Charles Rosenberg

Four genes of Medicago truncatula controlling components of a Nod factor transduction pathway.

Rhizobium nodulation (Nod) factors are lipo-chitooligosaccharides that act as symbiotic signals, eliciting several key developmental responses in the roots of legume hosts. Using nodulation-defective mutants of Medicago truncatula, we have started to dissect the genetic control of Nod factor transduction. Mutants in four genes (DMI1, DMI2, DMI3, and NSP) were pleiotropically affected in Nod factor responses, indicating that these genes are required for a Nod factor–activated signal transduction pathway that leads to symbiotic responses such as root hair deformations, expressions of nodulin genes, and cortical cell divisions. Mutant analysis also provides evidence that Nod factors have a dual effect on the growth of root hair: inhibition of endogenous (plant) tip growth, and elicitation of a novel tip growth dependent on (bacterial) Nod factors. dmi1, dmi2, and dmi3 mutants are also unable to establish a symbiotic association with endomycorrhizal fungi, indicating that there are at least three common steps to nodulation and endomycorrhization in M. truncatula and providing further evidence for a common signaling pathway between nodulation and mycorrhization.

Genes controlling early and late functions in symbiosis are located on a megaplasmid in Rhizobium meliloti

SummaryLarge plasmids of molecular weight varying from 90 to around 200×106 have earlier been detected in most Rhizobium meliloti strains using an alkaline denaturation-phenol extraction procedure. With a less destructive method (Eckhardt 1978) it was possible additionally to detect one plasmid of molecular weight clearly greater than 300×106 (=megaplasmid) in all of twenty-seven R. meliloti strains of various geographical origins and nodulation groupings investigated. Four strains (RCR 2011, A145, S26 and CC2013) were found to carry one megaplasmid and no smaller plasmids. Hybridization experiments with Klebsiella pneumoniae and R. meliloti cloned nitrogenase structural genes D and H showed that these genes are located on the megaplasmid and not on the smaller plasmids.All of the ten independent spontaneous non-nodulating derivatives of three strains of R. meliloti were shown to have suffered a deletion in the nif DH region of the megaplasmid. These results indicate that a gene controlling an early step in nodule formation is located in the nif DH region of the megaplasmid. This indicates that the same replicon carries genes controlling early and late functions in symbiosis.

Rhizobium meliloti Genes Encoding Catabolism of Trigonelline Are Induced under Symbiotic Conditions.

Rhizobium meliloti trc genes controlling the catabolism of trigonelline, a plant secondary metabolite often abundant in legumes, are closely linked to nif-nod genes on the symbiotic megaplasmid pSym [Boivin, C., Malpica, C., Rosenberg, C., Denarie, J., Goldman, A., Fleury, V., Maille, M., Message, B., and Tepfer, D. (1989). In Molecular Signals in the Microbe-Plant Symbiotic and Pathogenic Systems. (Berlin: Springer-Verlag), pp. 401-407]. To investigate the role of trigonelline catabolism in the Rhizobium-legume interaction, we studied the regulation of trc gene expression in free-living and in endosymbiotic bacteria using Escherichia coli lacZ as a reporter gene. Experiments performed with free-living bacteria indicated that trc genes were organized in at least four transcription units and that the substrate trigonelline was a specific inducer for three of them. Noninducing trigonelline-related compounds such as betaines appeared to antagonize the inducing effect of trigonelline. None of the general or symbiotic regulatory genes ntrA, dctB/D, or nodD seemed to be involved in trigonelline catabolism. trc fusions exhibiting a low basal and a high induced [beta]-galactosidase activity when present on pSym were used to monitor trc gene expression in alfalfa tissue under symbiotic conditions. Results showed that trc genes are induced during all the symbiotic steps, i.e., in the rhizosphere, infection threads, and bacteroids of alfalfa, suggesting that trigonelline is a nutrient source throughout the Rhizobium-legume association.

Molecular basis of lipo-chitooligosaccharide recognition by the lysin motif receptor-like kinase LYR3 in legumes.

LYR3 [LysM (lysin motif) receptor-like kinase 3] of Medicago truncatula is a high-affinity binding protein for symbiotic LCO (lipo-chitooligosaccharide) signals, produced by rhizobia bacteria and arbuscular mycorrhizal fungi. The present study shows that LYR3 from several other legumes, but not from two Lupinus species which are incapable of forming the mycorrhizal symbiosis, bind LCOs with high affinity and discriminate them from COs (chitooligosaccharides). The biodiversity of these proteins and the lack of binding to the Lupinus proteins were used to identify features required for high-affinity LCO binding. Swapping experiments between each of the three LysMs of the extracellular domain of the M. truncatula and Lupinus angustifolius LYR3 proteins revealed the crucial role of the third LysM in LCO binding. Site-directed mutagenesis identified a tyrosine residue, highly conserved in all LYR3 LCO-binding proteins, which is essential for high-affinity binding. Molecular modelling suggests that it may be part of a hydrophobic tunnel able to accommodate the LCO acyl chain. The lack of conservation of these features in the binding site of plant LysM proteins binding COs provides a mechanistic explanation of how LCO recognition might differ from CO perception by structurally related LysM receptors.

Metabolic Signals in the Rhizosphere: Catabolism of Calystegins and Trigonelline by Rhizobium Meliloti

Bacteria that associate with plants are generally saprophitic. They compete with other heterotrophic organisms for the products of plant photosynthesis. In certain cases their relationships with plants are commensalistic or symbiotic. Transfer of energy from plants to bacteria takes place primarily in the soil through the release of exudates by roots and the shedding of aerial and subterranean plant parts: leaves and roots (e.g. root cap). The ability to catabolize substances produced by the plant is surely crucial to the survival of soil bacteria, and these substances are likely objects of intense competition (Nutman 1965). Plants produce a variety of secondary metabolites that are potential carbon and/or nitrogen sources for soil bacteria. It would seem logical that bacteria would evolve catabolic functions to degrade and utilize these metabolites, and that exclusive nutritional relationships could co-evolve, i.e. plants would produce exotic metabolites, not generally catabolized by microorganisms, and certain bacteria would benefit from these substances, which we have called nutritional mediators (Tepfer et al 1988), by evolving the corresponding catabolic functions. These nutritional mediators can be thought of as signals that at the same time trigger and fuel catabolism leading to growth. It is also logical that selective microbial growth would be important to the specificity of plant-microorganism interactions.