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RECG maintains plastid and mitochondrial genome stability by suppressing extensive recombination between short dispersed repeats.

Odahara M, Masuda Y, Sato M, Wakazaki M, Harada C, Toyooka K, Sekine Y - PLoS Genet. (2015)

Bottom Line: This result suggests that mitochondrial genomic instability is responsible for the defective phenotypes of RECG KO plants.Such loci were sometimes associated with a decrease in the levels of normal mtDNA and significant decrease in the number of transcripts derived from the loci.These results suggest that RECG plays a role in the maintenance of both plastid and mitochondrial genome stability by suppressing aberrant recombination between dispersed short repeats; this role is crucial for plastid and mitochondrial functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, College of Science, Rikkyo (St. Paul's) University, Toshima-ku, Tokyo, Japan.

ABSTRACT
Maintenance of plastid and mitochondrial genome stability is crucial for photosynthesis and respiration, respectively. Recently, we have reported that RECA1 maintains mitochondrial genome stability by suppressing gross rearrangements induced by aberrant recombination between short dispersed repeats in the moss Physcomitrella patens. In this study, we studied a newly identified P. patens homolog of bacterial RecG helicase, RECG, some of which is localized in both plastid and mitochondrial nucleoids. RECG partially complements recG deficiency in Escherichia coli cells. A knockout (KO) mutation of RECG caused characteristic phenotypes including growth delay and developmental and mitochondrial defects, which are similar to those of the RECA1 KO mutant. The RECG KO cells showed heterogeneity in these phenotypes. Analyses of RECG KO plants showed that mitochondrial genome was destabilized due to a recombination between 8-79 bp repeats and the pattern of the recombination partly differed from that observed in the RECA1 KO mutants. The mitochondrial DNA (mtDNA) instability was greater in severe phenotypic RECG KO cells than that in mild phenotypic ones. This result suggests that mitochondrial genomic instability is responsible for the defective phenotypes of RECG KO plants. Some of the induced recombination caused efficient genomic rearrangements in RECG KO mitochondria. Such loci were sometimes associated with a decrease in the levels of normal mtDNA and significant decrease in the number of transcripts derived from the loci. In addition, the RECG KO mutation caused remarkable plastid abnormalities and induced recombination between short repeats (12-63 bp) in the plastid DNA. These results suggest that RECG plays a role in the maintenance of both plastid and mitochondrial genome stability by suppressing aberrant recombination between dispersed short repeats; this role is crucial for plastid and mitochondrial functions.

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Subcellular localization of the RECG-GFP protein in P. patens protoplast cells.A. Subcellular localization of GFP fused to RECG N-terminal region. The fluorescence of GFP was merged with Mito Tracker or chlorophyll autofluorescence. Plastids were distinguished by the distribution of their chlorophyll autofluorescence, while mitochondria were detected by staining with Mito Tracker Orange. The arrowheads with P and M denote examples of RECG-GFP localized to plastid and mitochondrion, respectively. B. Subcellular localization of GFP fused to full-length RECG. The fluorescence of GFP was merged with DAPI fluorescence. C and D. Localization of full-length RECG-GFP to plastid (C) and mitochondrial (D) nucleoids. GFP fluorescence was merged with DAPI fluorescence (left panels) and then GFP fluorescence was shifted to left (right panels). The arrowheads denote examples of correspondence between GFP and DAPI signals. Bars = 10 μm in A and B, and 1 μm in C and D.
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pgen.1005080.g001: Subcellular localization of the RECG-GFP protein in P. patens protoplast cells.A. Subcellular localization of GFP fused to RECG N-terminal region. The fluorescence of GFP was merged with Mito Tracker or chlorophyll autofluorescence. Plastids were distinguished by the distribution of their chlorophyll autofluorescence, while mitochondria were detected by staining with Mito Tracker Orange. The arrowheads with P and M denote examples of RECG-GFP localized to plastid and mitochondrion, respectively. B. Subcellular localization of GFP fused to full-length RECG. The fluorescence of GFP was merged with DAPI fluorescence. C and D. Localization of full-length RECG-GFP to plastid (C) and mitochondrial (D) nucleoids. GFP fluorescence was merged with DAPI fluorescence (left panels) and then GFP fluorescence was shifted to left (right panels). The arrowheads denote examples of correspondence between GFP and DAPI signals. Bars = 10 μm in A and B, and 1 μm in C and D.

