Simona Radutoiu

Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases

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.

A receptor kinase gene of the LysM type is involved in legumeperception of rhizobial signals

Plants belonging to the legume family develop nitrogen-fixing root nodules in symbiosis with bacteria commonly known as rhizobia. The legume host encodes all of the functions necessary to build the specialized symbiotic organ, the nodule, but the process is elicited by the bacteria. Molecular communication initiates the interaction, and signals, usually flavones, secreted by the legume root induce the bacteria to produce a lipochitin-oligosaccharide signal molecule (Nod-factor), which in turn triggers the plant organogenic process. An important determinant of bacterial host specificity is the structure of the Nod-factor, suggesting that a plant receptor is involved in signal perception and signal transduction initiating the plant developmental response. Here we describe the cloning of a putative Nod-factor receptor kinase gene (NFR5) from Lotus japonicus. NFR5 is essential for Nod-factor perception and encodes an unusual transmembrane serine/threonine receptor-like kinase required for the earliest detectable plant responses to bacteria and Nod-factor. The extracellular domain of the putative receptor has three modules with similarity to LysM domains known from peptidoglycan-binding proteins and chitinases. Together with an atypical kinase domain structure this characterizes an unusual receptor-like kinase.

LysM domains mediate lipochitin–oligosaccharide recognition and Nfr genes extend the symbiotic host range

Legume–Rhizobium symbiosis is an example of selective cell recognition controlled by host/non‐host determinants. Individual bacterial strains have a distinct host range enabling nodulation of a limited set of legume species and vice versa. We show here that expression of Lotus japonicus Nfr1 and Nfr5 Nod‐factor receptor genes in Medicago truncatula and L. filicaulis, extends their host range to include bacterial strains, Mesorhizobium loti or DZL, normally infecting L. japonicus. As a result, the symbiotic program is induced, nodules develop and infection threads are formed. Using L. japonicus mutants and domain swaps between L. japonicus and L. filicaulis NFR1 and NFR5, we further demonstrate that LysM domains of the NFR1 and NFR5 receptors mediate perception of the bacterial Nod‐factor signal and that recognition depends on the structure of the lipochitin–oligosaccharide Nod‐factor. We show that a single amino‐acid variation in the LysM2 domain of NFR5 changes recognition of the Nod‐factor synthesized by the DZL strain and suggests a possible binding site for bacterial lipochitin–oligosaccharide signal molecules.

Receptor-mediated exopolysaccharide perception controls bacterial infection

Surface polysaccharides are important for bacterial interactions with multicellular organisms, and some are virulence factors in pathogens. In the legume–rhizobium symbiosis, bacterial exopolysaccharides (EPS) are essential for the development of infected root nodules. We have identified a gene in Lotus japonicus, Epr3, encoding a receptor-like kinase that controls this infection. We show that epr3 mutants are defective in perception of purified EPS, and that EPR3 binds EPS directly and distinguishes compatible and incompatible EPS in bacterial competition studies. Expression of Epr3 in epidermal cells within the susceptible root zone shows that the protein is involved in bacterial entry, while rhizobial and plant mutant studies suggest that Epr3 regulates bacterial passage through the plant’s epidermal cell layer. Finally, we show that Epr3 expression is inducible and dependent on host perception of bacterial nodulation (Nod) factors. Plant–bacterial compatibility and bacterial access to legume roots is thus regulated by a two-stage mechanism involving sequential receptor-mediated recognition of Nod factor and EPS signals.

