Identification and characterization of factors for chloroplast RNA editing in Nicotiana tabacum

• Applicant: Dr. Michael Philipp Tillich
• Subject Area: Plant Physiology ; Plant Biochemistry and Biophysics
• Term: from 2014 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 250969564

Chloroplast RNA editing is essential for the proper expression of chloroplast genes and, thus, for chloroplast biogenesis. RNA editing evolves rapidly. This results in species-specific patterns of editing sites. Nicotiana tabacum (tobacco), for instance, possesses 40 chloroplast editing sites and only 19 are shared with Arabidopsis thaliana. In total, about 100 different editing sites have been found in the chloroplasts of seed plants. Tobacco has been early on employed in editing research. It was and is the model organism of choice for the mapping and mutagenesis of the cis-acting elements that define editing sites, because of the technical ability to manipulate the chloroplast genome in an efficient and directed manner. However, the nuclear encoded factors that carry out chloroplast RNA editing were identified in Arabidopsis, because of the availability its genomic sequence and the conductibility of genetic screens.This research program will revive tobacco as prime model organism for studying chloroplast RNA editing in higher plants. To facilitate this, the hitherto unknown gene sequences of the putative editing factors of tobacco were assembled. Furthermore, a bioinformatic framework for the prediction of editing factor/editing site relationships for 22 angiosperm species has been established. These advancements in combination with chloroplast transformation allow us to simultaneously manipulate both cis-elements and editing factor genes in the same species and thus, to systematically dissect the mechanism of chloroplast RNA editing. Within this research program, bioinformatically predicted chloroplast RNA editing factors will be confirmed by reverse genetics in tobacco and selected factors will be characterized by state-of-the-art molecular biological methods. Candidates for novel components of the chloroplast editosomes have been and will be isolated by RNA-affinity chromatography and characterized for their role in RNA editing. This set-up will be strongly supported by an efficient in vitro editing system.The obtained results will enable to design highly precise experiments aimed to study the interaction between trans-factors and their RNA targets by the manipulation of both interaction partners in the future. This includes also heterologous editing factor/editing site couples from other species. Likewise, based on the obtained results, it will be possible to employ chloroplast transformation to elucidate the interplay of RNA editing with splicing and translation. Finally, approaches will be established for the inducible or tissue-specific generation of translational start codons in order to control the expression of chloroplast (trans-)genes by employing heterologous RNA editing sites and their cognate editing factors.

• DFG Programme: Research Grants