Mentions: We identified a homolog of E. coli RecG in the P. patens nuclear genomic sequence [24] and named it RECG. Homologs of RecG are found in other plants, but not in fungi or animals, like bacterial-type RecA homologs [25]. Based on its cDNA sequence which we determined by rapid amplification of cDNA ends (RACE), the RECG protein is predicted to be 1152 amino acids in length and shares a high degree of sequence similarity with E. coli RecG, except for its extended N-terminal region (S1A Fig.). The extended N-terminal region, which is assumed to be a signal peptide that targets the protein to organelles, is potentially sufficient for localization to both plastid and mitochondrion (S1B Fig.) as judged by TargetP [26]. A similar N-terminal extension also exists in an annotated version of the RecG homolog in A. thaliana with a potential for localizing to both plastid and mitochondria (S1B Fig.). Fluorescent microscopy of protoplast cells expressing green fluorescent protein (GFP) gene fused to downstream of the 5’UTR and the N-terminus of RECG cDNA showed that the RECG-GFP localized to both plastids and mitochondria (Fig. 1A). Similar analysis with GFP gene fused to full-length RECG coding sequence demonstrated GFP fluorescence foci in both plastids and extra-plastid cytoplasmic space (Fig. 1B). 4′,6-diamidino-2-phenylindole (DAPI) staining of the cell showed that the GFP foci sometimes corresponded to some plastid and mitochondrial nucleoids (Fig. 1C and D), suggesting that RECG protein associates with these nucleoids.


RECG maintains plastid and mitochondrial genome stability by suppressing extensive recombination between short dispersed repeats.

Odahara M, Masuda Y, Sato M, Wakazaki M, Harada C, Toyooka K, Sekine Y - PLoS Genet. (2015)

Subcellular localization of the RECG-GFP protein in P. patens protoplast cells.A. Subcellular localization of GFP fused to RECG N-terminal region. The fluorescence of GFP was merged with Mito Tracker or chlorophyll autofluorescence. Plastids were distinguished by the distribution of their chlorophyll autofluorescence, while mitochondria were detected by staining with Mito Tracker Orange. The arrowheads with P and M denote examples of RECG-GFP localized to plastid and mitochondrion, respectively. B. Subcellular localization of GFP fused to full-length RECG. The fluorescence of GFP was merged with DAPI fluorescence. C and D. Localization of full-length RECG-GFP to plastid (C) and mitochondrial (D) nucleoids. GFP fluorescence was merged with DAPI fluorescence (left panels) and then GFP fluorescence was shifted to left (right panels). The arrowheads denote examples of correspondence between GFP and DAPI signals. Bars = 10 μm in A and B, and 1 μm in C and D.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4358946&req=5

