Nod factor signaling controlled genes in Medicago truncatula nodules

Legume nodule formation is induced by lipo-chito-oligosaccharides, known as Nod factors (NFs), that are secreted by rhizobium bacteria. NF induced transcriptional changes in roots have been intensively studied. Although it has been shown that NF receptors also accumulate in the apex of Medicago truncatula nodules, the transcriptional changes induced there by NF signaling have never been studied. Here, we studied this by using NF signaling mutant TE7, a weak allele of IPD3, blocked in rhizobial release. Nodule apices were isolated with laser microdissection and used for transcriptional analysis. By this we identified 1655 NF signaling controlled genes. To identify which of these genes depend on rhizobial release, we generated transcriptome data from apices of VAMP721d&e RNAi nodule, in which rhizobial release is blocked by reduced activity of the symbiotic exocytosis pathway. We identified a subset of 445 genes whose regulation of expression depends, directly or indirectly, on NF signaling and rhizobial release. Further, we compared the set of genes controlled by NF signaling in nodule apices with that controlled in root epidermis, and these were markedly different. Among the genes specifically regulated in nodules are genes required for rhizobial differentiation and intracellular accommodation, cell wall organization, and many transcription factors, the expression of some of which depend on rhizobial release. NIN is induced by NF signaling both in the root epidermis and in the nodule. By overexpression of NIN in TE7 and knock down of NIN in wildtype nodules we showed that NF signaling controlled rhizobial release depends on NIN.