Georges Truchet

Rhizobium meliloti lipooligosaccharide nodulation factors: different structural requirements for bacterial entry into target root hair cells and induction of plant symbiotic developmental responses.

Rhizobium meliloti produces lipochitooligosaccharide nodulation NodRm factors that are required for nodulation of legume hosts. NodRm factors are O-acetylated and N-acylated by specific C16-unsaturated fatty acids. nodL mutants produce non-O-acetylated factors, and nodFE mutants produce factors with modified acyl substituents. Both mutants exhibited a significantly reduced capacity to elicit infection thread (IT) formation in alfalfa. However, once initiated, ITs developed and allowed the formation of nitrogen-fixing nodules. In contrast, double nodF/nodL mutants were unable to penetrate into legume hosts and to form ITs. Nevertheless, these mutants induced widespread cell wall tip growth in trichoblasts and other epidermal cells and were also able to elicit cortical cell activation at a distance. NodRm factor structural requirements are thus clearly more stringent for bacterial entry than for the elicitation of developmental plant responses.

Molecular basis of symbiotic host specificity in rhizobium meliloti: nodH and nodPQ genes encode the sulfation of lipo-oligosaccharide signals

The symbiosis between Rhizobium and legumes is highly specific. For example, R. meliloti elicits the formation of root nodules on alfalfa and not on vetch. We recently reported that R. meliloti nodulation (nod) genes determine the production of acylated and sulfated glucosamine oligosaccharide signals. We now show that the biochemical function of the major host-range genes, nodH and nodPQ, is to specify the 6-O-sulfation of the reducing terminal glucosamine. Purified Nod factors (sulfated or not) from nodH+ or nodH- strains exhibited the same plant specificity in a variety of bioassays (root hair deformations, nodulation, changes in root morphology) as the bacterial cells from which they were purified. These results provide strong evidence that the molecular mechanism by which the nodH and nodPQ genes mediate host specificity is by determining the sulfation of the extracellular Nod signals.

Rhizobium meliloti elicits transient expression of the early nodulin gene ENOD12 in the differentiating root epidermis of transgenic alfalfa.

To study the molecular responses of the host legume during early stages of the symbiotic interaction with Rhizobium, we have cloned and characterized the infection-related early nodulin gene MtENOD12 from Medicago truncatula. In situ hybridization experiments have shown that, within the indeterminate Medicago nodule, transcription of the MtENOD12 gene begins in cell layers of meristematic origin that lie ahead of the infection zone, suggesting that these cells are undergoing preparation for bacterial infection. Histochemical analysis of transgenic alfalfa plants that express an MtENOD12 promoter-beta-glucuronidase gene fusion has confirmed this result and further revealed that MtENOD12 gene transcription occurs as early as 3 to 6 hr following inoculation with R. meliloti in a zone of differentiating root epidermal cells which lies close to the growing root tip. It is likely that this transient, nodulation (nod) gene-dependent activation of the ENOD12 gene also corresponds to the preparation of the plant for bacterial infection. We anticipate that this extremely precocious response to Rhizobium will provide a valuable molecular marker for studying early signal exchange between the two symbiotic organisms.

Alfalfa nodulation in the absence of Rhizobium

SummaryCertain alfalfa plants can develop non-nitrogen fixing structures on their root systems when grown in testtubes under strictly axenic conditions. We demonstrate that these structures possess all the histological features characteristic of indeterminate nodules and that their formation is inhibited by combined nitrogen. The nodule morphogenesis-related gene ENOD2 is expressed in these nodules whereas leghemoglobin transcripts cannot be detected. The capacity to Nodulate in the Absence of Rhizobium (NAR) is maintained during clonal propagation of these alfalfa plants. Our results show that Rhizobium is not absolutely required for nodule morphogenesis and suggest that plant genetic determinants are involved in the NAR phenomenon.

Saprophytic intracellular rhizobia in alfalfa nodules.

In indeterminate alfalfa nodules, the establishment of the senescent zone IV, in which both symbionts undergo simultaneous degeneration, has been considered, until now, as the end point of the symbiotic interaction. However, we now describe an additional zone, zone V, proximal to the senescent zone IV and present in alfalfa nodules more than 6 weeks old. In zone V, a new round of bacterial release occurs from remaining infection threads, leading to the reinvasion of plant cells that have completely senesced. These intracellular rhizobia are rod shaped and do not display the ultrastructural differentiation features of bacteroids observed in the more distal zones of the nodule. Interestingly, we have found that oxygen is available in zone V at a concentration compatible with both bacterial development and nitrogen fixation gene expression in newly released rhizobia. However, this expression is not correlated with acetylene reduction. Moreover, the pattern of nifH expression in this zone, as well as new data relating to expression in zone II, strongly suggest that nifH transcription in the nodule is under the control of a negative regulator in addition to oxygen. Our results support the conclusion that zone V is an ecological niche where intracellular rhizobia take advantage of the interaction for their exclusive benefit and live as parallel saprophytic partners. The demonstration of such an advantage for rhizobia in nodules was the missing evidence that Rhizobium-legume interactions are indeed symbiotic and, in particular, suggests that benefits to the two partners are associated with different developmental stages within the nodule.

