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Use of Green Fluorescent Protein Color Variants Expressed on Stable Broad-Host-Range Vectors to Visualize Rhizobia Interacting with Plants

November 2000 , Volume 13 , Number  11
Pages  1,163 - 1,169

Nico Stuurman , Cristina Pacios Bras , Helmi R. M. Schlaman , André H. M. Wijfjes , Guido Bloemberg , and Herman P. Spaink

Leiden University, Institute of Molecular Plant Sciences, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands

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Accepted 20 July 2000.

We developed two sets of broad-host-range vectors that drive expression of the green fluorescent protein (GFP) or color variants thereof (henceforth collectively called autofluorescent proteins [AFPs]) from the lac promoter. These two sets are based on different replicons that are maintained in a stable fashion in Escherichia coli and rhizobia. Using specific filter sets or a dedicated confocal laser scanning microscope setup in which emitted light is split into its color components through a prism, we were able to unambiguously identify bacteria expressing enhanced cyan fluorescent protein (ECFP) 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 found that progeny from different rhizobia can be found in the same nodule or even in the same infection thread. We also visualized movements of bacteroids within plant nodule cells.

© 2000 The American Phytopathological Society