Editing Factors Research Articles

The pentatricopeptide repeat protein EMP601 functions in maize seed development by affecting RNA editing of mitochondrial transcript ccmC

Although several pentatricopeptide repeat (PPR) proteins are involved in post-transcriptional processing of mitochondrial RNA, it is unclear which specific protein is involved in the RNA editing of ccmC in maize (Zea mays). Here we report the identification of the maize empty pericarp 601 (emp601) mutant and the map-based cloning of the Emp601 gene, which encodes an E2-type PPR protein that is targeted to mitochondria. A single-nucleotide deletion in the emp601 mutant caused a frameshift and introduced a premature stop codon into the predicted EMP601. This mutation was associated with reduced accumulation of mitochondrial complex III as well as with inhibition of growth and differentiation of basal endosperm transfer layer cells, leading to final degeneration of the embryo and endosperm. We determine that loss of EMP601 function prevents the C-to-U RNA editing of the mitochondrial transcript ccmC at position 358. EMP601 binds to the ccmC transcript and directly interacts with Multiple organellar RNA editing factor 8 and may be a component of the plant mitochondrial editosome. We conclude that EMP601 functions in RNA editing of mitochondrial ccmC transcripts and influences mitochondrial function and seed development.

MORF2-mediated plastidial retrograde signaling is involved in stress response and skotomorphogenesis beyond RNA editing.

Retrograde signaling modulates the expression of nuclear genome-encoded organelle proteins to adjust organelle function in response to environmental cues. MULTIPLE ORGANELLAR RNA EDITING FACTOR 2 (MORF2) was initially recognized as a plastidial RNA-editing factor but recently shown to interact with GUN1. Given the central role of GUN1 in chloroplast retrograde signaling and the unviable phenotype of morf2 mutants that is inconsistent with many viable mutants involved in RNA editing, we hypothesized that MORF2 has functions either dosage dependent or beyond RNA editing. Using an inducible Clustered Interspaced Short Palindromic Repeat interference (iCRISPRi) approach, we were able to reduce the MORF2 transcripts in a controlled manner. In addition to MORF2-dosage dependent RNA-editing errors, we discovered that reducing MORF2 by iCRISPRi stimulated the expression of stress responsive genes, triggered plastidial retrograde signaling, repressed ethylene signaling and skotomorphogenesis, and increased accumulation of hydrogen peroxide. These findings along with previous discoveries suggest that MORF2 is an effective regulator involved in plastidial metabolic pathways whose reduction can readily activate multiple retrograde signaling molecules possibly involving reactive oxygen species to adjust plant growth. In addition, our newly developed iCRISPRi approach provided a novel genetic tool for quantitative reverse genetics studies on hub genes in plants.

OsDXR interacts with OsMORF1 to regulate chloroplast development and the RNA editing of chloroplast genes in rice

Plant chlorophyll biosynthesis and chloroplast development are two complex processes regulated by exogenous and endogenous factors. In this study, we identified OsDXR , a gene expressing a reductoisomerase that positively regulates chlorophyll biosynthesis and chloroplast development in rice. OsDXR knock-out lines displayed the albino phenotype and could not complete the whole life cycle process. OsDXR was highly expressed in rice leaves, and subcellular localization indicated that OsDXR was a chloroplast protein. Many genes involved in chlorophyll biosynthesis and chloroplast development were differentially expressed in the OsDXR knock-out lines compared to the wild type. Moreover, we found that the RNA editing efficiency of ndhA -1019 and rpl2 -1 were significantly reduced in the OsDXR knock-out lines. Furthermore, OsDXR interacted with the RNA editing factor OsMORF1 in a yeast two-hybrid screen and bimolecular fluorescence complementation assay. We demonstrate that disruption of the plastidial 2-C-methyl-derythritol-4-phosphate pathway results in defects in chloroplast development and RNA editing of chloroplast genes.

