Widespread and transgenerational retrotransposon activation in inter- and intraspecies recombinant inbred populations of Lotus japonicus.

Transposable elements (TEs) constitute a large proportion of genomes of multicellular eukaryotes, including flowering plants. TEs are normally maintained in a silenced state and their transpositions rarely occur. Hybridization between distant species has been regarded as a 'shock' that stimulates genome reorganization, including TE mobilization. However, whether crosses between genetically close parents that result in viable and fertile offspring can induce TE transpositions has remained unclear. Here, we investigated the activation of long terminal repeat (LTR) retrotransposons in three Lotus japonicus recombinant inbred line (RIL) populations. We found that at least six LTR retrotransposon families were activated and transposed in 78% of the RILs investigated. LORE1a, one of the transposed LTR retrotransposons, showed transgenerational epigenetic activation, indicating the long-term effects of epigenetic instability induced by hybridization. Our study highlights TE activation as an unexpectedly common event in plant reproduction.

A Promiscuity Locus Confers Lotus burttii Nodulation with Rhizobia from Five Different Genera.

Legumes acquire access to atmospheric nitrogen through nitrogen fixation by rhizobia in root nodules. Rhizobia are soil-dwelling bacteria and there is a tremendous diversity of rhizobial species in different habitats. From the legume perspective, host range is a compromise between the ability to colonize new habitats, in which the preferred symbiotic partner may be absent, and guarding against infection by suboptimal nitrogen fixers. Here, we investigate natural variation in rhizobial host range across Lotus species. We find that Lotus burttii is considerably more promiscuous than Lotus japonicus, represented by the Gifu accession, in its interactions with rhizobia. This promiscuity allows Lotus burttii to form nodules with Mesorhizobium, Rhizobium, Sinorhizobium, Bradyrhizobium, and Allorhizobium species that represent five distinct genera. Using recombinant inbred lines, we have mapped the Gifu/burttii promiscuity quantitative trait loci (QTL) to the same genetic locus regardless of rhizobial genus, suggesting a general genetic mechanism for symbiont-range expansion. The Gifu/burttii QTL now provides an opportunity for genetic and mechanistic understanding of promiscuous legume-rhizobia interactions. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Three classes of hemoglobins are required for optimal vegetative and reproductive growth of Lotus japonicus: genetic and biochemical characterization of LjGlb2-1.

Legumes express two major types of hemoglobins, namely symbiotic (leghemoglobins) and non-symbiotic (phytoglobins), with the latter being categorized into three classes according to phylogeny and biochemistry. Using knockout mutants, we show that all three phytoglobin classes are required for optimal vegetative and reproductive development of Lotus japonicus. The mutants of two class 1 phytoglobins showed different phenotypes: Ljglb1-1 plants were smaller and had relatively more pods, whereas Ljglb1-2 plants had no distinctive vegetative phenotype and produced relatively fewer pods. Non-nodulated plants lacking LjGlb2-1 showed delayed growth and alterations in the leaf metabolome linked to amino acid processing, fermentative and respiratory pathways, and hormonal balance. The leaves of mutant plants accumulated salicylic acid and contained relatively less methyl jasmonic acid, suggesting crosstalk between LjGlb2-1 and the signaling pathways of both hormones. Based on the expression of LjGlb2-1 in leaves, the alterations of flowering and fruiting of nodulated Ljglb2-1 plants, the developmental and biochemical phenotypes of the mutant fed on ammonium nitrate, and the heme coordination and reactivity of the protein toward nitric oxide, we conclude that LjGlb2-1 is not a leghemoglobin but an unusual class 2 phytoglobin. For comparison, we have also characterized a close relative of LjGlb2-1 in Medicago truncatula, MtLb3, and conclude that this is an atypical leghemoglobin.

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.

Insights into the evolution of symbiosis gene copy number and distribution from a chromosome-scale Lotus japonicus Gifu genome sequence.

Lotus japonicus is a herbaceous perennial legume that has been used extensively as a genetically tractable model system for deciphering the molecular genetics of symbiotic nitrogen fixation. Our aim is to improve the L. japonicus reference genome sequence, which has so far been based on Sanger and Illumina sequencing reads from the L. japonicus accession MG-20 and contained a large fraction of unanchored contigs. Here, we use long PacBio reads from L. japonicus Gifu combined with Hi-C data and new high-density genetic maps to generate a high-quality chromosome-scale reference genome assembly for L. japonicus. The assembly comprises 554 megabases of which 549 were assigned to six pseudomolecules that appear complete with telomeric repeats at their extremes and large centromeric regions with low gene density. The new L. japonicus Gifu reference genome and associated expression data represent valuable resources for legume functional and comparative genomics. Here, we provide a first example by showing that the symbiotic islands recently described in Medicago truncatula do not appear to be conserved in L. japonicus.

Extreme genetic signatures of local adaptation during Lotus japonicus colonization of Japan.

