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Dernière mise à jour : Mai 2018

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Institut Sophia Agrobiotech

UMR INRA - Univ. Nice Sophia Antipolis - Cnrs

http://www.paca.inra.fr/institut-sophia-agrobiotech_eng/

Development of new cellular tools to analyze the symbiotic nitrogen fixing interaction in vivo (Symbiose Team)

The team focus on improving our knowledge of plant/nitrogen-fixing bacteria (Rhizobium) by studying the role of the cellular redox state during the symbiotic interaction or nodule senescence.

In order to better understand the nitrogen fixing symbiosis in Fabaceae (Fig. 1), we have developed new cellular tools. In this context, multiple probes analyzed using confocal microscope* imaging were developed and used in nitrogen fixing nodules. PBS pH was measured in vitro during the whole symbiotic process using ratiometric fluorescent probe* Lysosensor Yellow/Blue DND160. This analysis showed that nitrogen fixing zone maturation goes with the acidification of the peribacteroid space in the nitrogen-fixing organite, the symbiosomesa (Fig. 2). The viability of bacteroids was analyzed during their differentiation and throughout the symbiotic interaction in vivo using nodule sections with the Live/Dead® BacLightTM probe.

Finally, the in vivo quantification of H2O2 was measured in nodule using a protein probe HyPer b. This analysis correlates the production of H2O2 with the regulation of MtSpk1, an H2O2 regulated gene. 

Self explicit" Sequential steps of symbiosis between M. truncatula and S. meliloti from plant cell infection to senescence.

Fig 1: Self explicit" Sequential steps of symbiosis between M. truncatula and S. meliloti from plant cell infection to senescence.

A- Nodulated Medicago truncatula plant; B- Slice of functional nodule. Scale: 500 µm; C- Activation of CP6 gene at the interface between symbiosis and senescence (Red = Gus detection); D- Bacteroid of S. meliloti (Syto® 9 fluorecence); E- Detection of low pH in peribacteroid space (green: LysoTracker®) and bacteroid (red:Syto® 9).

Fig 2: A- Nodulated Medicago truncatula plant; B- Slice of functional nodule. Scale: 500 µm; C- Activation of CP6 gene at the interface between symbiosis and senescence (Red = Gus detection); D- Bacteroid of S. meliloti (Syto® 9 fluorecence); E- Detection of low pH in peribacteroid space (green: LysoTracker®) and bacteroid (red:Syto® 9).

*Confocal microscopy is an optical imaging technique used to increase optical resolution and contrast of a micrograph by using point illumination and a spatial pinhole to eliminate out-of-focus light in specimens that are thicker than the focal plane.

* Ratiometric fluorescent probe: Images are analyzed by measuring fluorescence intensity changes for two wavelengths expressed as a ratio

  • a) Pierre, O., Engler, G., Hopkins, J., Brau, F., Boncompagni, E., and Hérouart, D. (2013). Peribacteriod space acidification: a marker of mature bacteroid functioning in Medicago truncatula nodules. Plant Cell Environ. 36(11):2059-70
  • b) Andrio E, Marino D, Marmeys A, Dunoyer de Segonzac M, Damiani I, Genre A, Huguet S, Frendo P, Puppo A, Pauly N. (2013) Hydrogen peroxide-regulated genes in the Medicago truncatula – Sinorhizobium meliloti symbiosis. New Phytologist, 198, 179-89