Single-cell analysis identifies genes facilitating rhizobium infection in Lotus japonicus.

Legume-rhizobium signaling during establishment of symbiotic nitrogen fixation restricts rhizobium colonization to specific cells. A limited number of root hair cells allow infection threads to form, and only a fraction of the epidermal infection threads progress to cortical layers to establish functional nodules. Here we use single-cell analysis to define the epidermal and cortical cell populations that respond to and facilitate rhizobium infection. We then identify high-confidence nodulation gene candidates based on their specific expression in these populations, pinpointing genes stably associated with infection across genotypes and time points. We show that one of these, which we name SYMRKL1, encodes a protein with an ectodomain predicted to be nearly identical to that of SYMRK and is required for normal infection thread formation. Our work disentangles cellular processes and transcriptional modules that were previously confounded due to lack of cellular resolution, providing a more detailed understanding of symbiotic interactions.

The giant diploid faba genome unlocks variation in a global protein crop.

Increasing the proportion of locally produced plant protein in currently meat-rich diets could substantially reduce greenhouse gas emissions and loss of biodiversity1. However, plant protein production is hampered by the lack of a cool-season legume equivalent to soybean in agronomic value2. Faba bean (Vicia faba L.) has a high yield potential and is well suited for cultivation in temperate regions, but genomic resources are scarce. Here, we report a high-quality chromosome-scale assembly of the faba bean genome and show that it has expanded to a massive 13 Gb in size through an imbalance between the rates of amplification and elimination of retrotransposons and satellite repeats. Genes and recombination events are evenly dispersed across chromosomes and the gene space is remarkably compact considering the genome size, although with substantial copy number variation driven by tandem duplication. Demonstrating practical application of the genome sequence, we develop a targeted genotyping assay and use high-resolution genome-wide association analysis to dissect the genetic basis of seed size and hilum colour. The resources presented constitute a genomics-based breeding platform for faba bean, enabling breeders and geneticists to accelerate the improvement of sustainable protein production across the Mediterranean, subtropical and northern temperate agroecological zones.

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.

The Lotus japonicus AFB6 Gene Is Involved in the Auxin Dependent Root Developmental Program.

Auxin is essential for root development, and its regulatory action is exerted at different steps from perception of the hormone up to transcriptional regulation of target genes. In legume plants there is an overlap between the developmental programs governing lateral root and N2-fixing nodule organogenesis, the latter induced as the result of the symbiotic interaction with rhizobia. Here we report the characterization of a member of the L. japonicus TIR1/AFB auxin receptor family, LjAFB6. A preferential expression of the LjAFB6 gene in the aerial portion of L. japonicus plants was observed. Significant regulation of the expression was not observed during the symbiotic interaction with Mesorhizobium loti and the nodule organogenesis process. In roots, the LjAFB6 expression was induced in response to nitrate supply and was mainly localized in the meristematic regions of both primary and lateral roots. The phenotypic analyses conducted on two independent null mutants indicated a specialized role in the control of primary and lateral root elongation processes in response to auxin, whereas no involvement in the nodulation process was found. We also report the involvement of LjAFB6 in the hypocotyl elongation process and in the control of the expression profile of an auxin-responsive gene.

The Lotus japonicus NPF3.1 Is a Nodule-Induced Gene That Plays a Positive Role in Nodule Functioning.

Nitrogen-fixing nodules are new organs formed on legume roots as a result of the beneficial interaction with the soil bacteria, rhizobia. Proteins of the nitrate transporter 1/peptide transporter family (NPF) are largely represented in the subcategory of nodule-induced transporters identified in mature nodules. The role of nitrate as a signal/nutrient regulating nodule functioning has been recently highlighted in the literature, and NPFs may play a central role in both the permissive and inhibitory pathways controlling N2-fixation efficiency. In this study, we present the characterization of the Lotus japonicus LjNPF3.1 gene. LjNPF3.1 is upregulated in mature nodules. Promoter studies show transcriptional activation confined to the cortical region of both roots and nodules. Under symbiotic conditions, Ljnpf3.1-knockout mutant's display reduced shoot development and anthocyanin accumulation as a result of nutrient deprivation. Altogether, LjNPF3.1 plays a role in maximizing the beneficial outcome of the root nodule symbiosis.

VC1 catalyses a key step in the biosynthesis of vicine in faba bean.

Faba bean (Vicia faba L.) is a widely adapted and high-yielding legume cultivated for its protein-rich seeds1. However, the seeds accumulate the pyrimidine glucosides vicine and convicine, which can cause haemolytic anaemia (favism) in 400 million genetically predisposed individuals2. Here, we use gene-to-metabolite correlations, gene mapping and genetic complementation to identify VC1 as a key enzyme in vicine and convicine biosynthesis. We demonstrate that VC1 has GTP cyclohydrolase II activity and that the purine GTP is a precursor of both vicine and convicine. Finally, we show that cultivars with low vicine and convicine levels carry an inactivating insertion in the coding sequence of VC1. Our results reveal an unexpected, purine rather than pyrimidine, biosynthetic origin for vicine and convicine and pave the way for the development of faba bean cultivars that are free of these anti-nutrients.

