A glycan receptor kinase facilitates intracellular accommodation of arbuscular mycorrhiza and symbiotic rhizobia in the legume Lotus japonicus.

Receptors that distinguish the multitude of microbes surrounding plants in the environment enable dynamic responses to the biotic and abiotic conditions encountered. In this study, we identify and characterise a glycan receptor kinase, EPR3a, closely related to the exopolysaccharide receptor EPR3. Epr3a is up-regulated in roots colonised by arbuscular mycorrhizal (AM) fungi and is able to bind glucans with a branching pattern characteristic of surface-exposed fungal glucans. Expression studies with cellular resolution show localised activation of the Epr3a promoter in cortical root cells containing arbuscules. Fungal infection and intracellular arbuscule formation are reduced in epr3a mutants. In vitro, the EPR3a ectodomain binds cell wall glucans in affinity gel electrophoresis assays. In microscale thermophoresis (MST) assays, rhizobial exopolysaccharide binding is detected with affinities comparable to those observed for EPR3, and both EPR3a and EPR3 bind a well-defined ß-1,3/ß-1,6 decasaccharide derived from exopolysaccharides of endophytic and pathogenic fungi. Both EPR3a and EPR3 function in the intracellular accommodation of microbes. However, contrasting expression patterns and divergent ligand affinities result in distinct functions in AM colonisation and rhizobial infection in Lotus japonicus. The presence of Epr3a and Epr3 genes in both eudicot and monocot plant genomes suggest a conserved function of these receptor kinases in glycan perception.

Kinetic proofreading of lipochitooligosaccharides determines signal activation of symbiotic plant receptors.

Plants and animals use cell surface receptors to sense and interpret environmental signals. In legume symbiosis with nitrogen-fixing bacteria, the specific recognition of bacterial lipochitooligosaccharide (LCO) signals by single-pass transmembrane receptor kinases determines compatibility. Here, we determine the structural basis for LCO perception from the crystal structures of two lysin motif receptor ectodomains and identify a hydrophobic patch in the binding site essential for LCO recognition and symbiotic function. We show that the receptor monitors the composition of the amphiphilic LCO molecules and uses kinetic proofreading to control receptor activation and signaling specificity. We demonstrate engineering of the LCO binding site to fine-tune ligand selectivity and correct binding kinetics required for activation of symbiotic signaling in plants. Finally, the hydrophobic patch is found to be a conserved structural signature in this class of LCO receptors across legumes that can be used for in silico predictions. Our results provide insights into the mechanism of cell-surface receptor activation by kinetic proofreading of ligands and highlight the potential in receptor engineering to capture benefits in plant-microbe interactions.

A Lotus japonicus cytoplasmic kinase connects Nod factor perception by the NFR5 LysM receptor to nodulation.

The establishment of nitrogen-fixing root nodules in legume-rhizobia symbiosis requires an intricate communication between the host plant and its symbiont. We are, however, limited in our understanding of the symbiosis signaling process. In particular, how membrane-localized receptors of legumes activate signal transduction following perception of rhizobial signaling molecules has mostly remained elusive. To address this, we performed a coimmunoprecipitation-based proteomics screen to identify proteins associated with Nod factor receptor 5 (NFR5) in Lotus japonicus. Out of 51 NFR5-associated proteins, we focused on a receptor-like cytoplasmic kinase (RLCK), which we named NFR5-interacting cytoplasmic kinase 4 (NiCK4). NiCK4 associates with heterologously expressed NFR5 in Nicotiana benthamiana, and directly binds and phosphorylates the cytoplasmic domains of NFR5 and NFR1 in vitro. At the cellular level, Nick4 is coexpressed with Nfr5 in root hairs and nodule cells, and the NiCK4 protein relocates to the nucleus in an NFR5/NFR1-dependent manner upon Nod factor treatment. Phenotyping of retrotransposon insertion mutants revealed that NiCK4 promotes nodule organogenesis. Together, these results suggest that the identified RLCK, NiCK4, acts as a component of the Nod factor signaling pathway downstream of NFR5.

Receptor-mediated chitin perception in legume roots is functionally separable from Nod factor perception.

