CONTEXT

Plant mitochondrial genetics determines or contributes to a number of agronomically important traits, as for instance cytoplasmic male sterility. On the other hand, plant mitochondria have large genomes, with numerous unassigned regions, and complex genetic processes. Mastering the genetic system in plant mitochondria is thus of wide interest and has many applications. Unfortunately, the current conventional methods do not enable to transform these organelles, so that it is difficult to generate valuable mitochondrially-driven traits and to identify new mitochondrial genes or regulation functions.

Based on the natural transfer RNA (tRNA) import pathway of plant mitochondria, the inventors have developed the use of a tRNA mimic as a vector to drive into the organelles RNAs of interest expressed in the nucleus. The inventors have then designed a sequence comprising a trans-cleaving hammerhead ribozyme attached to a tRNA mimic through a special linker. The ribozyme activity of this chimeric RNA has been validated in vitro. Subsequent expression in plant cell suspensions and in whole plants from an inducible nuclear transgene demonstrated the in vivo import of the chimeric RNA into mitochondria and the specific cleavage of the target RNA in the organelles, yielding the first directed knockdown of a mitochondrial RNA in plants.

As a whole, this invention is a new method to genetically manipulate mitochondria in plant cells, using nuclear expression and organellar import of chimeric catalytic RNAs.

TECHNICAL DESCRIPTION

More precisely, the method is based on expressing in plant cells and importing intomitochondria a polyribonucleotide that comprises two main elements:

an aminoacylatable tRNA mimic,
and a hammerhead ribozyme able to specifically cleave a target RNA in trans within the mitochondria.

The ribozyme can be attached to the tRNA mimic through a specific linker. The method is based on nuclear transformation and expression of the transgene can be driven by any appropriate promoter, in particular an inducible or tissue-specific promoter. This gives to the strategy a full flexibility. It can be applied to any plant species which is amenable to nuclear transformation. Any target can be selected
in the organelles, as a specific trans-cleaving ribozyme can be designed against virtually any mitochondrial RNA.

DEVELOPMENT STAGE

This novel approach is already fully validated in vitro, in plant cell suspensions and in whole plants. It can be applied to specifically knock down any mitochondrial RNA, as upported by experiments carried out by the inventors in plant cell suspensions and in whole plants with ribozymes designed to trans-cleave further targets in the organelles.

BENEFITS & INDUSTRIAL APPLICATIONS

The present invention is particularly useful for research and applications in the agronomic field and for producing transgenic plants with altered mitochondrial properties. Through controlled manipulation of mitochondrial gene expression, the method has the potential to generate new mitochondrial traits and in particular to provide controlled cytoplasmic male sterility. The method works for both mono- and dicotyledoneae and the genetic transformation is stable in the progeny.