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NOD-FACTOR PERCEPTION IN MEDICAGO TRUNCATULA J. Cullimore1, B. Lefebvre1, J. F. Arrighi1, C. Gough1, A. Barre2, J. J. Bono2, P. Rougé2, E. Samain3, H. Driguez3, A. Imberty3, A. Untergasser4, R. Geurts4, T. W. J. Gadella, Jr.5, J. Cañada6 and J. Jimenez-Barbero6 1

Laboratoire des Interactions Plantes Microorganismes, INRA-CNRS; Surfaces Cellulaires et Signalisation chez les Végétaux, CNRS-UPS, 31326 Castanet-Tolosan, France; 3Centre de Recherches sur les Macromolécules Végétales, CNRS (affiliated with Université Joseph Fourier), 38041 Grenoble, France; 4Laboratory of Molecular Biology, Department of Plant Science, Wageningen University, 6703HA, The Netherlands; 5Section of Molecular Cytology and Centre for Advanced Microscopy, SILS, University of Amsterdam, 1098 SM Amsterdam, The Netherlands; 6CSIC, Centro de Investigaciones Biológicas, 28040 Madrid, Spain 2

Nod factors of Rhizobium have been described as the key to the legume door (Relic et al., 1994) and consequently, Nod factor receptors can be considered as the locks which allow Rhizobium access. This concept lies at the heart of the mechanisms of partner recognition leading to the establishment of the legume-Rhizobium symbiosis. Since the discovery of Rhizobium Nod factors in 1990 (see Dénarié et al., 1996), much attention has been paid to how these intriguing molecules interact with plants and this has led to the recent identification of legume symbiotic receptor genes. These studies were aided by focussing on model systems in which genetic and genomic approaches could be used. In this short review, we present our current knowledge of Nod factor perception in Medicago truncatula, a model legume that is nodulated by Sinorhizobium meliloti. Sinorhizobium meliloti and its Nod Factors The major Nod factor produced by S. meliloti is an O-sulphated, O-acetylated lipochito-tetra-oligosaccharide (LCO) termed NodSm-IV(Ac,S,C16:2). Studies on S. meliloti host-specificity mutants have shown that the nodH gene, encoding a sulphotransferase, is the major specificity determinant of nodulation on Medicago species, whereas the nodL and the nodFE genes, determining O-acetylation and the production

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of the specific C16:2 fatty acid, respectively, are required for infection. Studies on these mutants and with the corresponding LCOs led to the suggestion that .Mtruncatula may have two types of receptors for Nod factors: (i) the signalling receptor, which is required for initial responses; and (ii) the entry receptor, which is required for infection (Ardourel et al., 1994). The entry receptor is predicted to have more stringent requirements for the structure of the Nod factor (O-acetylation and the specific C16:2 acyl chain), whereas the signalling receptor requires the O-sulphate, but is less specific for these other structures. Because Nod-factor responses occur at concentrations as low as 10–13M, their receptors are expected to have high affinities for Nod factors and should have high specificities in order to discriminate between closely related molecules. Studies with fluorescent Nod factors have shown that they bind to cell walls of M. truncatula roots but this binding is relatively non-specific because it is independent of the O-sulphate (Goedhart et al., 2003). Biophysical studies have shown that these fluorescent LCOs rapidly incorporate into plant and artificial membranes (Goedhart et al., 1999), but it is not clear if this attribute is related to the mechanism of Nod-factor perception. In solution, Nod factors are monomers at sub-micromolar concentrations and a combination of NMR spectroscopy and molecular mechanics and dynamics calculations have shown that, in aqueous solution, they preferentially form structures in which the acyl chain and the oligosaccharide backbone are in a quasi-parallel orienttation (Gonzalez et al., 1999). However, differences in the saturation of the acyl chain and in the polarity of the solvent may lead to changes to this orientation. The importance of the acyl-chain structure for determining the shape of the Nod factors suggests that the lipid moiety may play an active role in receptor binding and ligand specificity rather than a more passive role such as membrane-anchoring (Groves et al., 2005). Nod-Factor Perception Involves LysM-Receptor-Like Kinases A combination of forward genetics and analysis of allelic variation has led to the identification of genes involved in Nod-factor perception and signal transduction (see Geurts et al., 2005). In M. truncatula, a single gene has been identified which is required for all Nod factor responses. This gene is called NOD FACTOR PERCEPTION (NFP) and has recently been shown to encode a lysin-motif receptor-like kinase (LysMRLK) (Arrighi et al., 2006). In pea, a gene, SYM2, has been identified in which different allelic variants control the interaction with Nod factors differing in the presence of an O-acetate on the reducing sugar. The corresponding genic region has been cloned from M. truncatula through genetic synteny and this region has been shown to contain a cluster of seven LysM-RLK genes, which have been termed the LYSM-RECEPTOR KINASE (LYK) genes. The gene encoding LYK3 is required for infection of M. truncatula by a rhizobial nodFE strain, but not for initial responses (Limpens et al., 2003). These results are consistent with NFP and LYK3 constituting part of the signalling and entry receptors, respectively.