Colonization of new habitats is expected to require genetic adaptations to overcome environmental challenges. Here, we use full genome re-sequencing and extensive common garden experiments to investigate demographic and selective processes associated with colonization of Japan by Lotus japonicus over the past ~20,000 years. Based on patterns of genomic variation, we infer the details of the colonization process where L. japonicus gradually spread from subtropical conditions to much colder climates in northern Japan. We identify genomic regions with extreme genetic differentiation between northern and southern subpopulations and perform population structure-corrected association mapping of phenotypic traits measured in a common garden. Comparing the results of these analyses, we find that signatures of extreme subpopulation differentiation overlap strongly with phenotype association signals for overwintering and flowering time traits. Our results provide evidence that these traits were direct targets of selection during colonization and point to associated candidate genes.

Altered Plant and Nodule Development and Protein S-Nitrosylation in Lotus japonicus Mutants Deficient in S-Nitrosoglutathione Reductases.

Nitric oxide (NO) is a crucial signaling molecule that conveys its bioactivity mainly through protein S-nitrosylation. This is a reversible post-translational modification (PTM) that may affect protein function. S-nitrosoglutathione (GSNO) is a cellular NO reservoir and NO donor in protein S-nitrosylation. The enzyme S-nitrosoglutathione reductase (GSNOR) degrades GSNO, thereby regulating indirectly signaling cascades associated with this PTM. Here, the two GSNORs of the legume Lotus japonicus, LjGSNOR1 and LjGSNOR2, have been functionally characterized. The LjGSNOR1 gene is very active in leaves and roots, whereas LjGSNOR2 is highly expressed in nodules. The enzyme activities are regulated in vitro by redox-based PTMs. Reducing conditions and hydrogen sulfide-mediated cysteine persulfidation induced both activities, whereas cysteine oxidation or glutathionylation inhibited them. Ljgsnor1 knockout mutants contained higher levels of S-nitrosothiols. Affinity chromatography and subsequent shotgun proteomics allowed us to identify 19 proteins that are differentially S-nitrosylated in the mutant and the wild-type. These include proteins involved in biotic stress, protein degradation, antioxidant protection and photosynthesis. We propose that, in the mutant plants, deregulated protein S-nitrosylation contributes to developmental alterations, such as growth inhibition, impaired nodulation and delayed flowering and fruiting. Our results highlight the importance of GSNOR function in legume biology.

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.

Sinorhizobium fredii HH103 nolR and nodD2 mutants gain capacity for infection thread invasion of Lotus japonicus Gifu and Lotus burttii.

Sinorhizobium fredii HH103 RifR , a broad-host-range rhizobial strain, forms ineffective nodules with Lotus japonicus but induces nitrogen-fixing nodules in Lotus burttii roots that are infected by intercellular entry. Here we show that HH103 RifR nolR or nodD2 mutants gain the ability to induce infection thread formation and to form nitrogen-fixing nodules in L. japonicus Gifu. Microscopy studies showed that the mode of infection of L. burttii roots by the nodD2 and nolR mutants switched from intercellular entry to infection threads (ITs). In the presence of the isoflavone genistein, both mutants overproduced Nod-factors. Transcriptomic analyses showed that, in the presence of Lotus japonicus Gifu root exudates, genes related to Nod factors production were overexpressed in both mutants in comparison to HH103 RifR . Complementation of the nodD2 and nolR mutants provoked a decrease in Nod-factor production, the incapacity to form nitrogen-fixing nodules with L. japonicus Gifu and restored the intercellular way of infection in L. burttii. Thus, the capacity of S. fredii HH103 RifR nodD2 and nolR mutants to infect L. burttii and L. japonicus Gifu by ITs and fix nitrogen L. japonicus Gifu might be correlated with Nod-factor overproduction, although other bacterial symbiotic signals could also be involved.

A plant chitinase controls cortical infection thread progression and nitrogen-fixing symbiosis.

Morphogens provide positional information and their concentration is key to the organized development of multicellular organisms. Nitrogen-fixing root nodules are unique organs induced by Nod factor-producing bacteria. Localized production of Nod factors establishes a developmental field within the root where plant cells are reprogrammed to form infection threads and primordia. We found that regulation of Nod factor levels by Lotus japonicus is required for the formation of nitrogen-fixing organs, determining the fate of this induced developmental program. Our analysis of plant and bacterial mutants shows that a host chitinase modulates Nod factor levels possibly in a structure-dependent manner. In Lotus, this is required for maintaining Nod factor signalling in parallel with the elongation of infection threads within the nodule cortex, while root hair infection and primordia formation are not influenced. Our study shows that infected nodules require balanced levels of Nod factors for completing their transition to functional, nitrogen-fixing organs.

A genetic screen for plant mutants with altered nodulation phenotypes in response to rhizobial glycan mutants.