pgen.1005080.g001: Subcellular localization of the RECG-GFP protein in P. patens protoplast cells.A. Subcellular localization of GFP fused to RECG N-terminal region. The fluorescence of GFP was merged with Mito Tracker or chlorophyll autofluorescence. Plastids were distinguished by the distribution of their chlorophyll autofluorescence, while mitochondria were detected by staining with Mito Tracker Orange. The arrowheads with P and M denote examples of RECG-GFP localized to plastid and mitochondrion, respectively. B. Subcellular localization of GFP fused to full-length RECG. The fluorescence of GFP was merged with DAPI fluorescence. C and D. Localization of full-length RECG-GFP to plastid (C) and mitochondrial (D) nucleoids. GFP fluorescence was merged with DAPI fluorescence (left panels) and then GFP fluorescence was shifted to left (right panels). The arrowheads denote examples of correspondence between GFP and DAPI signals. Bars = 10 μm in A and B, and 1 μm in C and D.
Mentions: We identified a homolog of E. coli RecG in the P. patens nuclear genomic sequence [24] and named it RECG. Homologs of RecG are found in other plants, but not in fungi or animals, like bacterial-type RecA homologs [25]. Based on its cDNA sequence which we determined by rapid amplification of cDNA ends (RACE), the RECG protein is predicted to be 1152 amino acids in length and shares a high degree of sequence similarity with E. coli RecG, except for its extended N-terminal region (S1A Fig.). The extended N-terminal region, which is assumed to be a signal peptide that targets the protein to organelles, is potentially sufficient for localization to both plastid and mitochondrion (S1B Fig.) as judged by TargetP [26]. A similar N-terminal extension also exists in an annotated version of the RecG homolog in A. thaliana with a potential for localizing to both plastid and mitochondria (S1B Fig.). Fluorescent microscopy of protoplast cells expressing green fluorescent protein (GFP) gene fused to downstream of the 5’UTR and the N-terminus of RECG cDNA showed that the RECG-GFP localized to both plastids and mitochondria (Fig. 1A). Similar analysis with GFP gene fused to full-length RECG coding sequence demonstrated GFP fluorescence foci in both plastids and extra-plastid cytoplasmic space (Fig. 1B). 4′,6-diamidino-2-phenylindole (DAPI) staining of the cell showed that the GFP foci sometimes corresponded to some plastid and mitochondrial nucleoids (Fig. 1C and D), suggesting that RECG protein associates with these nucleoids.

Bottom Line: This result suggests that mitochondrial genomic instability is responsible for the defective phenotypes of RECG KO plants.Such loci were sometimes associated with a decrease in the levels of normal mtDNA and significant decrease in the number of transcripts derived from the loci.These results suggest that RECG plays a role in the maintenance of both plastid and mitochondrial genome stability by suppressing aberrant recombination between dispersed short repeats; this role is crucial for plastid and mitochondrial functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, College of Science, Rikkyo (St. Paul's) University, Toshima-ku, Tokyo, Japan.

ABSTRACT
Maintenance of plastid and mitochondrial genome stability is crucial for photosynthesis and respiration, respectively. Recently, we have reported that RECA1 maintains mitochondrial genome stability by suppressing gross rearrangements induced by aberrant recombination between short dispersed repeats in the moss Physcomitrella patens. In this study, we studied a newly identified P. patens homolog of bacterial RecG helicase, RECG, some of which is localized in both plastid and mitochondrial nucleoids. RECG partially complements recG deficiency in Escherichia coli cells. A knockout (KO) mutation of RECG caused characteristic phenotypes including growth delay and developmental and mitochondrial defects, which are similar to those of the RECA1 KO mutant. The RECG KO cells showed heterogeneity in these phenotypes. Analyses of RECG KO plants showed that mitochondrial genome was destabilized due to a recombination between 8-79 bp repeats and the pattern of the recombination partly differed from that observed in the RECA1 KO mutants. The mitochondrial DNA (mtDNA) instability was greater in severe phenotypic RECG KO cells than that in mild phenotypic ones. This result suggests that mitochondrial genomic instability is responsible for the defective phenotypes of RECG KO plants. Some of the induced recombination caused efficient genomic rearrangements in RECG KO mitochondria. Such loci were sometimes associated with a decrease in the levels of normal mtDNA and significant decrease in the number of transcripts derived from the loci. In addition, the RECG KO mutation caused remarkable plastid abnormalities and induced recombination between short repeats (12-63 bp) in the plastid DNA. These results suggest that RECG plays a role in the maintenance of both plastid and mitochondrial genome stability by suppressing aberrant recombination between dispersed short repeats; this role is crucial for plastid and mitochondrial functions.

Show MeSH
Related in: MedlinePlus