Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases Simona Radutoiu 1 , Lene Heegaard Madsen 1 , Esben Bjørn Madsen 1 , Hubert H. Felle 2 , Yosuke Umehara 1 *, Mette Grønlund 1 , Shusei Sato 3 , Yasukazu Nakamura 3 , Satoshi Tabata 3 , Niels Sandal 1 & Jens Stougaard 1 1 Laboratory of Gene Expression, Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10, 8000 Aarhus C, Denmark 2 Botanisches Institut I, Justus-Liebig Universita ¨t, D-35390 Giessen, Germany 3 Kazusa DNA Research Institute, Kisarazu, Chiba 292-0812, Japan *Present address: National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan ........................................................................................................................................................................................................................... Although most higher plants establish a symbiosis with arbuscular mycorrhizal fungi, symbiotic nitrogen fixation with rhizobia is a salient feature of legumes. Despite this host range difference, mycorrhizal and rhizobial invasion shares a common plant-specified genetic programme controlling the early host interaction. One feature distinguishing legumes is their ability to perceive rhizobial- specific signal molecules. We describe here two LysM-type serine/threonine receptor kinase genes, NFR1 and NFR5, enabling the model legume Lotus japonicus to recognize its bacterial microsymbiont Mesorhizobium loti. The extracellular domains of the two transmembrane kinases resemble LysM domains of peptidoglycan- and chitin-binding proteins, suggesting that they may be involved directly in perception of the rhizobial lipochitin-oligosaccharide signal. We show that NFR1 and NFR5 are required for the earliest physiological and cellular responses to this lipochitin-oligosaccharide signal, and demonstrate their role in the mechanism establishing susceptibility of the legume root for bacterial infection. Legumes can acquire two important macronutrients—nitrogen and phosphorus—through symbiotic relationships with rhizobial bac- teria and mycorrhizal fungi, respectively. Carbon from photo- synthesis is exchanged for either nitrogen reduced by bacterial nitrogenase or phosphate extracted from the soil by the network of fungal hyphae. The host range of mycorrhizal fungi is broad, and the majority of all land plants form arbuscular mycorrhizal sym- biosis 1,2 . In contrast the host range of rhizobial symbiosis is narrow and, with one known exception, only legumes develop root nodules with rhizobia. Even within the legume family, specific and mutual recognition limits the host range of bacterial strains. Despite these differences, legume genetics connects fungal and rhizobial sym- bioses and provides evidence for the recruitment of components from a pre-existing plant–fungal pathway during evolution of the plant–bacterial interaction 1,3 . For instance, several classes of legume mutants arrested early in rhizobial interaction are also impaired in mycorrhizal colonization 4–6 . The genetic loci corresponding to these mutants define a common endosymbiotic pathway involved in legume–microsymbiont communication. In Lotus at least six genetic loci constitute the common pathway 6 , and the SYMRK gene was the first to be characterized molecularly. SYMRK encodes a leucine-rich repeat (LRR) receptor-like kinase that perceives both mycorrhizal and rhizobial signals, probably at the junction of the common pathway 7,8 . The nature of fungal signal molecules perceived by the plant root to allow hyphal entry and accommodation of the fungus is unknown 9 . In contrast, a combination of chemistry and bacterial genetics has identified lipochito-oligosaccharides (Nod-factors), consisting of substituted b,1-4 N-acetylglucosamine (chitin) back- bones, as the rhizobial morphogenic signal 10,11 inducing root hair deformation and cell division leading to development of nodule primordia 12–14 . The major Nod-factor secreted by the microsym- biont of Lotus, M. loti, is a pentameric N-acetylglucosamine, which carries a cis-vaccenic acid and a carbamoyl group at the non- reducing terminal residue together with a 4-O-acetylfucose at the reducing terminal residue 13,14 . Several lines of evidence demonstrate that rhizobial host range is determined by the type and position of Nod-factor substituents, suggesting the involvement of one or more receptors 15 . For example, addition of acetyl-fucose to the Rhizobium leguminosarum biovar viciae Nod-factor enables this rhizobial symbiont of pea to nodulate Lotus 16 . Further indications of recep- tor-mediated perception of Nod-factors come from physiological studies, where ion fluxes, membrane depolarization and Ca 2þ spiking in legume root hairs is elicited within minutes of application of purified Nod-factor in the picomolar to nanomolar range 17,18 . Lotus plants carrying mutations in genes of the common pathway, such as SYMRK, show a Nod-factor-dependent root hair defor- mation response on rhizobial inoculation 7 , indicating that they are still able to sense the presence of bacterial signal(s) through upstream genes or a parallel pathway. Two mutant classes in Lotus—nfr1 and nfr5—that have a wild-type mycorrhization pheno- type 4,19,20 but lack the root hair response to rhizobia, identify loci predicted to be involved in rhizobial signal perception or trans- duction. Here we show that NFR1 and NFR5 (ref. 21) are putative Nod-factor receptor genes necessary for Nod-factor perception upstream of SYMRK and the common pathway. Map-based cloning and characterization of NFR1 and NFR5 (ref. 21) reveals that both encode LysM-type serine/threonine receptor kinases. NFR1 and NFR5 mutant phenotypes Structural requirements of Nod-factor molecules and the nano- molar concentrations eliciting root hair deformation and root cell divisions indicate that legume perception of the bacterial signal is receptor mediated. As a first step towards understanding the molecular mechanisms involved, we have characterized in detail the non-responsive phenotypes of the monogenic recessive nfr1 and nfr5 mutants (formerly known as sym1 and sym5, respectively 19 ). Three different strains of M. loti, NZP2235, R7A and TONO, all failed to induce infection threads or nodule primordia in mutant plants carrying the nfr1-1, nfr1-2 or nfr5-1, nfr5-2, nfr5-3 alleles. The earliest visible cellular change in wild-type plants is root hair deformation and root hair curling, easily observable 24 h after M. loti inoculation (Fig. 1a, b). These cellular responses were not observed in nfr1 and nfr5 mutants (Fig. 1c, d). The fact that lipochitin-oligosaccharides purified from the M. loti R7A strain also failed to induce any deformation of mutant root hairs suggests that both the NFR1 and NFR5 genes act early in the rhizobial interaction and are involved in Nod-factor perception (Fig. 1b, d). articles NATURE | VOL 425 | 9 OCTOBER 2003 | www.nature.com/nature 585 © 2003 Nature Publishing Group