoded protein.This double mutant can survive,but grows very slowly and cannot set seeds.By detecting the editing efficiency in the weak phenotype double mutant and find that the editing efficiency of 129 editing sites is significantly reduced,among them,117 were not reported before.Further analysis find that PPR-DUF and MEF8/8S share 95 edited sites.Through EST search and RT-PCR analysis,the expression of DYW3 gene can not be detected.We use CRISPR-Cas9 technology to knock out DYW3 in DYW4/heterozygous plants,and obtain dyw3 and dyw3dyw4 mutants that growth was not affected.So DYW3 is determined to be a pseudogene.DYW4 encodes a mitochondrion-localized PPR-DYW protein.By analyzing the T-DNA insertion mutant,it is found that the plant growth had no obvious defects.The study shows that the editing efficiency is slightly reduced at 42 editing sites.Among this sites,34 overlap with PPR-DUF.In order to further prove the function of DYW4,we use CRISPR-Cas9 technology to obtain another two mutants dyw4-Cas1 and dyw4-Cas2,their growth and editing defects are consistent with T-DNA insertion mutants.In summary,PPR-DUF participates in mitochondrial RNA editing by connecting atypical PPR-DYW proteins DYW2,MEF8,MEF8S and DYW4,respectively.The specific function of PPR-DUF in mitochondrial editing and the function in chloroplast and nucleus need further analysis.