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

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.

Tissue-specific down-regulation of LjAMT1;1 compromises nodule function and enhances nodulation in Lotus japonicus

“…We followed the procedures described in Handberg and Stougaard (1992) and in Martirani et al (1999) for Agrobacterium tumefaciens and Agrobacterium rhizogenes mediated plant transformations, respectively.…”

A Medicago truncatula Tobacco Retrotransposon Insertion Mutant Collection with Defects in Nodule Development and Symbiotic Nitrogen Fixation

“…T-DNA tagging has been deployed most effectively in the nonlegume Arabidopsis (Arabidopsis thaliana) because this species is easy to transform and propagate in large numbers (hundreds of thousands of plants) in a limited area (Krysan et al, 1999;Alonso et al, 2003). Legumes are larger and more difficult to transform, so T-DNA mutagenesis has found limited application in this family of plants (Schauser et al, 1998;Martirani et al, 1999;Scholte et al, 2002;Brocard et al, 2006;Yano et al, 2009). DNA transposons have also been tested in legumes: the maize (Zea mays) Ac/Dc system in Lotus (Thykjaer et al, 1995;Pajuelo et al, 1996) and soybean (Glycine max; Mathieu et al, 2009); the En/Spm and Tag1 systems in Medicago (d'Erfurth et al, 2003b; and the rice (Oryza sativa) MITE mPing element in soybean (Hancock et al, 2011).…”

A MYB-related transcription factor affects nodule formation in <i>Lotus japonicus</i>

“…The expression pattern of Ljmybr, Enod40 and Nin are similar (Figure 1). Ljmybr-or Enod40-promoter induced GUS expression in the vascular bundles of the nodule ( Figure 2, Martirani et al 1999). Nin is expressed in the nodule primordia and in the central tissue and vascular bundles of mature nodules (Schauser et al 1999).…”