Use of T Dna Tagging to Identify Plant Genes Involved in Symbiotic Nitrogen Fixation

Abstract

The legume Lotus japonicus was used in a T-DNA tagging programme to identify plant genes expressed in root nodules. A T-DNA construct containing a promoter-less ß-glucuronidase reporter gene (GUS) was used to detect plant regulatory sequences directing GUS expression in nodules. More than 200 transformed plant lines were screened histochemically for GUS activity. Seven of those had GUS staining in developing and mature nodules. One also had GUS staining in root hairs and root epidermal cells after infection with Rhizobium loti. Using Inverse PCR a T-DNA border fragment was cloned from this line, which contained only one T-DNA copy. Between one and three copies of T-DNA were present in the genomes of the other six GUS positive lines. Nucleotide sequencing of the cloned plant DNA showed no significant homology to sequences in the databases, but it is likely that it contains the regulatory sequences directing the observed GUS expression. This work demonstrates the potential of promoter trapping to identify genes involved in the legume-Rhizobium symbiosis.