Co-localizing symbiont and endophytic bacteria in legumes by tagging with different fluorescent proteins.

Bacterial endophytes can promote plant growth and yield, suppress pathogens, solubilize nutrients or contribute to nitrogen uptake in plants. The use of fluorescent reporters is nowadays a key tool for studying microbe-plant interactions. In a previous work we assessed the presence of different endophytes in addition to rhizobia inside root nodules of wild legume plants and showed that rhizobia share nodules with a variety of different co-infecting taxa (Muresu et al., 2008). In the present report we explore the plant-endophyte relationships attempting the co-localization of endophytes (Pseudomonas sp., Enterobacter agglomerans) and rhizobia (Rhizobium leguminosarum bv. trifolii) by the introduction of different fluorescent proteins as bacterial markers for the different kinds of bacteria. This would help their localization throughout the plant and in particular the distinction of rhizobia from other endophytes coinfecting root nodules. Green fluorescent protein (GFP) and red fluorescent protein (RFP) markers were integrated into the bacterial chromosome. For the marked constructs, a suicide plasmid, carrying the gfp gene in a transposable element was mobilized into the endophytic species. To obtain rfp-tagged bacteria, a replacement of gfp with rfp was made starting from the pRL765gfp plasmid. The use of dual fluorescence markers will allow to co-localize different bacterial taxa within plant tissues and will enable to plan different innovative applications in the field of symbiosis, biocontrol and other endophytic plantmicrobe interactions.