Project

Gene targeting; Physcomitrella patens; DNA repair; moss; mutant analysis; homologous recombination; DNA double strand break

Lead
Lay summary
The possibility to generate precise modifications in a genome by gene targeting provides the ultimate genetic tool for functional studies and account for the success of yeast or mouse as model systems. Yet this methodology is not available in most plant and animal systems in which transgenes essentially integrate at random locations. This situation affects the predictability of genetic modifications and dramatically impedes gene therapeutic approaches. Numerous studies conducted in yeasts and animal cells strongly suggest that the ratio of targeted to random integration observed upon transgenesis reflects the relative activity of to distinct DNA double strand break (DSB) repair pathways in the cell: random integration being associated with the non homologous end-joining (NHEJ) pathway and targeted insertion with the homologous recombination (HR) pathway. The molecular mechanisms of both pathways are well described and highly conserved among eukaryotes, but the factors that control the balance between these two pathways are not fully understood, especially upon transgenesis.The moss Physcomitrella patens is the only plant proficient for gene targeting. The complete sequence of its genome has been established recently and this moss is being more and more recognised as a model "green yeast" in plant biology. A collection of P.patens deletion mutants in distinct DNA repair pathways has been generated ans is being characterised in collaboration with the INRA in Versailles. In this project, we shall combine molecular, cellular, biochemical and genetic experimental approaches to identify among the core genes of the DNA DSB repair pathways of P.patens the ones that are specifically involved in GT. The information acquired in this project will provide insight into the relative function of these genes during DSB repair and upon GT in P.patens. Genes identified as displaying a specific action on GT will be introduced in Arabidopsis to test if their overexpression can improve GT efficiencies in other plants.

Direct link to Lay Summary

Last update: 21.02.2013

Active participation

The possibility to generate precise modifications in a genome by gene targeting provides the ultimate genetic tool for functional studies and account for the success of yeast or mouse as model systems. Yet this methodology is not available in most plant and animal systems in which transgenes essentially integrate at random locations. This situation affects the predictability of genetic modifications and dramatically impedes gene therapeutic approaches. Numerous studies conducted in yeasts and animal cells strongly suggest that the ratio of targeted to random integration observed upon transgenesis reflects the relative activity of to distinct DNA double strand break (DSB) repair pathways in the cell: random integration being associated with the non homologous end-joining (NHEJ) pathway and targeted insertion with the homologous recombination (HR) pathway. The molecular mechanisms of both pathways are well described and highly conserved among eukaryotes, but the factors that control the balance between these two pathways are not fully understood, especially upon transgenesis.The moss Physcomitrella patens is the only plant proficient for gene targeting. The complete sequence of its genome has been established recently and this moss is being more and more recognised as a model “green yeast” in plant biology. In 2005, we have started to generate a collection of P.patens deletion mutants in distinct DNA repair pathways in collaboration with the INRA in Versailles. Characterisation of Msh2 and Rad51A1/2 deletion mutants has been completed. In this project, we shall combine complementary experimental approaches to identify among the core genes of the DNA DSB repair pathways of P.patens the ones that are specifically involved in GT. We shall perform•gene expression studies: genes from the NHEJ and HR pathways are differentially expressed during the cell cycle in yeast and animal cells. We will establish the expression profiles of the corresponding genes during the moss cell cycle and upon transformation•protein interaction studies: the Rad51paralogues form distinct complexes that are clearly involved in HR. P.patens has an incomplete set of these genes: protein interactions among the rad51 paralogues will be analysed to understand their function•in vivo double strand break repair: this assay is essential to correlate GT efficiency with HR-mediated repair. An in vivo DSB repair assay will be established to asses the HR / NHEJ ratio in moss cells•genetic analysis: a detailed analysis of deletion mutants in the NHEJ and HR pathway will be achieved. Half of the deletion mutants studied in his project have already been generated and partially characterised (Lig4, Rad51b, Rad51c, Rad51d, Rad54-1), whereas the others are currently being generated (Xrcc2, Rad54-2, Ku70, Ku80, CtIP). The mutants will be characterised phenotypically and in response to distinct DNA damaging agents (UV, ?-ray and genotoxic chemicals). Cross complementation experiments with Arabidopsis orthologues will be performed to assess the level of functional conservation between moss and Arabidopsis genes •transformation studies: transformation and GT frequencies upon PEG- or Agrobacterium-mediated transformation will be determined in deletion mutantsThe information acquired from these different approaches will provide insight into the relative function of each of these genes during DSB repair and upon GT in P.patens. Genes identified as displaying a specific action on GT will be tested in Arabidopsis to test if their overexpression can improve GT efficiencies in other plants.