The endosymbiosis-induced genes ENOD40 and CCS52a are involved in endoparasitic-nematode interactions in Medicago truncatula.

Plants associate with a wide range of mutualistic and parasitic biotrophic organisms. Here, we investigated whether beneficial plant symbionts and biotrophic pathogens induce distinct or overlapping regulatory pathways in Medicago truncatula. The symbiosis between Sinorhizobium meliloti and this plant results in the formation of nitrogen-fixing root nodules requiring the activation of specific genes in the host plant. We studied expression patterns of nodule-expressed genes after infection with the root-knot nematode Meloidogyne incognita. Two regulators induced during nodule organogenesis, the early nodulin gene ENOD40 involved in primordium formation and the cell cycle gene CCS52a required for cell differentiation and endoreduplication, are expressed in galls of the host plant. Expression analysis of promoter-uidA fusions indicates an accumulation of CCS52a transcripts in giant cells undergoing endoreduplication, while ENOD40 expression is localized in surrounding cell layers. Transgenic plants overexpressing ENOD40 show a significantly higher number of galls. In addition, out of the 192 nodule-expressed genes tested, 38 genes were upregulated in nodules at least threefold compared with control roots, but only two genes, nodulin 26 and cyclin D3, were found to be induced in galls. Taken together, these results suggest that certain events, such as endoreduplication, cell-to-cell communication with vascular tissues, or water transport, might be common between giant cell formation and nodule development.

Differentiation of symbiotic cells and endosymbionts in Medicago truncatula nodulation are coupled to two transcriptome-switches.

The legume plant Medicago truncatula establishes a symbiosis with the nitrogen-fixing bacterium Sinorhizobium meliloti which takes place in root nodules. The formation of nodules employs a complex developmental program involving organogenesis, specific cellular differentiation of the host cells and the endosymbiotic bacteria, called bacteroids, as well as the specific activation of a large number of plant genes. By using a collection of plant and bacterial mutants inducing non-functional, Fix(-) nodules, we studied the differentiation processes of the symbiotic partners together with the nodule transcriptome, with the aim of unravelling links between cell differentiation and transcriptome activation. Two waves of transcriptional reprogramming involving the repression and the massive induction of hundreds of genes were observed during wild-type nodule formation. The dominant features of this "nodule-specific transcriptome" were the repression of plant defense-related genes, the transient activation of cell cycle and protein synthesis genes at the early stage of nodule development and the activation of the secretory pathway along with a large number of transmembrane and secretory proteins or peptides throughout organogenesis. The fifteen plant and bacterial mutants that were analyzed fell into four major categories. Members of the first category of mutants formed non-functional nodules although they had differentiated nodule cells and bacteroids. This group passed the two transcriptome switch-points similarly to the wild type. The second category, which formed nodules in which the plant cells were differentiated and infected but the bacteroids did not differentiate, passed the first transcriptome switch but not the second one. Nodules in the third category contained infection threads but were devoid of differentiated symbiotic cells and displayed a root-like transcriptome. Nodules in the fourth category were free of bacteria, devoid of differentiated symbiotic cells and also displayed a root-like transcriptome. A correlation thus exists between the differentiation of symbiotic nodule cells and the first wave of nodule specific gene activation and between differentiation of rhizobia to bacteroids and the second transcriptome wave in nodules. The differentiation of symbiotic cells and of bacteroids may therefore constitute signals for the execution of these transcriptome-switches.

A novel family in Medicago truncatula consisting of more than 300 nodule-specific genes coding for small, secreted polypeptides with conserved cysteine motifs.

Transcriptome analysis of Medicago truncatula nodules has led to the discovery of a gene family named NCR (nodule-specific cysteine rich) with more than 300 members. The encoded polypeptides were short (60-90 amino acids), carried a conserved signal peptide, and, except for a conserved cysteine motif, displayed otherwise extensive sequence divergence. Family members were found in pea (Pisum sativum), broad bean (Vicia faba), white clover (Trifolium repens), and Galega orientalis but not in other plants, including other legumes, suggesting that the family might be specific for galegoid legumes forming indeterminate nodules. Gene expression of all family members was restricted to nodules except for two, also expressed in mycorrhizal roots. NCR genes exhibited distinct temporal and spatial expression patterns in nodules and, thus, were coupled to different stages of development. The signal peptide targeted the polypeptides in the secretory pathway, as shown by green fluorescent protein fusions expressed in onion (Allium cepa) epidermal cells. Coregulation of certain NCR genes with genes coding for a potentially secreted calmodulin-like protein and for a signal peptide peptidase suggests a concerted action in nodule development. Potential functions of the NCR polypeptides in cell-to-cell signaling and creation of a defense system are discussed.

The Medicago species A2-type cyclin is auxin regulated and involved in meristem formation but dispensable for endoreduplication-associated developmental programs.

Phytohormones as well as temporal and spatial regulation of the cell cycle play a key role in plant development. Here, we investigated the function and regulation of an alfalfa (Medicago sativa) A2-type cyclin in three distinct root developmental programs: in primary and secondary root development, nodule development, and nematode-elicited gall formation. Using transgenic plants carrying the Medsa;cycA2;2 promoter-beta-glucuronidase gene fusion, in combination with other techniques, cycA2;2 expression was localized in meristems and proliferating cells in the lateral root and nodule primordia. Rapid induction of cycA2;2 by Nod factors demonstrated that this gene is implicated in cell cycle activation of differentiated cells developing to nodule primordia. Surprisingly, cycA2;2 was repressed in the endoreduplicating, division-arrested cells both during nodule development and formation of giant cells in nematode-induced galls, indicating that CycA2;2 was dispensable for S-phase in endoreduplication cycles. Overexpression of cycA2;2 in transgenic plants corresponded to wild type protein levels and had no apparent phenotype. In contrast, antisense expression of cycA2;2 halted regeneration of somatic embryos, suggesting a role for CycA2;2 in the formation or activity of apical meristems. Expression of cycA2;2 was up-regulated by auxins, as expected from the presence of auxin response elements in the promoter. Moreover, auxin also affected the spatial expression pattern of this cyclin by shifting the cycA2;2 expression from the phloem to the xylem poles.