Fluorescence resonance energy transfer (FRET)-based technique for tracking of endophytic bacteria in rice roots

Visualization of endophytes inside the host tissue has remained a prime interest to the plant microbiologist throughout the last decade. Assessment of a root colonization pattern of a nitrogen-fixing polyvalent plant-growth-promoting endophyte, Azotobacter chroococcum Avi2 (16S rRNA gene NCBI acc. no. KP099933), of Oryza sativa var. Swarna, by a plasmid containing green fluorescent protein (GFP) gene failed because the bacteria formed a cyst on exposure to CaCl2 during the transformation process of the green fluorescent protein gene. A new technique based on fluorescence resonance energy transfer (FRET) was developed to visualize the intracellular location of the bacterium of rice root as an alternative to track the bacterium where the use of GFP is restricted. An Al3+-specific rhodamine-based novel fluorescent ligand (lambda(ex)=400 nm, lambda(em)=455 nm), (E)-3', 6'-bis(diethylamino)2-(2-(pyren-4-ylmethyleneamino) ethyl) spiro[isoindoline-1,9' xanthen]-3-one, was synthesized and used to visualize 100 mu M aluminium nitrate treated A. chroococcum inside rice root tissues. Within 6 h of incubation, a clear, stable and distinguishable green fluorescence was observed under the fluorescence microscope form Al3+-treated A. chroococcum colonized in the rice roots when incubated with a ligand. The results confirmed colonization of the bacteria inside the roots.