Isolation and Characterization of SPOTTED LEAF42 Encoding a Porphobilinogen Deaminase in Rice.

The formation and development of chloroplasts play a vital role in the breeding of high-yield rice (Oryza sativa L.). Porphobilinogen deaminases (PBGDs) act in the early stage of chlorophyll and heme biosynthesis. However, the role of PBGDs in chloroplast development and chlorophyll production remains elusive in rice. Here, we identified the spotted leaf 42 (spl42) mutant, which exhibited a reddish-brown spotted leaf phenotype. The mutant showed a significantly lower chlorophyll content, abnormal thylakoid morphology, and elevated activities of reactive oxygen species (ROS)-scavenging enzymes. Consistently, multiple genes related to chloroplast development and chlorophyll biosynthesis were significantly down-regulated, whereas many genes involved in leaf senescence, ROS production, and defense responses were upregulated in the spl42 mutant. Map-based cloning revealed that SPL42 encodes a PBGD. A C-to-T base substitution occurred in spl42, resulting in an amino acid change and significantly reduced PBGD enzyme activity. SPL42 targets to the chloroplast and interacts with the multiple organelle RNA editing factors (MORFs) OsMORF8-1 and OsMORF8-2 to affect RNA editing. The identification and characterization of spl42 helps in elucidating the molecular mechanisms associated with chlorophyll synthesis and RNA editing in rice.

Diosgenin enhances liposome-enabled nucleic acid delivery and CRISPR/Cas9-mediated gene editing by modulating endocytic pathways.

The CRISPR/Cas9 system holds great promise in treating genetic diseases, owing to its safe and precise genome editing. However, the major challenges to implementing the technology in clinics lie in transiently limiting the expression of genome editing factors and achieving therapeutically relevant frequencies with fidelity. Recent findings revealed that non-viral vectors could be a potential alternative delivery system to overcome these limitations. In our previous research, we demonstrated that liposomal formulations with amide linker-based cationic lipids and cholesterol were found to be effective in delivering a variety of nucleic acids. In the current study, we screened steroidal sapogenins as an alternative co-lipid to cholesterol in cationic liposomal formulations and found that liposomes with diosgenin (AD, Amide lipid: Diosgenin) further improved nucleic acid delivery efficacy, in particular, delivering Cas9 pDNA and mRNA for efficient genome editing at multiple loci, including AAVS1 and HBB, when compared to amide cholesterol. Mechanistic insights into the endocytosis of lipoplexes revealed that diosgenin facilitated the lipoplexes' cholesterol-independent and clathrin-mediated endocytosis, which in turn leads to increased intracellular delivery. Our study identifies diosgenin-doped liposomes as an efficient tool to deliver CRISPR/Cas9 system.

A Bibliometric Study for Plant RNA Editing Research: Trends and Future Challenges.

RNA editing is a post-transcriptional process that introduces changes in RNA sequences encoded by nuclear, mitochondrial, or plastid genomes. To understand the research progress of plant RNA editing, we comprehensively analyze the articles on plant RNA editing from 2001 to 2022 through bibliometric methods. Nucleic Acids Research, Plant Journal and Plant cell are the journals that deserve attention with their high production, total local citation scores (TLCS), and h-indexes. The USA, China, and Germany are the top three countries with highly productive publications. Ulm University, Cornell University, and Chinese Acad Sci are excellent cooperative institutions with a high level of influence in the field, and KNOOP V and TAKENAKA M are good partnership. Plant RNA editing researches concentrate on the subject categories of Biochemistry & Molecular Biology, Plant Sciences, Genetics & Heredity, etc. Plant mitochondria, genome editing and messenger-RNA may be the research hotspots in the future. The main plant RNA editing research tools are JACUSA, SPRINT, and REDO, and the main databases are REDIdb, PED, and dbRES. At present, the research streams are (1) RNA editing sites; (2) Pentapeptide repeat protein (PPR) involved in RNA editing; (3) RNA editing factors. Overall, this article summarizes the research overview of plant RNA editing until 2022 and provides theoretical implications for its possible future directions.