TheRhizobium-legume symbiosis Two methods to discriminate between nodules and other root-derived structures

SummaryTwo methods have been developed in order to discriminate between lateral roots, nodules and root-derived structures which exhibit both root and nodule histological features and which can develop on legumes inoculated with certainRhizobium mutants. The first method, known as the “clearing method”, allows the observation by light microscopy of cleared undissected root-structures. The second, known as the “slicing method”, is a complementary technique which provides a greater degree of structural information concerning such structures. The two methods have proved invaluable in defining unequivocally the nature of the interaction between a rhizobial strain and a legume host.

The Rhizobium - legume symbiosis: observation of root infection by bright-field microscopy after staining with methylene blue

Staining of infected legume roots with 0.01% methylene blue facilitated the observation of the initial steps of the Rhizobium—legume symbiosis. It allowed particularly the visualization by bright-field microscopy of infection threads in the root hairs and the root cortex of the host plant.

In Rhizobium meliloti, the operon associated with the nod box n5 comprises nodL, noeA and noeB, three host-range genes specifically required for the nodulation of particular Medicago species

In Rhizobium meliloti, the genes required for nodulation of legume hosts are under the control of DNA regulatory sequences called nod boxes. In this paper, we have characterized three host‐specific nodulation genes, which form a flavonoid‐inducible operon down‐stream of the nod box n5. The first gene of this operon is identical to the nodL gene identified by Baev and Kondorosi (1992) in R. meliloti strain AK631. The product of the second gene, NoeA, presents some homology with a methyl transferase. nodL mutants synthesize Nod factors lacking the O‐acetate substituent. In contrast, in strains carrying a mutation in either noeA or noeB, no modification in Nod‐factor structure or production could be detected. On particular hosts, such as Medicago littoralis, mutants of the n5 operon showed a very weak nodule‐forming ability, associated with a drastic decrease in the number of infection threads, while nodulation of Medicago truncatula or Melilotus alba was not affected. Thus, nodL, noeA and noeB are host‐specific nodulation genes. By using a gain‐of‐function approach, we showed that the presence of nodL, and hence of O‐acetylated Nod factors, is a major prerequisite for confering the ability to nodulate alfalfa upon the heterologous bacterium Rhizobium tropici.

Rhizobium and Legume Nodulation: A Molecular Dialogue

Soil bacteria belonging to the genera Rhizobium, Bradyrhirobium and Azorhizobium, collectively referred to as rhizobia, elicit the formation on legume roots (and stems of some species) of specific organs, the nodules, in which they fix nitrogen. The symbiosis between rhizobia and legumes produce about the same amount of ammonia worldwide as the chemical industry of nitrogen fertilizers and this fixation occurs in nodules. It is therefore important to understand by which mechanisms rhizobia induce nodule formation. These symbiotic associations are specific: every rhizobial strain has a definable host-range (Young, Johnston 1989). The mechanisms by which rhizobia infect legumes are varied. In the Rhizobium meliloti/alfalfa symbiosis, the bacteria induce the formation of marked curls at the tip of root hairs, and then the formation of tubular structures, the infection threads, which grow through the root hairs and the root cortex (see the review of Brewin 1991). At some distance from the advancing infection thread, the induction of cell divisions in the root cortex further leads to the formation of a nodule primordium and a nodule meristem. The functioning of the meristem gives rise to a nodule.

Expression of the Medicago Truncatula ENOD12 Gene in Response to R. Meliloti Nod Factors and during Spontaneous Nodulation in Transgenic Alfalfa

A complex interplay, involving multiple signal exchange between the legume host and its rhizobial partner, is required for the induction and subsequent development of the N2-fixing symbiotic root nodule. In particular, it has been shown that a sulphated lipo-oligosaccharide (NodRm), purified from the supernatant of Rhizobium meliloti (Lerouge et al., 1990), can act as a specific symbiotic signal to elicit root hair deformations and nodule organogenesis on alfalfa (Medicago sativa) plants (Truchet et al.,1991). It is now established that other Rhizobium species produce different specific symbiotic signal molecules, the so-called Nod factors, with a core structure similar to NodRm (Spaink et al., 1991). Rhizobial nodulation (nod) genes are responsible for the synthesis of Nod factors (Denarie, Roche, 1992). A detailed analysis of the host response to these Nod factors requires the identification of plant genes which can serve as molecular markers for the earliest stages of the recognition, infection and nodule organogenetic triggering processes. Recently, Scheres et al. (1990) have reported that transcripts of a pea gene (PsENOD12), which encodes a proline-rich protein, are present in a variety of cell types involved in the early stages of infection.