Regulatory Patterns of a Large Family of Defensin-Like Genes Expressed in Nodules of Medicago truncatula

Abstract

Root nodules are the symbiotic organ of legumes that house nitrogen-fixing bacteria. Many genes are specifically induced in nodules during the interactions between the host plant and symbiotic rhizobia. Information regarding the regulation of expression for most of these genes is lacking. One of the largest gene families expressed in the nodules of the model legume Medicago truncatula is the nodule cysteine-rich (NCR) group of defensin-like (DEFL) genes. We used a custom Affymetrix microarray to catalog the expression changes of 566 NCRs at different stages of nodule development. Additionally, bacterial mutants were used to understand the importance of the rhizobial partners in induction of NCRs. Expression of early NCRs was detected during the initial infection of rhizobia in nodules and expression continued as nodules became mature. Late NCRs were induced concomitantly with bacteroid development in the nodules. The induction of early and late NCRs was correlated with the number and morphology of rhizobia in the nodule. Conserved 41 to 50 bp motifs identified in the upstream 1,000 bp promoter regions of NCRs were required for promoter activity. These cis-element motifs were found to be unique to the NCR family among all annotated genes in the M. truncatula genome, although they contain sub-regions with clear similarity to known regulatory motifs involved in nodule-specific expression and temporal gene regulation.

An antimicrobial peptide essential for bacterial survival in the nitrogen-fixing symbiosis

“…The lack of symbiotic mutants in this gene family to date has been attributed to gene redundancy (16). Here we report that removing a single NCR peptide (NCR211) leads to defects in nitrogen fixation.…”

“…Microarray analysis of NCR genes during nodule development allowed us to search for the expression pattern of both DNF4 (NCR211) and NCR178 on Rm1021 inoculation (16). As shown in Fig.…”

Host plant peptides elicit a transcriptional response to control the Sinorhizobium meliloti cell cycle during symbiosis

“…Nodules of Medicago plants express hundreds of NCR peptides that vary in length, charge, number of cysteine residues, and spatiotemporal expression (4,5,7,8,48). It has been suggested that this chemical and structural diversity impacts peptide activity, as found in other families of small disulfide-linked peptides that exhibit rich chemical and structural diversity (4,49).…”

“…Indeed, experiments on modified versions of the NCR247 peptide indicated that the presence and order of disulfide bonds between cysteine residues significantly influences peptide antimicrobial activity (50). It has been proposed that NCR peptides (i) alter the S. meliloti cell cycle and metabolism to achieve a more efficient symbiosis (4), (ii) sanction non-nitrogen-fixing rhizobia (4), (iii) kill senescent bacteroids so that their nutrients can be absorbed by the plant (4,48), and (iv) serve as signals to the plant (48). In addition, the cytotoxicity of NCR peptides could help ensure that other bacterial species are unable to survive inside the nodule (12).…”

The anti-chlamydial activity of defensin-like proteins

“…This group includes major plant immunity effectors such as defensins, and also symbiotic peptides, including the nodule-specific cysteine rich (NCR) peptides, which are produced in Medicago Sinorhizobium meliloti symbiosis and provoke irreversible differentiation of the endosymbiont. The NCR family is composed of about 500 divergent peptides in Medicago truncatula [9][10][11]. Some cationic NCRs have been shown to possess genuine antimicrobial activities in vitro, killing various Gram-negative and Gram-positive bacteria highly efficiently [12].…”