Visualization of bioluminescence as a marker of gene expression in rhizobium-infected soybean root nodules

Abstract

The linked structural genes lux A and lux B, encoding bacterial luciferase of a marine bacterium Vibrio harveyi, were fused with the nitrogenase nifD promoter from Bradyrhizobium japonicum and with the P1 promoter of pBR322. Both fusions were integrated into the B. japonicum chromosome by site-specific recombination. Soybean roots infected with the two types of rhizobium transconjugants formed nitrogen-fixing nodules that produced bright blue-green light. Cells containing the P1 promoter/lux AB fusion resulted in continuously expressed bioluminescence in both free-living rhizobium and in nodule bacteriods. However, when under control of the nifD promoter, luciferase activity was found only in introgen-fixing nodules. Light emission from bacteroids allowed us to visualize and to photograph nodules expressing this marker gene fusion in vivo at various levels of resolution, including within single, living plant cells. Localization of host cells containing nitrogen-fixing bacteroids within nodule tissue was accomplished using low-light video microscopy aided by realtime image processing techniques developed specifically to enhance extreme low-level luminescent images.

Bacterial luciferase alpha beta fusion protein is fully active as a monomer and highly sensitive in vivo to elevated temperature.

“…The presence of the mutation(s) was confirmed by DNA sequencing using the dideoxynucleotide chain-termination method (13 (16). The wavelength of maximum emission was determined from the first derivative of the emission spectrum.…”

Imaging of light emission from the expression of luciferases in living cells and organisms: a review

“…LuxAB fusion expression in transformed B. japonicuminfected soybean root nodules was successfully visualized using photographic film (Fig. 9a, b) and low-light intensified video microscopy (211). Using N. tabacum transformed with an auxin-stimulated bidirectional mas promoter-luxAB gene fusion construct, it was possible to visualize the spread of Pseudomonas syringiae infection in N. tabacum leaves (Fig.…”

Mode d’action des rhizobactéries favorisant la croissance des plantes et potentiel de leur utilisation comme agent de lutte biologique

“…Les colonies de bactéries biolurninescentes peuvent être observées de diverses façons: visuellement dans une chambre noire, à l'aide de pellicule photographique (Beauchamp et al 1991(Beauchamp et al , 1993a(Beauchamp et al , 1993bGrant et al 1991;Legocki et al 1986;O'Kane et al 1988) et à rayons X (de Wegger et al 1991;Shaw et al 1987) ou à l'aide d'une caméra spécialisée pour détecter des quantités faibles de photons (caméra CCD) (Beauchamp et al 1993b;Mahaffee et al 1991;Shaw et al 1992). Des comptages photoniques peuvent 22 aussi être obtenus à l'aide d'un compteur à scintillation (Boivin et al 1988;Fravel ef al.…”