Extreme specificity of NCR gene expression in Medicago truncatula

Legumes form root nodules to house nitrogen fixing bacteria of the rhizobium family. The rhizobia are located intracellularly in the symbiotic nodule cells. In the legume Medicago truncatula these cells produce high amounts of Nodule-specific Cysteine-Rich (NCR) peptides which induce differentiation of the rhizobia into enlarged, polyploid and non-cultivable bacterial cells. NCRs are similar to innate immunity antimicrobial peptides. The NCR gene family is extremely large in Medicago with about 600 genes. Here we used the Medicago truncatula Gene Expression Atlas (MtGEA) and other published microarray data to analyze the expression of 334 NCR genes in 267 different experimental conditions. We find that all but five of these genes are expressed in nodules but in no other plant organ or in response to any other biotic interaction or abiotic stress tested. During symbiosis, none of the genes are induced by Nod factors. The NCR genes are activated in successive waves during nodule organogenesis, correlated with bacterial infection of the nodule cells and with a specific spatial localization of their transcripts from the apical to the proximal nodule zones. However, NCR expression is not associated with nodule senescence. According to their Shannon entropy, a measure expressing tissue specificity of gene expression, the NCR genes are among the most specifically expressed genes in M. truncatula. Moreover, when activated in nodules, their expression level is among the highest of all genes. Together, these data show that the NCR gene expression is subject to an extreme tight regulation and is only activated during nodule organogenesis in the polyploid symbiotic cells.