The ability of root cells to distinguish mutualistic microbes from pathogens is crucial for plants that allow symbiotic microorganisms to infect and colonize their internal root tissues. Here we show that Lotus japonicus and Medicago truncatula possess very similar LysM pattern-recognition receptors, LjLYS6/MtLYK9 and MtLYR4, enabling root cells to separate the perception of chitin oligomeric microbe-associated molecular patterns from the perception of lipochitin oligosaccharide by the LjNFR1/MtLYK3 and LjNFR5/MtNFP receptors triggering symbiosis. Inactivation of chitin-receptor genes in Ljlys6, Mtlyk9, and Mtlyr4 mutants eliminates early reactive oxygen species responses and induction of defense-response genes in roots. Ljlys6, Mtlyk9, and Mtlyr4 mutants were also more susceptible to fungal and bacterial pathogens, while infection and colonization by rhizobia and arbuscular mycorrhizal fungi was maintained. Biochemical binding studies with purified LjLYS6 ectodomains further showed that at least six GlcNAc moieties (CO6) are required for optimal binding efficiency. The 2.3-Å crystal structure of the LjLYS6 ectodomain reveals three LysM ßααß motifs similar to other LysM proteins and a conserved chitin-binding site. These results show that distinct receptor sets in legume roots respond to chitin and lipochitin oligosaccharides found in the heterogeneous mixture of chitinaceous compounds originating from soil microbes. This establishes a foundation for genetic and biochemical dissection of the perception and the downstream responses separating defense from symbiosis in the roots of the 80-90% of land plants able to develop rhizobial and/or mycorrhizal endosymbiosis.

LYS12 LysM receptor decelerates Phytophthora palmivora disease progression in Lotus japonicus.

Phytophthora palmivora is a devastating oomycete plant pathogen. We found that P. palmivora induces disease in Lotus japonicus and used this interaction to identify cellular and molecular events in response to this oomycete, which has a broad host range. Transcript quantification revealed that Lys12 was highly and rapidly induced during P. palmivora infection. Mutants of Lys12 displayed accelerated disease progression, earlier plant death and a lower level of defence gene expression than the wild type, while the defence program after chitin, laminarin, oligogalacturonide or flg22 treatment and the root symbioses with nitrogen-fixing rhizobia and arbuscular mycorrhiza were similar to the wild type. On the microbial side, we found that P. palmivora encodes an active chitin synthase-like protein, and mycelial growth is impaired after treatment with a chitin-synthase inhibitor. However, wheat germ agglutinin-detectable N-acetyl-glucosamine (GlcNAc) epitopes were not identified when the oomycete was grown in vitro or while infecting the roots. This indicates that conventional GlcNAc-mers are unlikely to be produced and/or accumulate in P. palmivora cell walls and that LYS12 might perceive an unknown carbohydrate. The impact of Lys12 on progression of root rot disease, together with the finding that similar genes are present in other P. palmivora hosts, suggests that LYS12 might mediate a common early response to this pathogen.

Dissection of Ramularia Leaf Spot Disease by Integrated Analysis of Barley and Ramularia collo-cygni Transcriptome Responses.

Ramularia leaf spot disease (RLS), caused by the ascomycete fungus Ramularia collo-cygni, has emerged as a major economic disease of barley. No substantial resistance has been identified, so far, among barley genotypes and, based on the epidemiology of the disease, a quantitative genetic determinacy of RLS has been suggested. The relative contributions of barley and R. collo-cygni genetics to disease infection and epidemiology are practically unknown. Here, we present an integrated genome-wide analysis of host and pathogen transcriptome landscapes identified in a sensitive barley cultivar following infection by an aggressive R. collo-cygni isolate. We compared transcriptional responses in the infected and noninfected leaf samples in order to identify which molecular events are associated with RLS symptom development. We found a large proportion of R. collo-cygni genes to be expressed in planta and that many were also closely associated with the infection stage. The transition from surface to apoplastic colonization was associated with downregulation of cell wall-degrading genes and upregulation of nutrient uptake and resistance to oxidative stresses. Interestingly, the production of secondary metabolites was dynamically regulated within the fungus, indicating that R. collo-cygni produces a diverse panel of toxic compounds according to the infection stage. A defense response against R. collo-cygni was identified in barley at the early, asymptomatic infection and colonization stages. We found activation of ethylene signaling, jasmonic acid signaling, and phenylpropanoid and flavonoid pathways to be highly induced, indicative of a classical response to necrotrophic pathogens. Disease development was found to be associated with gene expression patterns similar to those found at the onset of leaf senescence, when nutrients, possibly, are used by the infecting fungus. These analyses, combining both barley and R. collo-cygni transcript profiles, demonstrate the activation of complex transcriptional programs in both organisms.

Characterizing standard genetic parts and establishing common principles for engineering legume and cereal roots.

