The Medicago truncatula N5 gene encoding a root-specific lipid transfer protein is required for the symbiotic interaction with Sinorhizobium meliloti

The Medicago truncatula N5 gene is induced in roots after Sinorhizobium meliloti infection and it codes for a putative Lipid Transfer Protein (LTP), a family of plant small proteins capable of binding and transferring lipids between membranes in vitro. Various biological roles for plant LTP in vivo has been proposed including defence against pathogens and modulation of plant development. The aim of this study was to shed light on the role of MtN5 in the symbiotic interaction between M. truncatula and S. meliloti. MtN5 cDNA was cloned and the mature MtN5 protein expressed in E. coli. The lipid binding capacity and antimicrobial activity of the recombinant MtN5 protein were tested in vitro. MtN5 showed the capacity to bind lysophospholipids and to inhibit M. truncatula pathogens and symbiont growth in vitro. Furthermore, MtN5 was up-regulated in roots after infection with either the fungal pathogen Fusarium semitectum or the symbiont S. meliloti. Upon S. meliloti infection MtN5 was induced starting from 1 day after inoculation (dpi). It reached the highest concentration at 3 dpi and it was localized in the mature nodules. MtN5-silenced roots resulted impaired in nodulation, showing a 50% of reduction in the number of nodules as compared to control roots. On the other hand, transgenic roots over-expressing MtN5 developed 3-fold more nodules with respect to control roots. Here we demonstrate that MtN5 possesses biochemical features typical of LTPs and that it is required for the successful symbiotic association between M. truncatula and S. meliloti.