Autophosphorylation is essential for the in vivo function of the Lotus japonicus Nod factor receptor 1 and receptor‐mediated signalling in cooperation with Nod factor receptor 5

Soil-living rhizobia secrete lipochitin oligosaccharides known as Nod factors, which in Lotus japonicus are perceived by at least two Nod-factor receptors, NFR1 and NFR5. Despite progress in identifying molecular components critical for initial legume host recognition of the microsymbiont and cloning of downstream components, little is known about the activation and signalling mechanisms of the Nod-factor receptors themselves. Here we show that both receptor proteins localize to the plasma membrane, and present evidence for heterocomplex formation initiating downstream signalling. Expression of NFR1 and NFR5 in Nicotiana benthamiana and Allium ampeloprasum (leek) cells caused a rapid cell-death response. The signalling leading to cell death was abrogated using a kinase-inactive variant of NFR1. In these surviving cells, a clear interaction between NFR1 and NFR5 was detected in vivo through bimolecular fluorescence complementation (BiFC). To analyse the inter- and intramolecular phosphorylation events of the kinase complex, the cytoplasmic part of NFR1 was assayed for in vitro kinase activity, and autophosphorylation on 24 amino acid residues, including three tyrosine residues, was found by mass spectrometry. Substitution of the phosphorylated amino acids of NFR1 identified a single phosphorylation site to be essential for NFR1 Nod-factor signalling in vivo and kinase activity in vitro. In contrast to NFR1, no in vitro kinase activity of the cytoplasmic domain of NFR5 was detected. This is further supported by the fact that a mutagenized NFR5 construct, substituting an amino acid essential for ATP binding, restored nodulation of nfr5 mutant roots.

Dissection of symbiosis and organ development by integrated transcriptome analysis of Lotus japonicus mutant and wild-type plants.

Genetic analyses of plant symbiotic mutants has led to the identification of key genes involved in Rhizobium-legume communication as well as in development and function of nitrogen fixing root nodules. However, the impact of these genes in coordinating the transcriptional programs of nodule development has only been studied in limited and isolated studies. Here, we present an integrated genome-wide analysis of transcriptome landscapes in Lotus japonicus wild-type and symbiotic mutant plants. Encompassing five different organs, five stages of the sequentially developed determinate Lotus root nodules, and eight mutants impaired at different stages of the symbiotic interaction, our data set integrates an unprecedented combination of organ- or tissue-specific profiles with mutant transcript profiles. In total, 38 different conditions sampled under the same well-defined growth regimes were included. This comprehensive analysis unravelled new and unexpected patterns of transcriptional regulation during symbiosis and organ development. Contrary to expectations, none of the previously characterized nodulins were among the 37 genes specifically expressed in nodules. Another surprise was the extensive transcriptional response in whole root compared to the susceptible root zone where the cellular response is most pronounced. A large number of transcripts predicted to encode transcriptional regulators, receptors and proteins involved in signal transduction, as well as many genes with unknown function, were found to be regulated during nodule organogenesis and rhizobial infection. Combining wild type and mutant profiles of these transcripts demonstrates the activation of a complex genetic program that delineates symbiotic nitrogen fixation. The complete data set was organized into an indexed expression directory that is accessible from a resource database, and here we present selected examples of biological questions that can be addressed with this comprehensive and powerful gene expression data set.

Rearrangement of Actin Cytoskeleton Mediates Invasion of Lotus japonicus Roots by Mesorhizobium loti

Infection thread–dependent invasion of legume roots by rhizobia leads to internalization of bacteria into the plant cells, which is one of the salient features of root nodule symbiosis. We found that two genes, Nap1 (for Nck-associated protein 1) and Pir1 (for 121F-specific p53 inducible RNA), involved in actin rearrangements were essential for infection thread formation and colonization of Lotus japonicus roots by its natural microsymbiont, Mesorhizobium loti. nap1 and pir1 mutants developed an excess of uncolonized nodule primordia, indicating that these two genes were not essential for the initiation of nodule organogenesis per se. However, both the formation and subsequent progression of infection threads into the root cortex were significantly impaired in these mutants. We demonstrate that these infection defects were due to disturbed actin cytoskeleton organization. Short root hairs of the mutants had mostly transverse or web-like actin filaments, while bundles of actin filaments in wild-type root hairs were predominantly longitudinal. Corroborating these observations, temporal and spatial differences in actin filament organization between wild-type and mutant root hairs were also observed after Nod factor treatment, while calcium influx and spiking appeared unperturbed. Together with various effects on plant growth and seed formation, the nap1 and pir1 alleles also conferred a characteristic distorted trichome phenotype, suggesting a more general role for Nap1 and Pir1 in processes establishing cell polarity or polar growth in L. japonicus.

