The Nod factor-independent symbiotic signaling pathway: development of Agrobacterium rhizogenes-mediated transformation for the legume Aeschynomene indica.

The nitrogen-fixing symbiosis between Aeschynomene indica and photosynthetic bradyrhizobia is the only legume-rhizobium association described to date that does not require lipochito-oligosaccharide Nod factors (NF). To assist in deciphering the molecular basis of this NF-independent interaction, we have developed a protocol for Agrobacterium rhizogenes-mediated transformation of A. indica. The cotransformation frequency (79%), the nodulation efficiency of transgenic roots (90%), and the expression pattern of the 35S Cauliflower mosaic virus promoter in transgenic nodules were all comparable to those obtained for model legumes. We have made use of this tool to monitor the heterologous spatio-temporal expression of the pMtENOD11-β-glucuronidase fusion, a widely used molecular reporter for rhizobial infection and nodulation in both legumes and actinorhizal plants. While MtENOD11 promoter activation was not observed in A. indica roots prior to nodulation, strong reporter-gene expression was observed in the invaded cells of young nodules and in the cell layers bordering the central zone of older nodules. We conclude that pMtENOD11 expression can be used as an infection-related marker in A. indica and that Agrobacterium rhizogenes-mediated root transformation of Aeschynomene spp. will be an invaluable tool for determining the molecular basis of the NF-independent symbiosis.