Plant synthetic biology and cereal engineering depend on the controlled expression of transgenes of interest. Most engineering in plant species to date has relied heavily on the use of a few, well-established constitutive promoters to achieve high levels of expression; however, the levels of transgene expression can also be influenced by the use of codon optimization, intron-mediated enhancement and varying terminator sequences. Most of these alternative approaches for regulating transgene expression have only been tested in small-scale experiments, typically testing a single gene of interest. It is therefore difficult to interpret the relative importance of these approaches and to design engineering strategies that are likely to succeed in different plant species, particularly if engineering multigenic traits where the expression of each transgene needs to be precisely regulated. Here, we present data on the characterization of 46 promoters and 10 terminators in Medicago truncatula, Lotus japonicus, Nicotiana benthamiana and Hordeum vulgare, as well as the effects of codon optimization and intron-mediated enhancement on the expression of two transgenes in H. vulgare. We have identified a core set of promoters and terminators of relevance to researchers engineering novel traits in plant roots. In addition, we have shown that combining codon optimization and intron-mediated enhancement increases transgene expression and protein levels in barley. Based on our study, we recommend a core set of promoters and terminators for broad use and also propose a general set of principles and guidelines for those engineering cereal species.

Root nodule symbiosis in Lotus japonicus drives the establishment of distinctive rhizosphere, root, and nodule bacterial communities.

Lotus japonicus has been used for decades as a model legume to study the establishment of binary symbiotic relationships with nitrogen-fixing rhizobia that trigger root nodule organogenesis for bacterial accommodation. Using community profiling of 16S rRNA gene amplicons, we reveal that in Lotus, distinctive nodule- and root-inhabiting communities are established by parallel, rather than consecutive, selection of bacteria from the rhizosphere and root compartments. Comparative analyses of wild-type (WT) and symbiotic mutants in Nod factor receptor5 (nfr5), Nodule inception (nin) and Lotus histidine kinase1 (lhk1) genes identified a previously unsuspected role of the nodulation pathway in the establishment of different bacterial assemblages in the root and rhizosphere. We found that the loss of nitrogen-fixing symbiosis dramatically alters community structure in the latter two compartments, affecting at least 14 bacterial orders. The differential plant growth phenotypes seen between WT and the symbiotic mutants in nonsupplemented soil were retained under nitrogen-supplemented conditions that blocked the formation of functional nodules in WT, whereas the symbiosis-impaired mutants maintain an altered community structure in the nitrogen-supplemented soil. This finding provides strong evidence that the root-associated community shift in the symbiotic mutants is a direct consequence of the disabled symbiosis pathway rather than an indirect effect resulting from abolished symbiotic nitrogen fixation. Our findings imply a role of the legume host in selecting a broad taxonomic range of root-associated bacteria that, in addition to rhizobia, likely contribute to plant growth and ecological performance.

N-glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research.

Studies of protein N-glycosylation are important for answering fundamental questions on the diverse functions of glycoproteins in plant growth and development. Here we generated and characterised a comprehensive collection of Lotus japonicusLORE1 insertion mutants, each lacking the activity of one of the 12 enzymes required for normal N-glycan maturation in the glycosylation machinery. The inactivation of the individual genes resulted in altered N-glycan patterns as documented using mass spectrometry and glycan-recognising antibodies, indicating successful identification of null mutations in the target glyco-genes. For example, both mass spectrometry and immunoblotting experiments suggest that proteins derived from the α1,3-fucosyltransferase (Lj3fuct) mutant completely lacked α1,3-core fucosylation. Mass spectrometry also suggested that the Lotus japonicus convicilin 2 was one of the main glycoproteins undergoing differential expression/N-glycosylation in the mutants. Demonstrating the functional importance of glycosylation, reduced growth and seed production phenotypes were observed for the mutant plants lacking functional mannosidase I, N-acetylglucosaminyltransferase I, and α1,3-fucosyltransferase, even though the relative protein composition and abundance appeared unaffected. The strength of our N-glycosylation mutant platform is the broad spectrum of resulting glycoprotein profiles and altered physiological phenotypes that can be produced from single, double, triple and quadruple mutants. This platform will serve as a valuable tool for elucidating the functional role of protein N-glycosylation in plants. Furthermore, this technology can be used to generate stable plant mutant lines for biopharmaceutical production of glycoproteins displaying relative homogeneous and mammalian-like N-glycosylation features.

Regulation of Nod factor biosynthesis by alternative NodD proteins at distinct stages of symbiosis provides additional compatibility scrutiny.

