Manipulation du système génétique des mitochondries dans les cellules végétales – MITOMANIP

In eukaryotic cells, mitochondria ensure fundamental functions in energy production, redox processes, metabolic pathways and cell death. The mitochondrial genetic system provides a number of polypeptides which are essential for cell survival, as they are components of the respiratory chain or contribute to its biogenesis. In plants, mitochondrial genetic processes are complex and influence several traits of great agronomical importance, including fertility, plant vigor, chloroplast function and cross-compatibility. When not lethal, mutations affecting plant mitochondrial DNA mainly lead to cytoplasmic male sterility (CMS), a trait which is widely used in crops for breeding and hybrid maintenance. Manipulating the plant mitochondrial genetic system is thus of both fundamental and applied interest. However, genetic transformation of mitochondria in plant cells turned out to be impossible with the current conventional methods. In such a context, we have developed alternative strategies for the genetic manipulation of plant mitochondria, based on the physiological mechanisms of nucleic acid trafficking between cell compartments. In many organisms, the population of transfer RNAs (tRNAs) encoded by the mitochondrial genome is not sufficient to support protein synthesis and mitochondria import from the cytosol nuclear-encoded tRNAs. Our laboratory has demonstrated the existence of this specific process in higher plants. Together with extensive investigation of the import pathway, we have subsequently developed two different strategies to introduce RNAs of interest into the organelles. In the first strategy, RNA import is mediated by a shuttle protein. This approach is a potent tool for the efficient import of foreign RNAs into isolated mitochondria and in organello functional tests. It was successfully used to import precursor RNAs and messenger RNAs (mRNAs) that were subsequently processed and post-transcriptionally modified. The second strategy allows to transport RNAs into the mitochondria in stably transformed plant cells. We have shown that a "passenger" RNA attached to the 5' end of a tRNA mimic and expressed from a nuclear transgene is transported into the mitochondria of the transformed cells. This approach allowed us to obtain the import of a specific trans-cleaving ribozyme into the mitochondria of transgenic cells and to produce the efficient knockdown of a major mitochondrial mRNA (CNRS patent pending). Based on these two efficient strategies, the project aims to manipulate plant mitochondrial genetics in vitro, in plant cell suspensions and in whole plants. We expect to further characterize mitochondrial regulation processes, to identify new mitochondrial functions, to investigate the CMS mechanism and to develop RNA import-directed generation of CMS lines. The first objective is to determine the impact of a trans-ribozyme-mediated knockdown of major mitochondrial mRNAs (nad9 and atp9) on mitochondrial and nuclear gene expression. As very little is known about mitochondrial translation in plants, the second objective is to validate with a marker protein (GFP) the in organello translation of an exogenous imported mRNA. Subsequently, regular mitochondrial proteins (NAD9 and ATP9) will be overexpressed upon import of their mRNAs into the organelles in vivo, and the molecular and physiological consequences of the overexpression will be analyzed. Running inverse genetics in plant mitochondria has so far been impossible. The third objective is thus to target into mitochondria trans-ribozymes destined to knockdown conserved transcripts encoding polypeptides of still unknown function (mat-r and mttB) and candidate non-coding RNAs (mnc1 and mnc2). Analysis of the resulting molecular and physiological phenotype will give the clues to the functions of these mitochondrial components. Through the impairment of mitochondrial functions, most of the approaches in the first three objectives may lead to a CMS phenotype in whole plants. On the other hand, CMS due to mutations in the mitochondrial genome is generally associated with the presence, in the organelles, of particular transcripts carrying chimeric open reading frames. The fourth objective is to target into mitochondria of wild-type cells and whole plants RNAs normally found in CMS lines and to obtain the expression of the chimeric polypeptides (ORF77 and ORF138) in the organelles. This approach will allow to better understand the CMS mechanisms and to establish a new way to generate CMS lines.