C-to-U and U-to-C: RNA editing in plant organelles and beyond.

The genomes in the two energy-converting organelles of plant cells, chloroplasts and mitochondria, contain numerous 'errors' that are corrected at the level of RNA transcript copies. The genes encoded in the two endosymbiotic organelles would not function properly if their transcripts were not altered by site-specific cytidine-to-uridine (C-to-U) exchanges and by additional reverse U-to-C exchanges in hornworts, lycophytes, and ferns. These peculiar processes of plant RNA editing, re-establishing genetic information that could alternatively be present at the organelle genome level, has spurred much research over >30 years. Lately new studies have revealed numerous interesting insights, notably on the biochemical machinery identifying specific pyrimidine nucleobases for conversion from C to U and vice versa. Here, I will summarize prominent research findings that lately have contributed to our better understanding of these phenomena introducing an added layer of information processing in plant cells. Some of this recent progress is based on the successful functional expression of plant RNA editing factors in bacteria and mammalian cells. These research approaches have recapitulated natural processes of horizontal gene transfer through which some protist lineages seem to have acquired plant RNA editing factors and adapted them functionally for their own purposes.

Genome-wide identification and expression analysis of peach multiple organellar RNA editing factors reveals the roles of RNA editing in plant immunity

BackgroundMultiple organellar RNA editing factor (MORF) genes play key roles in chloroplast developmental processes by mediating RNA editing of Cytosine-to-Uracil conversion. However, the function of MORF genes in peach (Prunus persica), a perennial horticultural crop species of Rosaceae, is still not well known, particularly the resistance to biotic and abiotic stresses that threaten peach yield seriously.ResultsIn this study, to reveal the regulatory roles of RNA editing in plant immunity, we implemented genome-wide analysis of peach MORF (PpMORF) genes in response to biotic and abiotic stresses. The chromosomal and subcellular location analysis showed that the identified seven PpMORF genes distributed on three peach chromosomes were mainly localized in the mitochondria and chloroplast. All the PpMORF genes were classified into six groups and one pair of PpMORF genes was tandemly duplicated. Based on the meta-analysis of two types of public RNA-seq data under different treatments (biotic and abiotic stresses), we observed down-regulated expression of PpMORF genes and reduced chloroplast RNA editing, especially the different response of PpMORF2 and PpMORF9 to pathogens infection between resistant and susceptible peach varieties, indicating the roles of MORF genes in stress response by modulating the RNA editing extent in plant immunity. Three upstream transcription factors (MYB3R-1, ZAT10, HSFB3) were identified under both stresses, they may regulate resistance adaption by modulating the PpMORF gene expression.ConclusionThese results provided the foundation for further analyses of the functions of MORF genes, in particular the roles of RNA editing in plant immunity. In addition, our findings will be conducive to clarifying the resistance mechanisms in peaches and open up avenues for breeding new cultivars with high resistance.

SlRIP1b is a global organellar RNA editing factor, required for normal fruit development in tomato plants.

RNA editing in plant organelles involves numerous C-U conversions, which often restore evolutionarily conserved codons and may generate new translation initiation and termination codons. These RNA maturation events rely on a subset of nuclear-encoded protein cofactors. Here, we provide evidence of the role of SlRIP1b on RNA editing of mitochondrial transcripts in tomato (Solanum lycopersicum) plants. SlRIP1b is a RIP/MORF protein that was originally identified as an interacting partner of the organellar editing factor SlORRM4. Mutants of SlRIP1b, obtained by CRISPR/Cas9 strategy, exhibited abnormal carpel development and grew into fruit with more locules. RNA-sequencing revealed that SlRIP1b affects the C-U editing of numerous mitochondrial pre-RNA transcripts and in particular altered RNA editing of various cytochrome c maturation (CCM)-related genes. The slrip1b mutants display increased H2 O2 and aberrant mitochondrial morphologies, which are associated with defects in cytochrome c biosynthesis and assembly of respiratory complex III. Taken together, our results indicate that SlRIP1b is a global editing factor that plays a key role in CCM and oxidative phosphorylation system biogenesis during fruit development in tomato plants. These data provide important insights into the molecular roles of organellar RNA editing factors during fruit development.