The Lotus japonicus symbiont Mesorhizobium loti R7A encodes two copies of nodD and here we identify striking differences in Nod factor biosynthesis gene induction by NodD1 and NodD2 both in vitro and in planta. We demonstrate that induction of Nod factor biosynthesis genes is preferentially controlled by NodD1 and NodD2 at specific stages of symbiotic infection. NodD2 is primarily responsible for induction in the rhizosphere and within nodules, while NodD1 is primarily responsible for induction within root hair infection threads. nodD1 and nodD2 mutants showed significant symbiotic phenotypes and competition studies establish that nodD1 and nodD2 mutants were severely outcompeted by wild-type R7A, indicating that both proteins are required for proficient symbiotic infection. These results suggest preferential activation of NodD1 and NodD2 by different inducing compounds produced at defined stages of symbiotic infection. We identified Lotus chalcone isomerase CHI4 as a root hair induced candidate involved in the biosynthesis of an inducer compound that may be preferentially recognized by NodD1 within root hair infection threads. We propose an alternative explanation for the function of multiple copies of nodD that provides the host plant with another level of compatibility scrutiny at the stage of infection thread development.

A Lotus japonicus E3 ligase interacts with the Nod Factor Receptor 5 and positively regulates nodulation.

BACKGROUND: Post-translational modification of receptor proteins is involved in activation and de-activation of signalling systems in plants. Both ubiquitination and deubiquitination have been implicated in plant interactions with pathogens and symbionts. RESULTS: Here we present LjPUB13, a PUB-ARMADILLO repeat E3 ligase that specifically ubiquitinates the kinase domain of the Nod Factor receptor NFR5 and has a direct role in nodule organogenesis events in Lotus japonicus. Phenotypic analyses of three LORE1 retroelement insertion plant lines revealed that pub13 plants display delayed and reduced nodulation capacity and retarded growth. LjPUB13 expression is spatially regulated during symbiosis with Mesorhizobium loti, with increased levels in young developing nodules. CONCLUSION: LjPUB13 is an E3 ligase with a positive regulatory role during the initial stages of nodulation in L. japonicus.

A legume genetic framework controls infection of nodules by symbiotic and endophytic bacteria.

Legumes have an intrinsic capacity to accommodate both symbiotic and endophytic bacteria within root nodules. For the symbionts, a complex genetic mechanism that allows mutual recognition and plant infection has emerged from genetic studies under axenic conditions. In contrast, little is known about the mechanisms controlling the endophytic infection. Here we investigate the contribution of both the host and the symbiotic microbe to endophyte infection and development of mixed colonised nodules in Lotus japonicus. We found that infection threads initiated by Mesorhizobium loti, the natural symbiont of Lotus, can selectively guide endophytic bacteria towards nodule primordia, where competent strains multiply and colonise the nodule together with the nitrogen-fixing symbiotic partner. Further co-inoculation studies with the competent coloniser, Rhizobium mesosinicum strain KAW12, show that endophytic nodule infection depends on functional and efficient M. loti-driven Nod factor signalling. KAW12 exopolysaccharide (EPS) enabled endophyte nodule infection whilst compatible M. loti EPS restricted it. Analysis of plant mutants that control different stages of the symbiotic infection showed that both symbiont and endophyte accommodation within nodules is under host genetic control. This demonstrates that when legume plants are exposed to complex communities they selectively regulate access and accommodation of bacteria occupying this specialized environmental niche, the root nodule.

Compositionally aware estimation of cross-correlations for microbiome data.

In the field of microbiome studies, it is of interest to infer correlations between abundances of different microbes (here referred to as operational taxonomic units, OTUs). Several methods taking the compositional nature of the sequencing data into account exist. However, these methods cannot infer correlations between OTU abundances and other variables. In this paper we introduce the novel methods SparCEV (Sparse Correlations with External Variables) and SparXCC (Sparse Cross-Correlations between Compositional data) for quantifying correlations between OTU abundances and either continuous phenotypic variables or components of other compositional datasets, such as transcriptomic data. SparCEV and SparXCC both assume that the average correlation in the dataset is zero. Iterative versions of SparCEV and SparXCC are proposed to alleviate bias resulting from deviations from this assumption. We compare these new methods to empirical Pearson cross-correlations after applying naive transformations of the data (log and log-TSS). Additionally, we test the centered log ratio transformation (CLR) and the variance stabilising transformation (VST). We find that CLR and VST outperform naive transformations, except when the correlation matrix is dense. SparCEV and SparXCC outperform CLR and VST when the number of OTUs is small and perform similarly to CLR and VST for large numbers of OTUs. Adding the iterative procedure increases accuracy for SparCEV and SparXCC for all cases, except when the average correlation in the dataset is close to zero or the correlation matrix is dense. These results are consistent with our theoretical considerations.

