Characterization and genome-wide expression analysis of Medicago truncatula N5-overexpressing and N5-silencing plants at early nodulation phase.

MtN5 is a non-specific lipid transfer protein expressed at a very early phase of the rhizobial symbiosis in epidermal cells, root hairs and root nodules. Using composite plants overexpressing and silencing MtN5 in the roots, it was shown that this protein positively regulates the nodulation process (Pii et al., 2009, dpi:10.1094/MPMI-22-12-1577; Pii et al., 2012, www.biomedcentral.com/1471-2229/12/23). To get more information about MtN5 function during the symbiosis, we also overexpressed and silenced N5 gene via Agrobacterium tumefaciens stable genetic transformation. Microscopic observations of the roots inoculated with Sinorhizobium meliloti, revealed a significantly higher number of root hair curling events in both N5-overexpressing and N5-silenced plants compared to wild type plants. Although N5-overexpressing and N5-silenced plants displayed high responsiveness during the epidermal phase of symbiosis, N5-silenced plants were impaired in nodulation (40% reduction in the number of both primordia and mature nodules compared with wild type), whereas N5-overexpressing developed 34% more primordia and nodules with respect to wild type plants. To get deeper insight about the function of MtN5, we have undertaken a transcriptome analysis comparing wild type, silenced and overexpressing plants at 72 hours post inoculation. We obtained approximately 3,000 differentially expressed transcripts as a consequence of inoculation of wild type roots, while the inoculation of N5-overexpressing and N5-silenced plants resulted in about 1,400 and 180 differentially expressed genes, respectively, as compared with inoculated wild type. Taking into account the differentially expressed genes with a fold change ≥ 2.0, we detected 413 up-regulated and 579 down-regulated transcripts in N5-overexpressing inoculated roots, and 78 up-regulated and 14 down-regulated mRNAs in N5-silenced inoculated roots. Detailed transcripts analysis is still ongoing. Evidence from stably transformed plants confirms a crucial role for MtN5 in controlling bacterial invasion and nodulation in M. truncatula.