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Mediated plastid RNA editing in plant immunity.

García-Andrade J, Ramírez V, López A, Vera P - PLoS Pathog. (2013)

Bottom Line: Furthermore, we observed that following a pathogenic challenge, wild type plants respond with editing inhibition of ndhB transcript.In parallel, rapid destabilization of the plastidial NDH complex is also observed in the plant following perception of a pathogenic cue.Therefore, NDH complex activity and plant immunity appear as interlinked processes.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-C.S.I.C, Ciudad Politécnica de la Innovación, Ingeniero Fausto Elio, Valencia, Spain.

ABSTRACT
Plant regulatory circuits coordinating nuclear and plastid gene expression have evolved in response to external stimuli. RNA editing is one of such control mechanisms. We determined the Arabidopsis nuclear-encoded homeodomain-containing protein OCP3 is incorporated into the chloroplast, and contributes to control over the extent of ndhB transcript editing. ndhB encodes the B subunit of the chloroplast NADH dehydrogenase-like complex (NDH) involved in cyclic electron flow (CEF) around photosystem I. In ocp3 mutant strains, ndhB editing efficiency decays, CEF is impaired and disease resistance to fungal pathogens substantially enhanced, a process recapitulated in plants defective in editing plastid RNAs encoding NDH complex subunits due to mutations in previously described nuclear-encoded pentatricopeptide-related proteins (i.e. CRR21, CRR2). Furthermore, we observed that following a pathogenic challenge, wild type plants respond with editing inhibition of ndhB transcript. In parallel, rapid destabilization of the plastidial NDH complex is also observed in the plant following perception of a pathogenic cue. Therefore, NDH complex activity and plant immunity appear as interlinked processes.

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Related in: MedlinePlus

Editing defects in ocp3 plants and in vivo association of OCP3 with ndhB RNA.(A) Nucleotide sequences surrounding the RNA editing sites of ndhB-6 (95644), ndhB-5 (95650), ndhB-4 (96419), ndhB-3 (96579), ndhB-2 (96698) and ndhD-2 (116494) are shown as sequence chromatograms. The editing sites are specified relative to the nucleotide sequence of the complete Arabidopsis chloroplast genome (Genebank accession number AP000423). Editing sites are indicated by a red C residue in the genomic (gDNA) sequence and its conversion or not to a U(T) residue in Col-0 and ocp3 derived RNA samples. Editing defects in ocp3 are indicated by a blue mark above the corresponding peaks. (B–E) Poisoned primer extension (PPE) assays were conducted on the editing sites ndhB-6 (B), ndhB-5 (C), ndhB-4 (D) and ndhB-3 (E). RT-PCR products were obtained with labeled 6-carboxyfluorescein primers that anneals next to the target editing site (forward PPE primers were used for all sites). Acrylamide gels (below panels) were visualized under UV light, and intensity of bands quantified calculated and plotted. Bars represent mean ± SD, n = 3 independent replicates. Experiments were repeated at least three times with similar results. E, edited; U, unedited, P; primer. (F) Comparative RNA editing efficiency in Col-0 and ocp3 plants as quantified from direct DNA sequencing of 100 independent cDNAs per genotype encompassing each of the indicated editing sites. (G) RNA immunoprecipitation (RIP) of anti-HA precipitated protein complexes from leaves derived from Col-0 and a 35S::OCP3:YFP:HA transgenic line. The upper panel shows a Western blot of protein present in crude leaf extracts and proteins immunoprecipitated (IP) with anti-HA antibody. The blot was developed with anti-GFP antibody and shows enrichment of the OCP3:YFP:HA protein in samples derived from the transgenic line. In the lower panel RT-PCR was used to detect association of ndhB transcripts with OCP3-enriched complexes in comparison to the corresponding input sample. Lack of association of ndhD transcripts with OCP3-enriched complexes is shown as a negative control.
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ppat-1003713-g004: Editing defects in ocp3 plants and in vivo association of OCP3 with ndhB RNA.(A) Nucleotide sequences surrounding the RNA editing sites of ndhB-6 (95644), ndhB-5 (95650), ndhB-4 (96419), ndhB-3 (96579), ndhB-2 (96698) and ndhD-2 (116494) are shown as sequence chromatograms. The editing sites are specified relative to the nucleotide sequence of the complete Arabidopsis chloroplast genome (Genebank accession number AP000423). Editing sites are indicated by a red C residue in the genomic (gDNA) sequence and its conversion or not to a U(T) residue in Col-0 and ocp3 derived RNA samples. Editing defects in ocp3 are indicated by a blue mark above the corresponding peaks. (B–E) Poisoned primer extension (PPE) assays were conducted on the editing sites ndhB-6 (B), ndhB-5 (C), ndhB-4 (D) and ndhB-3 (E). RT-PCR products were obtained with labeled 6-carboxyfluorescein primers that anneals next to the target editing site (forward PPE primers were used for all sites). Acrylamide gels (below panels) were visualized under UV light, and intensity of bands quantified calculated and plotted. Bars represent mean ± SD, n = 3 independent replicates. Experiments were repeated at least three times with similar results. E, edited; U, unedited, P; primer. (F) Comparative RNA editing efficiency in Col-0 and ocp3 plants as quantified from direct DNA sequencing of 100 independent cDNAs per genotype encompassing each of the indicated editing sites. (G) RNA immunoprecipitation (RIP) of anti-HA precipitated protein complexes from leaves derived from Col-0 and a 35S::OCP3:YFP:HA transgenic line. The upper panel shows a Western blot of protein present in crude leaf extracts and proteins immunoprecipitated (IP) with anti-HA antibody. The blot was developed with anti-GFP antibody and shows enrichment of the OCP3:YFP:HA protein in samples derived from the transgenic line. In the lower panel RT-PCR was used to detect association of ndhB transcripts with OCP3-enriched complexes in comparison to the corresponding input sample. Lack of association of ndhD transcripts with OCP3-enriched complexes is shown as a negative control.