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Both NFP and LYK3 are predicted to have similar overall structures with three LysM domains in the extracellular region, separated from an intracellular kinase-like domain by a single transmembrane spanning helix. Studies on NFP have shown that it is a membrane-located highly glycosylated protein (Mulder et al., 2006). LysM domains are small domains of about 45 residues and, because they occur in proteins and enzymes that interact with GlcNAc oligomers including a plant chitin receptor (Kaku et al., 2006), it has been postulated that the symbiotic LysM-RLKs directly bind Nod factors. Using molecular modelling, structures of the three LysM domains of NFP have been proposed and surface analysis and docking calculations have been used to predict the most favoured binding modes for chitooligosaccharides and Nod factors (Mulder et al., 2006; Arrighi et al., 2006). The relative size of a LysM domain and a Nod factor suggests that a maximum of one Nod factor could bind per domain. Studies are in progress to determine whether this protein and/or LYK3 bind Nod factors. Studies on the extensive sequence data bases of M. truncatula (expressed sequence tags and genomic sequences) have shown that this model legume contains at least 17 LysM-RLK genes, most of which can be divided between two clades, based on kinase domains; these are the LYK genes and the LYK-related (LYR) genes, which includes NFP. Unlike the LYK genes, the LYR genes seem to have aberrant kinase domain structures. Indeed, studies on the intracellular domains of NFP and LYK3 have shown that only LYK3 has autophosphorylation activity, thus raising the question of how NFP transmits a signal to downstream components if it is enzymatically inactive (Arrighi et al., 2006). Studies on Nod Factor-Binding Sites Suggest a Role for DMI2/DMI1 in LCO Perception Forward genetic studies have identified three genes that are required for infection by Rhizobium and for Nod factor-induced gene expression, but not for early Nod factor responses, such as root-hair deformation or calcium uptake. These genes are also required for infection by arbuscular mycorrhizal fungi (leading to the beneficial AM symbiosis) and have been termed DOES NOT MAKE INFECTIONS (DMI) genes. DMI2 encodes a leucine-rich-repeat receptor-like kinase, whereas DMI1 encodes a channellike protein. Both these genes are required for a calcium spiking response, whereas DMI3, which encodes a calcium- and calmodulin-dependent protein kinase, probably interprets this response (see Geurts et al., 2005). Using a radiolabelled Nod factor ligand, Bono and colleagues have characterised three different binding sites for Nod factors in roots and cell cultures of Medicago. Remarkably, a high affinity Nod- factor binding site in roots of M. truncatula, NFBS3, is absent in roots of dmi1 and dmi2 mutants, thus suggesting that DMI1/DMI2 are involved in LCO binding (Hogg et al., 2006). Further work is required to test whether the binding is related to the symbiosis with Rhizobium and whether the binding occurs directly to DMI1/DMI2 or involves another protein, such as a LysM-RLK.

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A Model for Nod-Factor Perception in M. Truncatula Current work favours a two-step model of Nod-factor perception in root epidermal cells of M. truncatula (Figure 1). The first step requires NFP and leads to calcium uptake and root-hair deformation (Had). Due to the aberrant kinase domain of NFP, another LysMRLK may also act at this step in order to transmit the signal to downstream effectors. DMI1/DMI2 apparently act downstream of NFP and are required for the induction of calcium spiking and nodulin-gene expression, which are non-stringent early responses. Initiation of infection threads by S. meliloti is a stringent response and requires wildtype bacteria that produce Nod factors with the C16:2 acyl chain. This second step requires LYK3 and DMI2 and recent studies on RNAi lines of NFP suggest that this gene also intervenes during this step. Whether these proteins form multimeric complexes is an interesting possibility. Note DMI1 is not included in the model because it has recently been reported to be located in the nuclear membrane (Riely et al., 2006).

Figure 1. The two-step model for nod-factor perception in M. trunculata.

Acknowledgements The authors gratefully acknowledge funding from the European Union Marie Curie Research Training Network programme (contract NODPERCEPTION) and from the French Agence Nationale de Recherche (contract NodBindsLysM). References Ardourel M et al. (1994) Plant Cell 6, 1357–1374. Arrighi JF et al. (2006) Plant Physiol. 142, 265–279. Dénarié J et al. (1996) Annu. Rev. Biochem. 65, 503–535. Geurts R et al. (2005) Curr. Opin. Plant Biol. 8, 346–352.

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