Mechanism Research Of Rice Pentatricopeptide Repeat Proteins:PPS1 And OsPGL1 In Organelle RNA Editing

RNA editing is a kind of post-transcriptional processing that is widely spreade in higher plant organelles,this process changes the genetic information by changing RNA.In higher plants,RNA editing is primarily the transformation of cytosine(C)to uracil(U)occurred in mitochondria and chloroplasts.This biological process is closely related to PPR proteins.PPR protein is a newly discovered protein family in recent years,which is composed of 2-27 PPR motifs with 35 amino acids as a repeating unit.In terrestrial plants,the PPR proteoins are mainly distributed in Arabidopsis and Rice,with 450 and 477 members respectively.In addition to PPR proteins,there are another proteins(MORFs)that involved in RNA editing along with PPR.As few PPR proteins reported in rice,therefore our study aim to find new PPR proteins in rice and investigate its role in the process of RNA post-transcription.Through reverse genetics,using RNAi,antisense inhibition and CRISPR/Cas9 technology,we obtained two PPR mutant plant with different phenotype,respectively.Named ppsl and Ospgll.Our data show that PPSl is a mitochondrial localized PPR protein,while OsPGLl is a PPR protein that is localized both into mitochondria and chloroplast.PPS1 involves in 5 consecutive editing sites of nad3 transcript in mitochondrial,while OsPGL1 involves in RNA editing not only in chloroplast transcript but also in mitochondrial transcript,both for a single site.The function of these two PPR proteins resulted in the decrease of the RNA editing efficiency of the specific site and ultimately resulted in the defective phenotype,including vegetative growth and reproductive growth in rice plants.The specific research results of these two new PPR proteins are summarized as follows:(1)Through RNAi and antisense inhibition technique,we obtained the transgenic interference strains of ppsl and Ospgll respectively.Phenotypic identification of the transgenic TO lines showed that two lines of ppsl characterized by dwarfing,short anther,some infertility in pollen,low setting percentage and so on.To perform phenotypic identification to the transgenic T1 line of Ospgll,we found an independent line characterized by leaf color becomes pale green.With the advent of CRISPR/Cas9 technique,we did the CRISPR gene knockout and got the Ospgll mutant characterized by the same phenotype with the antisense inhibition line and study it in detail.(2)Because both of the two PPR proteins in our study belong to DYW type PPR in PLS subfamily,and sequence analysis found that DYW domain is similar to deaminase domain,these kind of protein were considered to be a class of mitochondrial or chloroplast RNA editing factors.Therefore,we analyze the editing efficiency within all of the editing sites both in mitochondria and chloriplast and found that the mitochondrial gene nad3 transcript in ppsl showed significant decrease in editing efficiency at 5 sites,while in Ospgll,we found that the editing efficiency were reduced to zero either in mitochondrial gene ccmFc or in chloroplast gene ndhD,thus the two PPR proteins were shown to be involved in the editing of organelle RNA.(3)By the RNA EMSA assay,the two PPR proteins can be combined directly with the cis-acting elements adjacent to the editing site in vitro.In addition,we found that the interval between the editing site and the nearest binding site was 4-5 nucleotides.We hypothesized that this interval may play an indispensable role in the embedding of deaminase molecules.(4)By BN-PAGE assay and the complex activity staining experiment we found that in ppsl,three of the five complexes of the mitochondrial electron transport chain(complex I,IV,V)were significantly less active than those in the WT.The decrease of complex activity leads to severe disorder in the mitochondrial morphology structure and finally results in pollen partial sterility.In Ospgll,the morphological structure of chloroplast appeared abnormal,while the mitochondria were almost undistinguishable compared with the WT,we speculated that among the two editing sites involved in OsPGLl,the "retention" of the ndhD-878 site editing event in chloroplast gene causes the 293 amino acids encoded in it not to be changed from serine(Ser)to Leucine(Leu),which results in abnormal development of chloroplast.In the mitochondrial gene ccmFc-543 locus,because of the codon degeneracy,the editing event is not changing the amino acid encoded by it,so that the structure of the mitochondria is not affected.(5)PPR protein is involved in RNA editing sometimes with the help of MORF proteins.In rice,there are seven MORF proteins localized in the organelles,including one MORF1-like(Os11g1020),two MORF2-like(Os06g02600,Os04g51280),one MORF3-like(Os03g38490),two MORF8-like(Os09g04670,Os09g33480)and one MORF9-like(Os08g04450).In Arabidopsis,MORF1 and MORF3 are localized in the mitochondria,MORF2 and MORF9 are localized in chloroplast,while MORF8 is mitochondrial and chloroplast dual-localized protein.Based on the characterization that PPS1 is only involved in the editing of mitochondrial transcripts,we found that PPS1 did not inteact with any MORF proteins through yeast two hybridization.But OsPGL1 can interact with three OsMORF(OsMORF2,OsMORF8 and OsMORF9)proteins.This interaction was confirmed by a series of experiments in vitro and in vivo.These results also confirmed the previous research conclusion:As organelles RNA-binding factor,the PPR proteins,along with MORF proteins and other unknown factors to form editing complex on its target RNA.In conclusion,this study reveals two DYW domain-containing PPR proteins:PPS1 and OsPGL1.They functioned commonly in organellar RNA editing.The changes of the proteins caused by the editing of these sites play a key role in assembly of the mitochondrial complex and the development of chloroplasts,and then play a crucial role in regulation of rice vegetative and reproductive growth,our study also provides a new perspective for the exploitation and utilization of male sterile resources in rice.