Distinct signaling routes mediate intercellular and intracellular rhizobial infection in Lotus japonicus.

Rhizobial infection of legume roots during the development of nitrogen-fixing root nodules can occur intracellularly, through plant-derived infection threads traversing cells, or intercellularly, via bacterial entry between epidermal plant cells. Although it is estimated that around 25% of all legume genera are intercellularly infected, the pathways and mechanisms supporting this process have remained virtually unexplored due to a lack of genetically amenable legumes that exhibit this form of infection. In this study, we report that the model legume Lotus japonicus is infected intercellularly by the IRBG74 strain, recently proposed to belong to the Agrobacterium clade of the Rhizobiaceae. We demonstrate that the resources available for L. japonicus enable insight into the genetic requirements and fine-tuning of the pathway governing intercellular infection in this species. Inoculation of L. japonicus mutants shows that Ethylene-responsive factor required for nodulation 1 (Ern1) and Leu-rich Repeat Receptor-Like Kinase (RinRK1) are dispensable for intercellular infection in contrast to intracellular infection. Other symbiotic genes, including nod factor receptor 5 (NFR5), symbiosis receptor-like kinase (SymRK), Ca2+/calmodulin dependent kinase (CCaMK), exopolysaccharide receptor 3 (Epr3), Cyclops, nodule inception (Nin), nodulation signaling pathway 1 (Nsp1), nodulation signaling pathway 2 (Nsp2), cystathionine-ß-synthase (Cbs), and Vapyrin are equally important for both entry modes. Comparative RNAseq analysis of roots inoculated with IRBG74 revealed a distinctive transcriptome response compared with intracellular colonization. In particular, several cytokinin-related genes were differentially regulated. Corroborating this observation, cyp735A and ipt4 cytokinin biosynthesis mutants were significantly affected in their nodulation with IRBG74, whereas lhk1 cytokinin receptor mutants formed no nodules. These results indicate a differential requirement for cytokinin signaling during intercellular rhizobial entry and highlight distinct modalities of inter- and intracellular infection mechanisms in L. japonicus.

Natural variation identifies a Pxy gene controlling vascular organisation and formation of nodules and lateral roots in Lotus japonicus.

Forward and reverse genetics using the model legumes Lotus japonicus and Medicago truncatula have been instrumental in identifying the essential genes governing legume-rhizobia symbiosis. However, little information is known about the effects of intraspecific variation on symbiotic signalling. Here, we use quantitative trait locus sequencing (QTL-seq) to investigate the genetic basis of the differentiated phenotypic responses shown by the Lotus accessions Gifu and MG20 to inoculation with the Mesorhizobium loti exoU mutant that produces truncated exopolysaccharides. We identified through genetic complementation the Pxy gene as a component of this differential exoU response. Lotus Pxy encodes a leucine-rich repeat receptor-like kinase similar to Arabidopsis thaliana PXY, which regulates stem vascular development. We show that Lotus pxy insertion mutants displayed defects in root and stem vascular organisation, as well as lateral root and nodule formation. Our work links Pxy to de novo organogenesis in the root, highlights the genetic overlap between regulation of lateral root and nodule formation, and demonstrates that natural variation in Pxy affects nodulation signalling.

A Toolkit for High Resolution Imaging of Cell Division and Phytohormone Signaling in Legume Roots and Root Nodules.

Legume plants benefit from a nitrogen-fixing symbiosis in association with rhizobia hosted in specialized root nodules. Formation of root nodules is initiated by de novo organogenesis and coordinated infection of these developing lateral root organs by rhizobia. Both bacterial infection and nodule organogenesis involve cell cycle activation and regulation by auxin and cytokinin is tightly integrated in the process. To characterize the hormone dynamics and cell division patterns with cellular resolution during nodulation, sensitive and specific sensors suited for imaging of multicellular tissues are required. Here we report a modular toolkit, optimized in the model legume Lotus japonicus, for use in legume roots and root nodules. This toolkit includes synthetic transcriptional reporters for auxin and cytokinin, auxin accumulation sensors and cell cycle progression markers optimized for fluorescent and bright field microscopy. The developed vectors allow for efficient one-step assembly of multiple units using the GoldenGate cloning system. Applied together with a fluorescence-compatible clearing approach, these reporters improve imaging depth and facilitate fluorescence examination in legume roots. We additionally evaluate the utility of the dynamic gravitropic root response in altering the timing and location of auxin accumulation and nodule emergence. We show that alteration of auxin distribution in roots allows for preferential nodule emergence at the outer side of the bend corresponding to a region of high auxin signaling capacity. The presented tools and procedures open new possibilities for comparative mutant studies and for developing a more comprehensive understanding of legume-rhizobia 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.

Epidermal auxin biosynthesis facilitates rhizobial infection in Lotus japonicus.