Structural insight into the activation of an Arabidopsis organellar C-to-U RNA editing enzyme by active site complementation.

RNA-binding pentatricopeptide repeat (PPR) proteins catalyze hundreds of cytidine to uridine RNA editing events in plant organelles; these editing events are essential for proper gene expression. More than half of the PPR-type RNA editing factors, however, lack the DYW cytidine deaminase domain. Genetic analyses have suggested that their cytidine deaminase activity arises by association with a family of DYW1-like proteins that contain an N-terminally truncated DYW domain, but their molecular mechanism has been unclear. Here, we report the crystal structure of the Arabidopsis thaliana DYW1 deaminase domain at 1.8 Å resolution. DYW1 has a cytidine deaminase fold lacking the PG box. The internal insertion within the deaminase fold shows an α-helical fold instead of the β-finger reported for the gating domain of the A. thaliana ORGANELLE TRANSCRIPT PROCESSING 86. The substrate-binding pocket is incompletely formed and appears to be complemented in the complex by the E2 domain and the PG box of the interacting PPR protein. In vivo RNA editing assays corroborate the activation model for DYW1 deaminase. Our study demonstrates the common activation mechanism of the DYW1-like proteins by molecular complementation of the DYW domain and reconstitution of the substrate-binding pocket.

Maize PPR-E proteins mediate RNA C-to-U editing in mitochondria by recruiting the trans deaminase PCW1.

RNA C-to-U editing in organelles is essential for plant growth and development; however, the underlying mechanism is not fully understood. Here, we report that pentatricopeptide repeat (PPR)-E subclass proteins carry out RNA C-to-U editing by recruiting the trans deaminase PPR motifs, coiled-coil, and DYW domain-containing protein 1 (PCW1) in maize (Zea mays) mitochondria. Loss-of-function of bZIP and coiled-coil domain-containing PPR 1 (bCCP1) or PCW1 arrests seed development in maize. bCCP1 encodes a bZIP and coiled-coil domain-containing PPR protein, and PCW1 encodes an atypical PPR-DYW protein. bCCP1 is required for editing at 66 sites in mitochondria and PCW1 is required for editing at 102 sites, including the 66 sites that require bCCP1. The PCW1-mediated editing sites are exclusively associated with PPR-E proteins. bCCP1 interacts with PCW1 and the PPR-E protein Empty pericarp7 (EMP7). Two multiple organellar RNA editing factor (MORF) proteins, ZmMORF1 and ZmMORF8, interact with PCW1, EMP7, and bCCP1. ZmMORF8 enhanced the EMP7-PCW1 interaction in a yeast three-hybrid assay. C-to-U editing at the ccmFN-1553 site in maize required EMP7, bCCP1, and PCW1. These results suggest that PPR-E proteins function in RNA editing by recruiting the trans deaminase PCW1 and bCCP1, and MORF1/8 assist this recruitment through protein-protein interactions.

Genome-wide identification of RNA editing sites in chloroplast transcripts and multiple organellar RNA editing factors in tea plant (Camellia sinensis L.): Insights into the albinism mechanism of tea leaves

RNA editing is a post-transcriptional modification process, the chloroplast genes of which are involved in the process of chloroplast development in plant. However, the RNA editing sites of chloroplast genes remains unknown. In this study, we identified 39 RNA editing sites in 18 chloroplast genes from chloroplast genome of C. sinensis. Furthermore, the feature, structures and specificity of RNA editing sites were systematic analyzed. The differential editing efficiency were examined at 11 RNA editing sites among C. sinensis var. sinensis ‘Huabai 1′, ‘Baiye 1′ and ‘Longjing 43′. Meanwhile, we identified 10 C. sinensis MORFs from five subgroups and performed comparative analyses of chromosome locations, duplication model and expression profiles. Expression analysis showed that the expression level of CsMORF9.2 was down-regulated significantly in ‘Huabai 1′ albino tea cultivar. This study provides a foundation for further reveal in the role of chloroplast RNA editing in albinism process of tea leaves.