Integrative transcript to proteome analysis of barley during Ramularia collo-cygni leaf spot development identified several proteins that are related to fungal recognition and infection responses.

Introduction: Ramularia leaf spot (RLS) disease is a growing threat to barley cultivation, but with no substantial resistance identified to date. Similarly, the understanding of the lifestyle of Ramularia collo-cygni (Rcc) and the prediction of RLS outbreak severity remain challenging, with Rcc displaying a rather untypical long endophytic phase and a sudden change to a necrotrophic lifestyle. The aim of this study was to provide further insights into the defense dynamics during the different stages of colonization and infection in barley in order to identify potential targets for resistance breeding. Methods: Utilizing the strength of proteomics in understanding plant-pathogen interactions, we performed an integrative analysis of a published transcriptome dataset with a parallel generated proteome dataset. Therefore, we included two spring barley cultivars with contrasting susceptibilities to Rcc and two fungal isolates causing different levels of RLS symptoms. Results: Interestingly, early responses in the pathogen recognition phase of the host were driven by strong responses differing between isolates. An important enzyme in this process is a xylanase inhibitor, which protected the plant from cell wall degradation by the fungal xylanase. At later time points, the differences were driven by cultivar-specific responses, affecting mostly features contributing to the pathogenesis- and senescence-related pathways or photosynthesis. Discussion: This supports the hypothesis of a hemibiotrophic lifestyle of Rcc, with slight differences in trophism of the two analyzed isolates. The integration of these data modalities highlights a strength of protein-level analysis in understanding plant-pathogen interactions and reveals new features involved in fungal recognition and susceptibility in barley cultivars.

Benchmarking long-read sequencing strategies for obtaining ASV-resolved rRNA operons from environmental microeukaryotes.

The use of short-read metabarcoding for classifying microeukaryotes is challenged by the lack of comprehensive 18S rRNA reference databases. While recent advances in high-throughput long-read sequencing provide the potential to greatly increase the phylogenetic coverage of these databases, the performance of different sequencing technologies and subsequent bioinformatics processing remain to be evaluated, primarily because of the absence of well-defined eukaryotic mock communities. To address this challenge, we created a eukaryotic rRNA operon clone-library and turned it into a precisely defined synthetic eukaryotic mock community. This mock community was then used to evaluate the performance of three long-read sequencing strategies (PacBio circular consensus sequencing and two Nanopore approaches using unique molecular identifiers) and three tools for resolving amplicons sequence variants (ASVs) (USEARCH, VSEARCH, and DADA2). We investigated the sensitivity of the sequencing techniques based on the number of detected mock taxa, and the accuracy of the different ASV-calling tools with a specific focus on the presence of chimera among the final rRNA operon ASVs. Based on our findings, we provide recommendations and best practice protocols for how to cost-effectively obtain essentially error-free rRNA operons in high-throughput. An agricultural soil sample was used to demonstrate that the sequencing and bioinformatic results from the mock community also translates to highly diverse natural samples, which enables us to identify previously undescribed microeukaryotic lineages.

Nitrogen and Nod factor signaling determine Lotus japonicus root exudate composition and bacterial assembly.

Symbiosis with soil-dwelling bacteria that fix atmospheric nitrogen allows legume plants to grow in nitrogen-depleted soil. Symbiosis impacts the assembly of root microbiota, but it is unknown how the interaction between the legume host and rhizobia impacts the remaining microbiota and whether it depends on nitrogen nutrition. Here, we use plant and bacterial mutants to address the role of Nod factor signaling on Lotus japonicus root microbiota assembly. We find that Nod factors are produced by symbionts to activate Nod factor signaling in the host and that this modulates the root exudate profile and the assembly of a symbiotic root microbiota. Lotus plants with different symbiotic abilities, grown in unfertilized or nitrate-supplemented soils, display three nitrogen-dependent nutritional states: starved, symbiotic, or inorganic. We find that root and rhizosphere microbiomes associated with these states differ in composition and connectivity, demonstrating that symbiosis and inorganic nitrogen impact the legume root microbiota differently. Finally, we demonstrate that selected bacterial genera characterizing state-dependent microbiomes have a high level of accurate prediction.