Symbiotic nitrogen fixation in legumes requires nodule organogenesis to be coordinated with infection by rhizobia. The plant hormone auxin influences symbiotic infection, but the precise timing of auxin accumulation and the genetic network governing it remain unclear. We used a Lotus japonicus optimised variant of the DII-based auxin accumulation sensor and identified a rapid accumulation of auxin in the epidermis, specifically in the root hair cells. This auxin accumulation occurs in the infected root hairs during rhizobia invasion, while Nod factor application induces this response across a broader range of root hairs. Using the DR5 auxin responsive promoter, we demonstrate that activation of auxin signalling also occurs specifically in infected root hairs. Analysis of root hair transcriptome data identified induction of an auxin biosynthesis gene of the Tryptophan Amino-transferase Related (LjTar1) family following both bacteria inoculation and Nod factor treatment. Genetic analysis showed that both expression of the LjTar1 biosynthesis gene and the auxin response requires Nod factor perception, while common symbiotic pathway transcription factors are only partially required or act redundantly to initiate auxin accumulation. Using a chemical genetics approach, we confirmed that auxin biosynthesis has a functional role in promoting symbiotic infection events in the epidermis.

Cytokinin Biosynthesis Promotes Cortical Cell Responses during Nodule Development.

Legume mutants have shown the requirement for receptor-mediated cytokinin signaling in symbiotic nodule organogenesis. While the receptors are central regulators, cytokinin also is accumulated during early phases of symbiotic interaction, but the pathways involved have not yet been fully resolved. To identify the source, timing, and effect of this accumulation, we followed transcript levels of the cytokinin biosynthetic pathway genes in a sliding developmental zone of Lotus japonicus roots. LjIpt2 and LjLog4 were identified as the major contributors to the first cytokinin burst. The genetic dependence and Nod factor responsiveness of these genes confirm that cytokinin biosynthesis is a key target of the common symbiosis pathway. The accumulation of LjIpt2 and LjLog4 transcripts occurs independent of the LjLhk1 receptor during nodulation. Together with the rapid repression of both genes by cytokinin, this indicates that LjIpt2 and LjLog4 contribute to, rather than respond to, the initial cytokinin buildup. Analysis of the cytokinin response using the synthetic cytokinin sensor, TCSn, showed that this response occurs in cortical cells before spreading to the epidermis in L. japonicus While mutant analysis identified redundancy in several biosynthesis families, we found that mutation of LjIpt4 limits nodule numbers. Overexpression of LjIpt3 or LjLog4 alone was insufficient to produce the robust formation of spontaneous nodules. In contrast, overexpressing a complete cytokinin biosynthesis pathway leads to large, often fused spontaneous nodules. These results show the importance of cytokinin biosynthesis in initiating and balancing the requirement for cortical cell activation without uncontrolled cell proliferation.

Fine tuning chloroplast movements through physical interactions between phototropins.

Phototropins are plant photoreceptors which regulate numerous responses to blue light, including chloroplast relocation. Weak blue light induces chloroplast accumulation, whereas strong light leads to an avoidance response. Two Arabidopsis phototropins are characterized by different light sensitivities. Under continuous light, both can elicit chloroplast accumulation, but the avoidance response is controlled solely by phot2. As well as continuous light, brief light pulses also induce chloroplast displacements. Pulses of 0.1s and 0.2s of fluence rate saturating the avoidance response lead to transient chloroplast accumulation. Longer pulses (up to 20s) trigger a biphasic response, namely transient avoidance followed by transient accumulation. This work presents a detailed study of transient chloroplast responses in Arabidopsis. Phototropin mutants display altered chloroplast movements as compared with the wild type: phot1 is characterized by weaker responses, while phot2 exhibits enhanced chloroplast accumulation, especially after 0.1s and 0.2s pulses. To determine the cause of these differences, the abundance and phosphorylation levels of both phototropins, as well as the interactions between phototropin molecules are examined. The formation of phototropin homo- and heterocomplexes is the most plausible explanation of the observed phenomena. The physiological consequences of this interplay are discussed, suggesting the universal character of this mechanism that fine-tunes plant reactions to blue light. Additionally, responses in mutants of different protein phosphatase 2A subunits are examined to assess the role of protein phosphorylation in signaling of chloroplast movements.

The LORE1 insertion mutant resource.

Long terminal repeat (LTR) retrotransposons are closely related to retroviruses, and their activities shape eukaryotic genomes. Here, we present a complete Lotus japonicus insertion mutant collection generated by identification of 640 653 new insertion events following de novo activation of the LTR element Lotus retrotransposon 1 (LORE1) (http://lotus.au.dk). Insertion preferences are critical for effective gene targeting, and we exploit our large dataset to analyse LTR element characteristics in this context. We infer the mechanism that generates the consensus palindromes typical of retroviral and LTR retrotransposon insertion sites, identify a short relaxed insertion site motif, and demonstrate selective integration into CHG-hypomethylated genes. These characteristics result in a steep increase in deleterious mutation rate following activation, and allow LORE1 active gene targeting to approach saturation within a population of 134 682 L. japonicus lines. We suggest that saturation mutagenesis using endogenous LTR retrotransposons with germinal activity can be used as a general and cost-efficient strategy for generation of non-transgenic mutant collections for unrestricted use in plant research.