Plant mitochondrial RNA editing factors can perform targeted C-to-U editing of nuclear transcripts in human cells.

RNA editing processes are strikingly different in animals and plants. Up to thousands of specific cytidines are converted into uridines in plant chloroplasts and mitochondria whereas up to millions of adenosines are converted into inosines in animal nucleo-cytosolic RNAs. It is unknown whether these two different RNA editing machineries are mutually incompatible. RNA-binding pentatricopeptide repeat (PPR) proteins are the key factors of plant organelle cytidine-to-uridine RNA editing. The complete absence of PPR mediated editing of cytosolic RNAs might be due to a yet unknown barrier that prevents its activity in the cytosol. Here, we transferred two plant mitochondrial PPR-type editing factors into human cell lines to explore whether they could operate in the nucleo-cytosolic environment. PPR56 and PPR65 not only faithfully edited their native, co-transcribed targets but also different sets of off-targets in the human background transcriptome. More than 900 of such off-targets with editing efficiencies up to 91%, largely explained by known PPR-RNA binding properties, were identified for PPR56. Engineering two crucial amino acid positions in its PPR array led to predictable shifts in target recognition. We conclude that plant PPR editing factors can operate in the entirely different genetic environment of the human nucleo-cytosol and can be intentionally re-engineered towards new targets.

Conserved and transcript-specific functions of the RESC factors, RESC13 and RESC14, in kinetoplastid RNA editing.

Uridine insertion/deletion RNA editing is an extensive post-transcriptional modification of mitochondrial mRNAs in kinetoplastid organisms, including Trypanosoma brucei This process is carried out using trans-acting gRNAs and complex protein machinery. The essential RNA editing substrate binding complex (RESC) serves as the scaffold that modulates protein and RNA interactions during editing, and contains the guide RNA binding complex (GRBC), the RNA editing mediator complexes (REMCs), and organizer proteins. Despite the importance of RESC in editing, the functions of each protein comprising this complex are not completely understood. Here, we further define the roles of a REMC protein, RESC13, and a RESC organizer, RESC14, using high-throughput sequencing on two large pan-edited mRNAs, A6 and COIII. When comparing our analyses to that of a previously published small pan-edited mRNA, RPS12, we find that RESC13 has conserved functions across the three transcripts with regard to editing initiation, gRNA utilization, gRNA exchange, and restricting the formation of long misedited junctions that likely arise from its ability to modulate RNA structure. However, RESC13 does have transcript-specific effects on the types of long junctions whose formation it restricts. RESC14 has a conserved effect on gRNA utilization across the three transcripts analyzed, but has transcript-specific effects on editing initiation, gRNA exchange, and junction formation. Our data suggest that transcript-specific effects of both proteins are due to differences in transcript length and sequences as well as transcript-specific protein interactions. These findings highlight the importance of studying multiple transcripts to determine the function of editing factors.

DYW deaminase domain has a distinct preference for neighboring nucleotides of the target RNA editing sites.

