T-DNA tagging in Medicago truncatula

Symbiotic interaction between rhizobia and leguminous plants leads to the formation of N 2 -fixing root nodules. This interaction shows a high degree of host specificity based on the exchange of chemical signals between the symbiotic partners. Although much is known about the bacterial genes required for the establishment of this interaction, less is known about the plant genes involved.Symbiotic plant mutants serve as a tool to identify genes involved in nodulation and to study their function. However, the isolation of the affected gene(s) found in natural populations or induced by 'classical' methods like EMS treatment or irradiation is difficult, and so far only few of such genetically identified genes resulting in a mutant phenotype have been isolated. Problems to isolate a mutated gene can be overcome by the use of T-DNA as a mutagen, because the known nucleotide sequence of the T-DNA can serve as a starting point for the identification of the flanking sequences. T-DNA tagging not only facilitates the cloning of new genes, but at the same time provides insight in the function of the identified genes. In addition, when the T-DNA tag contains a promoter-less reporter gene transcriptional and translational gene fusions may provide new marker genes for the different stages of nodule development.The aim of the thesis work was to assess the possibilities of T-DNA tagging as a tool to discover M. truncatula genes involved in symbiosis and to study their function.For this, a T-DNA insertion mutagenized M. truncatula population was produced and screened for GUS staining patterns and mutant phenotypes. Nineteen out of 187 lines showed GUS staining in the roots or the nodules and one line showed, in addition to the GUS staining, a dwarf phenotype.The isolation and characterization of the T-DNA flanking regions of a selection of the GUS expressing lines showed that in two lines the T-DNAs had inserted in the ORF of a Narf -like gene (causing the dwarf phenotype in homozygous knock-outs) and in the ORF of a MtN3 -like gene (probably causing letality), respectively.T-DNA tagging thus can be used as a tool for the discovery of genes or the production of new markers in M.truncatula