Medicago truncatula, belonging to the inverted repeat-lacking clade (IRLC) of legumes is able to establish one of the most effective nitrogen-fixing symbiotic interaction with the symbiotic partner Sinorhizobium spp. In the infected cells of the M. truncatula nodules, rhizobial genome undergo morphological and metabolic transformation. This differentiation is irreversible in species belonging to the IRLC legumes and this process is driven by the family of the nodule-specific cysteine rich (NCR) peptides produced by the plant. In the M. truncatula genome, approximately 700 genes encode NCR peptides. The high number of genes can imply that the function of NCR peptides may overlap or partially overlap and the NCRs act redundantly. Recent studies revealed that certain NCR peptides, NCR169 and NCR211 are essential for terminal bacteroid differentiation and hence for effective symbiotic nitrogen fixation. Others NCRs were also identified regulating the nitrogen-fixing symbiotic interaction in a strain-specific manner in M. truncatula. In the case of incompatible interaction, NCR peptides induce lysis of rhizobia and nodule senescence. In order to further dissect the bacteroid differentiation regulated by essential NCRs, we analysed the bacterial morphology and the degree of the bacteroid elongation in four M. truncatula mutant lines defective in production of NCR genes. To study the structural alterations in bacterial colonization, elongation and membrane development, mutant nodules were analysed by laser scanning confocal microscopy and scanning electron microscopy. To assess the progress of the bacteroid differentiation, we measured the length of bacteria in the mutant lines and wild type plant. The process of bacterial elongation and differentiation process is accompanied by successive waves of expression changes of several plant symbiotic genes. We monitored the activity of NCR and other symbiotic genes in the three ncr mutant lines using qRT-PCR to reveal differences in the expression pattern of these genes and define what stage of the bacterial differentiation is blocked in the mutant lines.