C-to-U RNA editing sites in plant organelles show a strong bias for neighboring nucleotides. The nucleotide upstream of the target cytidine is typically C or U, whereas A and G are less common and rare, respectively. In pentatricopeptide repeat (PPR)-type RNA editing factors, the PPR domain specifically binds to the 5' sequence of target cytidines, whereas the DYW domain catalyzes the C-to-U deamination. We comprehensively analyzed the effects of neighboring nucleotides of the target cytidines using an Escherichia coli orthogonal system. Physcomitrium PPR56 efficiently edited target cytidines when the nucleotide upstream was U or C, whereas it barely edited when the position was G or the nucleotide downstream was C. This preference pattern, which corresponds well with the observed nucleotide bias for neighboring nucleotides in plant organelles, was altered when the DYW domain of OTP86 or DYW1 was adopted. The PPR56 chimeric proteins edited the target sites even when the -1 position was G. Our results suggest that the DYW domain possesses a distinct preference for the neighboring nucleotides of the target sites, thus contributing to target selection in addition to the existing selection determined by the PPR domain.

Two chloroplast-localized MORF proteins act as chaperones to maintain tetrapyrrole biosynthesis.

Tetrapyrroles have essential functions as pigments and cofactors during plant growth and development, and the tetrapyrrole biosynthesis pathway is tightly controlled. Multiple organellar RNA editing factors (MORFs) are required for editing of a wide variety of RNA sites in chloroplasts and mitochondria, but their biochemical properties remain elusive. Here, we uncovered the roles of chloroplast-localized MORF2 and MORF9 in modulating tetrapyrrole biosynthesis and embryogenesis in Arabidopsis thaliana. The lack or reduced transcripts of MORF2 or MORF9 significantly affected biosynthesis of the tetrapyrrole precursor 5-aminolevulinic acid and accumulation of Chl and other tetrapyrrole intermediates. MORF2 directly interacts with multiple tetrapyrrole biosynthesis enzymes and regulators, including NADPH:PROTOCHLOROPHYLLIDE OXIDOREDUCTASE B (PORB) and GENOMES UNCOUPLED4 (GUN4). Strikingly, MORF2 and MORF9 display holdase chaperone activity, alleviate the aggregation of PORB in vitro, and are essential for POR accumulation in vivo. Moreover, both MORF2 and MORF9 significantly stimulate magnesium chelatase activity. Our findings reveal a previously unknown biochemical property of MORF proteins as chaperones and point to a new layer of post-translational control of the tightly regulated tetrapyrrole biosynthesis in plants.

Decoding RNA Editing Sites Through Transcriptome Analysis in Rice Under Alkaline Stress.

Ribonucleic acid editing (RE) is a post-transcriptional process that altered the genetics of RNA which provide the extra level of gene expression through insertion, deletions, and substitutions. In animals, it converts nucleotide residues C-U. Similarly in plants, the role of RNA editing sites (RES) in rice under alkaline stress is not fully studied. Rice is a staple food for most of the world population. Alkaline stress cause reduction in yield. Here, we explored the effect of alkaline stress on RES in the whole mRNA from rice chloroplast and mitochondria. Ribonucleic acid editing sites in both genomes (3336 RESs) including chloroplast (345 RESs) and mitochondria (2991 RESs) with average RES efficiency ∼55% were predicted. Our findings showed that majority of editing events found in non-synonymous codon changes and change trend in amino acids was hydrophobic. Four types of RNA editing A-G (A-I), C-T (C-U), G-A, and T-C were identified in treated and untreated samples. Overall, RNA editing efficiency was increased in the treated samples. Analysis of Gene Ontology revealed that mapped genes were engaged in many biological functions and molecular processes. We also checked the expression of pentatricopeptide repeat (PPR), organelle zinc-finger (OZI), and multiple organellar RNA editing factors/RNA editing factor interacting proteins genes in control and treatment, results revealed upregulation of PPR and OZ1 genes in treated samples. This induction showed the role of these genes in RNA editing. The current findings report that RNA editing increased under alkaline stress which may contribute in adaptation for rice by changing amino acids in edited genes (88 genes). These findings will provide basis for identification of RES in other crops and also will be useful in alkaline tolerance development in rice.

Structural Basis for the Peptidoglycan-Editing Activity of YfiH.

