Mediated Plastid RNA Editing in Plant Immunity
Figure 4
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.