Unraveling the secrets of plant roots: Simplified method for large scale root exudate sampling and analysis in Arabidopsis thaliana.

Background: Plants exude a plethora of compounds to communicate with their environment. Although much is known about above-ground plant communication, we are only beginning to fathom the complexities of below-ground chemical communication channels. Studying root-exuded compounds and their role in plant communication has been difficult due to the lack of standardized methodologies. Here, we develop an interdisciplinary workflow to explore the natural variation in root exudate chemical composition of the model plant Arabidopsis thaliana. We highlight key challenges associated with sampling strategies and develop a framework for analyzing both narrow- and broad-scale patterns of root exudate composition in a large set of natural A. thaliana accessions. Methods: Our method involves cultivating individual seedlings in vitro inside a plastic mesh, followed by a short hydroponic sampling period in small quantities of ultrapure water. The mesh makes it easy to handle plants of different sizes and allows for large-scale characterization of individual plant root exudates under axenic conditions. This setup can also be easily extended for prolonged temporal exudate collection experiments. Furthermore, the short sampling time minimizes the duration of the experiment while still providing sufficient signal even with small volume of the sampling solution. We used ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) for untargeted metabolic profiling, followed by tentative compound identification using MZmine3 and SIRIUS 5 software, to capture a broad overview of root exudate composition in A. thaliana accessions. Results: Based on 28 replicates of the Columbia genotype (Col-0) compared with 10 random controls, MZmine3 annotated 354 metabolites to be present only in Col-0 by negative ionization. Of these, 254 compounds could be annotated by SIRIUS 5 software. Conclusions: The methodology developed in this study can be used to broadly investigate the role of root exudates as chemical signals in plant belowground interactions.

Plants exude many compounds to communicate with their surroundings. For decades, our understanding of this chemical communication was limited to studying the aboveground parts of plants, as roots are hidden within the soil, which makes them difficult to study. We are only beginning to comprehend the complexities and importance of below ground communication channels (including plant-microbes, plant-insects, and plant-plants). Identifying the chemical compounds plant exude belowground (called root exudates) is important for us to fully comprehend their potential roles in a plant´s life. Here, we developed a simplified and easy-to-manage setup for collecting and analyzing root exudates from individual Arabidopsis thaliana plants.

A simple and efficient protocol for generating transgenic hairy roots using Agrobacterium rhizogenes.

For decades, Agrobacterium rhizogenes (now Rhizobium rhizogenes), the causative agent of hairy root disease, has been harnessed as an interkingdom DNA delivery tool for generating transgenic hairy roots on a wide variety of plants. One of the strategies involves the construction of transconjugant R. rhizogenes by transferring gene(s) of interest into previously constructed R. rhizogenes pBR322 acceptor strains; little has been done, however, to improve upon this system since its implementation. We developed a simplified method utilising bi-parental mating in conjunction with effective counterselection for generating R. rhizogenes transconjugants. Central to this was the construction of a new Modular Cloning (MoClo) compatible pBR322-derived integration vector (pIV101). Although this protocol remains limited to pBR322 acceptor strains, pIV101 facilitated an efficient construction of recombinant vectors, effective screening of transconjugants, and RP4-based mobilisation compatibility that enabled simplified conjugal transfer. Transconjugants from this system were tested on Lotus japonicus and found to be efficient for the transformation of transgenic hairy roots and supported infection of nodules by a rhizobia symbiont. The expedited protocol detailed herein substantially decreased both the time and labour for creating transconjugant R. rhizogenes for the subsequent transgenic hairy root transformation of Lotus, and it could readily be applied for the transformation of other plants.

Nanobody-driven signaling reveals the core receptor complex in root nodule symbiosis.

Understanding the composition and activation of multicomponent receptor complexes is a challenge in biology. To address this, we developed a synthetic approach based on nanobodies to drive assembly and activation of cell surface receptors and apply the concept by manipulating receptors that govern plant symbiosis with nitrogen-fixing bacteria. We show that the Lotus japonicus Nod factor receptors NFR1 and NFR5 constitute the core receptor complex initiating the cortical root nodule organogenesis program as well as the epidermal program controlling infection. We find that organogenesis signaling is mediated by the intracellular kinase domains whereas infection requires functional ectodomains. Finally, we identify evolutionarily distant barley receptors that activate root nodule organogenesis, which could enable engineering of biological nitrogen-fixation into cereals.

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.