ABSTRACTBacterial cells are encased in peptidoglycan (PG), a polymer of disaccharide N-acetylglucosamine (GlcNAc) and N-acetyl-muramic acid (MurNAc) cross-linked by peptide stems. PG is synthesized in the cytoplasm as UDP-MurNAc-peptide precursors, of which the amino acid composition of the peptide is unique, with l-Ala added at the first position in most bacteria but with l-Ser or Gly in some bacteria. YfiH is a PG-editing factor whose absence causes misincorporation of l-Ser instead of l-Ala into peptide stems, but its mechanistic function is unknown. Here, we report the crystal structures of substrate-bound and product-bound YfiH, showing that YfiH is a cytoplasmic amidase that controls the incorporation of the correct amino acid to the nucleotide precursors by preferentially cleaving the nucleotide precursor by-product UDP–MurNAc–l-Ser. This work reveals an editing mechanism in the cytoplasmic steps of peptidoglycan biosynthesis.

Genome-Wide Analysis of Multiple Organellar RNA Editing Factor (MORF) Family in Kiwifruit (Actinidia chinensis) Reveals Its Roles in Chloroplast RNA Editing and Pathogens Stress.

Kiwifruit (Actinidia chinensis) is well known for its high vitamin C content and good taste. Various diseases, especially bacterial canker, are a serious threat to the yield of kiwifruit. Multiple organellar RNA editing factor (MORF) genes are pivotal factors in the RNA editosome that mediates Cytosine-to-Uracil RNA editing, and they are also indispensable for the regulation of chloroplast development, plant growth, and response to stresses. Although the kiwifruit genome has been released, little is known about MORF genes in kiwifruit at the genome-wide level, especially those involved in the response to pathogens stress. In this study, we identified ten MORF genes in the kiwifruit genome. The genomic structures and chromosomal locations analysis indicated that all the MORF genes consisted of three conserved motifs, and they were distributed widely across the seven linkage groups and one contig of the kiwifruit genome. Based on the structural features of MORF proteins and the topology of the phylogenetic tree, the kiwifruit MORF gene family members were classified into six groups (Groups A–F). A synteny analysis indicated that two pairs of MORF genes were tandemly duplicated and five pairs of MORF genes were segmentally duplicated. Moreover, based on analysis of RNA-seq data from five tissues of kiwifruit, we found that both expressions of MORF genes and chloroplast RNA editing exhibited tissue-specific patterns. MORF2 and MORF9 were highly expressed in leaf and shoot, and may be responsible for chloroplast RNA editing, especially the ndhB genes. We also observed different MORF expression and chloroplast RNA editing profiles between resistant and susceptible kiwifruits after pathogen infection, indicating the roles of MORF genes in stress response by modulating the editing extend of mRNA. These results provide a solid foundation for further analyses of the functions and molecular evolution of MORF genes, in particular, for clarifying the resistance mechanisms in kiwifruits and breeding new cultivars with high resistance.

Cofactor-independent RNA editing by a synthetic S-type PPR protein.

Pentatricopeptide repeat (PPR) proteins are RNA-binding proteins that are attractive tools for RNA processing in synthetic biology applications given their modular structure and ease of design. Several distinct types of motifs have been described from natural PPR proteins, but almost all work so far with synthetic PPR proteins has focused on the most widespread P-type motifs. We have investigated synthetic PPR proteins based on tandem repeats of the more compact S-type PPR motif found in plant organellar RNA editing factors and particularly prevalent in the lycophyte Selaginella. With the aid of a novel plate-based screening method, we show that synthetic S-type PPR proteins are easy to design and bind with high affinity and specificity and are functional in a wide range of pH, salt and temperature conditions. We find that they outperform a synthetic P-type PPR scaffold in many situations. We designed an S-type editing factor to edit an RNA target in E. coli and demonstrate that it edits effectively without requiring any additional cofactors to be added to the system. These qualities make S-type PPR scaffolds ideal for developing new RNA processing tools.