T-DNA Tagging of Nodulation- and Root-Related Genes in Lotus japonicus: Expression Patterns and Potential for Promoter Trapping and Insertional Mutagenesis

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Abstract
High-efficiency transformation of the autogamous diploid legume Lotus japonicus by means of Agrobacterium rhizogenes was used to develop plant lines expressing a promoter-less gusA gene in a nodulation- or lateral root-associated manner. The approach exploits the putatively preferential integration of T-DNA into actively transcribed regions, thereby providing an enrichment for gene tagging events associated with the quickly assayable activation of a gusA promoter-less construct. Taking advantage of this enrichment and selection strategy, a T-DNA tagging program was initiated and screening for β-glucuronidase (GUS) activity was performed on root clones isolated after transformation with a gusA-promoter-less binary vector. The aim of this approach is the identification of genes involved in nodule formation induced by Mesorhizobium loti, lateral root organogenesis, and the eventual isolation of corresponding mutants. A large collection (220) of GUS-positive transformants showing a variety of expression patterns in different regions of roots and nodules was obtained; a preliminary molecular characterization of these plants is presented.