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EARLY EVENTS IN NODULATION OF CASUARINA GLAUCA BY FRANKIA

Laplaze L1, Svistoonoff S1, Obertello M1, Peret B1, Auguy F1, Sy MO1,

Hocher V1, Autran D1, Nicole M2, Franche C1, Bogusz D1

1UMR 1098, and 2UMR 1097, Institut de Recherche pour le Développement, 911 Avenue Agropolis, 34394 Montpellier Cedex 5, France. http://www.mpl.ird.fr/rhizo

Two groups of plants can enter nitrogen-fixing root nodule symbioses with soil bacteria: legumes associate with rhizobia, whereas the so-called actinorhizal plants belonging to eight angiosperm families interact with the actinomycete Frankia. Recent molecular phylogeny studies (Soltis et al., 1995) indicate that those plants belong to a same clade. This suggests that a predisposition to form nitrogen-fixing root nodule symbioses originated once in the history of flowering plants. Our group is working on Casuarina

glauca/Frankia interaction as a model system to study the molecular mechanisms of plant infection by Frankia.

1. cg12 is specifically expressed during plant cell infection by Frankia

The cg12 gene isolated from C. glauca encodes a susbtilisin-like protease, or subtilase, specifically expressed in plant cells infected by Frankia (Laplaze et al., 2000). Subtilases are a superfamily of proteases widely distributed in diverse organisms including bacteria, fungi and higher eucaryotes (Siezen and Leunissen, 1997). The precise function of plant subtilases is only known for two Arabidopsis genes, Ale1 and Sdd1, which are involved in epidermal surface formation and stomatal distribution respectively (Tanaka et al., 2001; Berger and Altman, 2000). Different functions have been proposed for other plant subtilases including involvement in programmed cell death, cell wall loosening during lateral root development, fruit ripening and response to pathogen attacks. cg12 is one of the earliest actinorhizal gene induced after Frankia

infection. Using transgenic Casuarinaceae containing the cg12 promoter fused to a reporter gene, we showed that (a) cg12 expression is specifically linked to the infection of root hairs and cortical cells by Frankia and (b) is not induced in endo- or ectomycorrhizae or by Frankia root hair deforming factors (Svistoonoff et al., 2003). This suggests that CG12 plays an important role during plant cell infection by Frankia.

Infection-related activation of the cg12 promoter is conserved between actinorhizal and legume-rhizobia root nodule symbiosis

Y-P. Wang et al, (eds.), Biological Nitrogen Fixation, Sustainable Agriculture and the Environment, 205-206.© 2005 Springer. Printed in the Netherlands.

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Transcriptional fusions between the cg12 promoter and gus or gfp reporter genes were introduced in the model legume Medicago truncatula. We showed that the cg12

promoter directs reporter gene expression specifically in plant cells infected or about to be infected by Sinorhizobium meliloti, but not in mycorrhized roots (Svistoonoff et al., 2004). Using purified Nod factors and bacterial mutants, we also demonstrated that Nod factors are necessary but not sufficient for cg12 promoter activation (Svistoonoff et al., 2004). This indicates that at least part of the transcriptional environment in plant cells infected by endosymbiotic nitrogen-fixing bacteria is conserved between legume and actinorhizal plants.

2. CG12 is localized at the interface between the plant cell and Frankia

In order to cytolocalize CG12 during root invasion by Frankia, antibodies were raised against two synthetic peptides corresponding to putative antigenic sites in the predicted protein. In western-blot experiments, a 95-kDa protein was recognized in nodules but not in uninfected roots or aerial part extracts. CG12 immunolabeling was found in infected cells in Frankia filaments, in the matrix surrounding Frankia infection-threads and vegetative hyphae. A strong labeling was also found in the cell wall of infected cells (Svistoonoff et al., unpublished).

Taken together, our results suggest that CG12 is a subtilase involved either in (a) cell wall remodeling to allow Frankia penetration or (b) signal exchanges between the plant cell and the bacteria.

3. Future prospects cg12 promoter activation in response to bacterial infection is conserved in the model legume M. truncatula. We will use genetics in this model plant in order to identify genes involved in the signaling cascade leading to cg12 expression. Moreover, we are currently setting up a hairy-root based RNAi system for C. glauca. This system will enable us to silence cg12 expression and analyze the effects of the absence of this gene on the infection phenotype.

4. References Laplaze L et al. (2000) Mol. Plant Microbe Interact. 13, 113-117. Soltis DE et al. (1995) Proc. Natl. Acad. Sci. USA 92, 2647-2651. Svistoonoff S et al. (2003) Mol. Plant Microbe Interact. 16, 600-607. Svistoonoff S et al. (2004) Plant Physiol. 136, 3191-3197. Siezen RJ and Leunissen JAM (1997) Protein Sci. 6, 501-523. Tanaka H et al. (2001) Development 128, 4681-4689. Berger D and Altmann T (2000) Genes Dev. 14, 1119-1131.