Mentions: To test the involvement of OCP3 in RNA editing, we systematically examined the editing status of chloroplast transcripts derived from wild type and ocp3 plants using high-resolution melting (HRM) screen of the 34 sites undergoing editing in Arabidopsis [7]. We identified major defects in the RNA editing of ndhB-6, nhdB-4, ndhB-3, and ndhB-2 sites in ocp3 plants (Figure S4). The comparison of the sequencing electrophoregrams of the RT-PCR products surrounding the editing sites confirmed that editing was compromised at the four indicated sites, if not totally at least partially, in ocp3 plants (Figure 4A and Figure S6). All other known sites appeared similarly edited in ocp3 plants as in Col-0 plants. Editing defects were further confirmed by poisoned primer extension (PPE) assays (Figure 4B–E). ndhB-6, ndhB-4, and ndhB-3 sites were edited in Col-0 at estimated efficiencies of approximately 72%, 95%, and 88%, respectively, while in ocp3 plants efficiencies were reduced approximately to 55%, 89%, and 72%, respectively (Figure 4B and 4D). The ndhB-2 editing site possessed a contiguous cytosine residue adjacent to the cytosine to be edited, which impeded a reliable PPE assay. Therefore, ndhB-2 was not further studied by this method. The ndhB-5 site exhibited no editing variation between Col-0 and ocp3, with efficiencies in the range of 82.5% and 81%, respectively; therefore, it served as an internal editing control site for the ndhB transcript not affected in ocp3 plants.


Mediated plastid RNA editing in plant immunity.

García-Andrade J, Ramírez V, López A, Vera P - PLoS Pathog. (2013)

Editing defects in ocp3 plants and in vivo association of OCP3 with ndhB RNA.(A) Nucleotide sequences surrounding the RNA editing sites of ndhB-6 (95644), ndhB-5 (95650), ndhB-4 (96419), ndhB-3 (96579), ndhB-2 (96698) and ndhD-2 (116494) are shown as sequence chromatograms. The editing sites are specified relative to the nucleotide sequence of the complete Arabidopsis chloroplast genome (Genebank accession number AP000423). Editing sites are indicated by a red C residue in the genomic (gDNA) sequence and its conversion or not to a U(T) residue in Col-0 and ocp3 derived RNA samples. Editing defects in ocp3 are indicated by a blue mark above the corresponding peaks. (B–E) Poisoned primer extension (PPE) assays were conducted on the editing sites ndhB-6 (B), ndhB-5 (C), ndhB-4 (D) and ndhB-3 (E). RT-PCR products were obtained with labeled 6-carboxyfluorescein primers that anneals next to the target editing site (forward PPE primers were used for all sites). Acrylamide gels (below panels) were visualized under UV light, and intensity of bands quantified calculated and plotted. Bars represent mean ± SD, n = 3 independent replicates. Experiments were repeated at least three times with similar results. E, edited; U, unedited, P; primer. (F) Comparative RNA editing efficiency in Col-0 and ocp3 plants as quantified from direct DNA sequencing of 100 independent cDNAs per genotype encompassing each of the indicated editing sites. (G) RNA immunoprecipitation (RIP) of anti-HA precipitated protein complexes from leaves derived from Col-0 and a 35S::OCP3:YFP:HA transgenic line. The upper panel shows a Western blot of protein present in crude leaf extracts and proteins immunoprecipitated (IP) with anti-HA antibody. The blot was developed with anti-GFP antibody and shows enrichment of the OCP3:YFP:HA protein in samples derived from the transgenic line. In the lower panel RT-PCR was used to detect association of ndhB transcripts with OCP3-enriched complexes in comparison to the corresponding input sample. Lack of association of ndhD transcripts with OCP3-enriched complexes is shown as a negative control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814343&req=5