Genetics of Symbiosis in Lotus japonicus: Recombinant Inbred Lines, Comparative Genetic Maps, and Map Position of 35 Symbiotic Loci

Development of molecular tools for the analysis of the plant genetic contribution to rhizobial and mycorrhizal symbiosis has provided major advances in our understanding of plant-microbe interactions, and several key symbiotic genes have been identified and characterized. In order to increase the efficiency of genetic analysis in the model legume Lotus japonicus, we present here a selection of improved genetic tools. The two genetic linkage maps previously developed from an interspecific cross between L. japonicus Gifu and L. filicaulis, and an intraspecific cross between the two ecotypes L. japonicus Gifu and L. japonicus MG-20, were aligned through a set of anchor markers. Regions of linkage groups, where genetic resolution is obtained preferentially using one or the other parental combination, are highlighted. Additional genetic resolution and stabilized mapping populations were obtained in recombinant inbred lines derived by a single seed descent from the two populations. For faster mapping of new loci, a selection of reliable markers spread over the chromosome arms provides a common framework for more efficient identification of new alleles and new symbiotic loci among uncharacterized mutant lines. Combining resources from the Lotus community, map positions of a large collection of symbiotic loci are provided together with alleles and closely linked molecular markers. Altogether, this establishes a common genetic resource for Lotus spp. A web-based version will enable this resource to be curated and updated regularly.

Evolution and Regulation of the Lotus japonicus LysM Receptor Gene Family

LysM receptor kinases were identified as receptors of acylated chitin (Nod factors) or chitin produced by plant-interacting microbes. Here, we present the identification and characterization of the LysM receptor kinase gene (Lys) family (17 members) in Lotus japonicus. Comprehensive phylogenetic analysis revealed a correlation between Lys gene structure and phylogeny. Further mapping coupled with sequence-based anchoring on the genome showed that the family has probably expanded by a combination of tandem and segmental duplication events. Using a sliding-window approach, we identified distinct regions in the LysM and kinase domains of recently diverged Lys genes where positive selection may have shaped ligand interaction. Interestingly, in the case of NFR5 and its closest paralog, LYS11, one of these regions coincides with the predicted Nod-factor binding groove and the suggested specificity determining area of the second LysM domain. One hypothesis for the evolutionary diversification of this receptor family in legumes is their unique capacity to decipher various structures of chitin-derived molecules produced by an extended spectrum of interacting organisms: symbiotic, associative, endophytic, and parasitic. In a detailed expression analysis, we found several Lotus Lys genes regulated not only during the symbiotic association with Mesorhizobium loti but also in response to chitin treatment.

T-DNA tagging of nodulation- and root-related genes in Lotus japonicus : Expression patterns and potential for promoter trapping and insertional mutagenesis

High-efficiency transformation of the autogamous diploid legume Lotus japonicus by means of Agrobacterium rhizogenes was used to develop plant lines expressing a promoter-less gusA gene in a nodulation- or lateral root-associated manner. The approach exploits the putatively preferential integration of T-DNA into actively transcribed regions, thereby providing an enrichment for gene tagging events associated with the quickly assayable activation of a gusA promoter-less construct. Taking advantage of this enrichment and selection strategy, a T-DNA tagging program was initiated and screening for β-glucuronidase (GUS) activity was performed on root clones isolated after transformation with a gusA-promoter-less binary vector. The aim of this approach is the identification of genes involved in nodule formation induced by Mesorhizobium loti, lateral root organogenesis, and the eventual isolation of corresponding mutants. A large collection (220) of GUS-positive transformants showing a variety of expression patte...