Use of dual marker transposons to identify new symbiosis genes in Rhizobium

Rhizobium etli elicits nitrogen-fixing nodules on the roots of Phaseolus vu lgaris. Using a composite dual-marker mini-Tn5 transposon carrying combinat ions of a constitutively expressed gfp gene and a promoterless gusA gene, w e identified novel genes required for an efficient symbiosis. The induction of the gusA gene was used to determine the expression level of the differe nt target genes under conditions partly mimicking the symbiotic environment ex planta. The green fluorescence was used to localize the bacteria in inf ection threads or inside the plant cells. hmong the identified R. etli muta nts, several produced a Nod(-) phenotype, whereas others were Fix(-) or dis played a reduced acetylene reduction activity during symbiosis. Partial seq uence analysis of the mutated genes allowed us to classify them as nodulati on genes, nitrogen fixation genes, genes possessing various enzymatic funct ions previously not yet associated with symbiosis, and genes displaying no similarity to any other sequence in the database. This methodology can be u sed to screen large numbers of mutants in the search for novel genes import ant for Rhizobium-legume symbiosis, and may be adapted to study other plant -bacterium interactions.