ppat-1003713-g004: Editing defects in ocp3 plants and in vivo association of OCP3 with ndhB RNA.(A) Nucleotide sequences surrounding the RNA editing sites of ndhB-6 (95644), ndhB-5 (95650), ndhB-4 (96419), ndhB-3 (96579), ndhB-2 (96698) and ndhD-2 (116494) are shown as sequence chromatograms. The editing sites are specified relative to the nucleotide sequence of the complete Arabidopsis chloroplast genome (Genebank accession number AP000423). Editing sites are indicated by a red C residue in the genomic (gDNA) sequence and its conversion or not to a U(T) residue in Col-0 and ocp3 derived RNA samples. Editing defects in ocp3 are indicated by a blue mark above the corresponding peaks. (B–E) Poisoned primer extension (PPE) assays were conducted on the editing sites ndhB-6 (B), ndhB-5 (C), ndhB-4 (D) and ndhB-3 (E). RT-PCR products were obtained with labeled 6-carboxyfluorescein primers that anneals next to the target editing site (forward PPE primers were used for all sites). Acrylamide gels (below panels) were visualized under UV light, and intensity of bands quantified calculated and plotted. Bars represent mean ± SD, n = 3 independent replicates. Experiments were repeated at least three times with similar results. E, edited; U, unedited, P; primer. (F) Comparative RNA editing efficiency in Col-0 and ocp3 plants as quantified from direct DNA sequencing of 100 independent cDNAs per genotype encompassing each of the indicated editing sites. (G) RNA immunoprecipitation (RIP) of anti-HA precipitated protein complexes from leaves derived from Col-0 and a 35S::OCP3:YFP:HA transgenic line. The upper panel shows a Western blot of protein present in crude leaf extracts and proteins immunoprecipitated (IP) with anti-HA antibody. The blot was developed with anti-GFP antibody and shows enrichment of the OCP3:YFP:HA protein in samples derived from the transgenic line. In the lower panel RT-PCR was used to detect association of ndhB transcripts with OCP3-enriched complexes in comparison to the corresponding input sample. Lack of association of ndhD transcripts with OCP3-enriched complexes is shown as a negative control.
Mentions: To test the involvement of OCP3 in RNA editing, we systematically examined the editing status of chloroplast transcripts derived from wild type and ocp3 plants using high-resolution melting (HRM) screen of the 34 sites undergoing editing in Arabidopsis [7]. We identified major defects in the RNA editing of ndhB-6, nhdB-4, ndhB-3, and ndhB-2 sites in ocp3 plants (Figure S4). The comparison of the sequencing electrophoregrams of the RT-PCR products surrounding the editing sites confirmed that editing was compromised at the four indicated sites, if not totally at least partially, in ocp3 plants (Figure 4A and Figure S6). All other known sites appeared similarly edited in ocp3 plants as in Col-0 plants. Editing defects were further confirmed by poisoned primer extension (PPE) assays (Figure 4B–E). ndhB-6, ndhB-4, and ndhB-3 sites were edited in Col-0 at estimated efficiencies of approximately 72%, 95%, and 88%, respectively, while in ocp3 plants efficiencies were reduced approximately to 55%, 89%, and 72%, respectively (Figure 4B and 4D). The ndhB-2 editing site possessed a contiguous cytosine residue adjacent to the cytosine to be edited, which impeded a reliable PPE assay. Therefore, ndhB-2 was not further studied by this method. The ndhB-5 site exhibited no editing variation between Col-0 and ocp3, with efficiencies in the range of 82.5% and 81%, respectively; therefore, it served as an internal editing control site for the ndhB transcript not affected in ocp3 plants.

Bottom Line: Furthermore, we observed that following a pathogenic challenge, wild type plants respond with editing inhibition of ndhB transcript.In parallel, rapid destabilization of the plastidial NDH complex is also observed in the plant following perception of a pathogenic cue.Therefore, NDH complex activity and plant immunity appear as interlinked processes.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-C.S.I.C, Ciudad Politécnica de la Innovación, Ingeniero Fausto Elio, Valencia, Spain.

ABSTRACT
Plant regulatory circuits coordinating nuclear and plastid gene expression have evolved in response to external stimuli. RNA editing is one of such control mechanisms. We determined the Arabidopsis nuclear-encoded homeodomain-containing protein OCP3 is incorporated into the chloroplast, and contributes to control over the extent of ndhB transcript editing. ndhB encodes the B subunit of the chloroplast NADH dehydrogenase-like complex (NDH) involved in cyclic electron flow (CEF) around photosystem I. In ocp3 mutant strains, ndhB editing efficiency decays, CEF is impaired and disease resistance to fungal pathogens substantially enhanced, a process recapitulated in plants defective in editing plastid RNAs encoding NDH complex subunits due to mutations in previously described nuclear-encoded pentatricopeptide-related proteins (i.e. CRR21, CRR2). Furthermore, we observed that following a pathogenic challenge, wild type plants respond with editing inhibition of ndhB transcript. In parallel, rapid destabilization of the plastidial NDH complex is also observed in the plant following perception of a pathogenic cue. Therefore, NDH complex activity and plant immunity appear as interlinked processes.

Show MeSH
Related in: MedlinePlus