Use of green fluorescent protein color variants expressed on stable broad-host-range vectors to visualize rhizobia interacting with plants

We developed two sets of broad-host-range vectors that drive expression of the green fluorescent protein (GFP) or color variants thereof (henceforth c ollectively called autofluorescent proteins [AFPs]) from the lac promoter, These two sets are based on different replicons that are maintained in a st able fashion in Escherichia coli and rhizobia. Using specific filter sets o r a dedicated confocal laser scanning microscope setup in which emitted lig ht is split into its color components through a prism, we were able to unam biguously identify bacteria expressing enhanced cyan fluorescent protein (E CFP) or enhanced yellow fluorescent protein (EYFP) in mixtures of the two. Clearly, these vectors will be valuable tools for competition, cohabitation , and rescue studies and will also allow the visualization of interactions between genetically marked bacteria in vivo. Here, we used these vectors to visualize the interaction between rhizobia and plants. Specifically, we fo und that progeny from different rhizobia can be found in the same nodule or even in the same infection thread, We also visualized movements of bactero ids within plant nodule cells.