Nodule primordia induced by rhizobial glycan mutants often remain uninfected. To identify processes involved in infection and organogenesis we used forward genetics to identify plant genes involved in perception and responses to bacterial glycans. To dissect the mechanisms underlying the negative plant responses to the Mesorhizobium loti R7AexoU and ML001cep mutants, a screen for genetic suppressors of the nodulation phenotypes was performed on a chemically mutagenized Lotus population. Two mutant lines formed infected nitrogen-fixing pink nodules, while five mutant lines developed uninfected large white nodules, presumably altered in processes controlling organogenesis. Genetic mapping identified a mutation in the cytokinin receptor Lhk1 resulting in an alanine to valine substitution adjacent to a coiled-coil motif in the juxta-membrane region of LHK1. This results in a spontaneous nodulation phenotype and increased ethylene production. The allele was renamed snf5, and segregation studies of snf5 together with complementation studies suggest that snf5 is a gain-of-function allele. This forward genetic approach to investigate the role of glycans in the pathway synchronizing infection and organogenesis shows that a combination of plant and bacterial genetics opens new possibilities to study glycan responses in plants as well as identification of mutant alleles affecting nodule organogenesis.

Lotus japonicus Genetic, Mutant, and Germplasm Resources.

A quarter of a century has passed since Lotus japonicus was proposed as a model legume because of its suitability for molecular genetic studies. Since then, a comprehensive set of genetic resources and tools has been developed, including recombinant inbred lines, a collection of wild accessions, published mutant lines, a large collection of mutant lines tagged with LORE1 insertions, cDNA clones with expressed sequence tag (EST) information, genomic clones with end-sequence information, and a reference genome sequence. Resource centers in Japan and Denmark ensure easy access to data and materials, and the resources have greatly facilitated L. japonicus research, thereby contributing to the molecular understanding of characteristic legume features such as endosymbiosis. Here, we provide detailed instructions for L. japonicus cultivation and describe how to order materials and access data using the resource center websites. The comprehensive overview presented here will make L. japonicus more easily accessible as a model system, especially for research groups new to L. japonicus research. © 2018 by John Wiley & Sons, Inc.

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.

User Guide for the LORE1 Insertion Mutant Resource.

Lotus japonicus is a model legume used in the study of plant-microbe interactions, especially in the field of biological nitrogen fixation due to its ability to enter into a symbiotic relationship with a soil bacterium, Mesorhizobium loti. The LORE1 mutant population is a valuable resource for reverse genetics in L. japonicus due to its non-transgenic nature, high tagging efficiency, and low copy count. Here, we outline a workflow for identifying, ordering, and establishing homozygous LORE1 mutant lines for a gene of interest, LjFls2, including protocols for growth and genotyping of a segregating LORE1 population.

SNOWY COTYLEDON 2 Promotes Chloroplast Development and Has a Role in Leaf Variegation in Both Lotus japonicus and Arabidopsis thaliana.

Plants contain various factors that transiently interact with subunits or intermediates of the thylakoid multiprotein complexes, promoting their stable association and integration. Hence, assembly factors are essential for chloroplast development and the transition from heterotrophic to phototrophic growth. Snowy cotyledon 2 (SCO2) is a DNAJ-like protein involved in thylakoid membrane biogenesis and interacts with the light-harvesting chlorophyll-binding protein LHCB1. In Arabidopsis thaliana, SCO2 function was previously reported to be restricted to cotyledons. Here we show that disruption of SCO2 in Lotus japonicus results not only in paler cotyledons but also in variegated true leaves. Furthermore, smaller and pale-green true leaves can also be observed in A. thaliana sco2 (atsco2) mutants under short-day conditions. In both species, SCO2 is required for proper accumulation of PSII-LHCII complexes. In contrast to other variegated mutants, inhibition of chloroplastic translation strongly affects L. japonicus sco2 mutant development and fails to suppress their variegated phenotype. Moreover, inactivation of the suppressor of variegation AtClpR1 in the atsco2 background results in an additive double-mutant phenotype with variegated true leaves. Taken together, our results indicate that SCO2 plays a distinct role in PSII assembly or repair and constitutes a novel factor involved in leaf variegation.

The Ethylene Responsive Factor Required for Nodulation 1 (ERN1) Transcription Factor Is Required for Infection-Thread Formation in Lotus japonicus.

Several hundred genes are transcriptionally regulated during infection-thread formation and development of nitrogen-fixing root nodules. We have characterized a set of Lotus japonicus mutants impaired in root-nodule formation and found that the causative gene, Ern1, encodes a protein with a characteristic APETALA2/Ethylene Responsive Factor (AP2/ERF) transcription-factor domain. Phenotypic characterization of four ern1 alleles shows that infection pockets are formed but root-hair infection threads are absent. Formation of root-nodule primordia is delayed and no normal transcellular infection threads are found in the infected nodules. Corroborating the role of ERN1 (ERF Required for Nodulation1) in nodule organogenesis, spontaneous nodulation induced by an autoactive CCaMK and cytokinin-induced nodule primordia were not observed in ern1 mutants. Expression of Ern1 is induced in the susceptible zone by Nod factor treatment or rhizobial inoculation. At the cellular level, the pErn1:GUS reporter is highly expressed in root epidermal cells of the susceptible zone and in the cortical cells that form nodule primordia. The genetic regulation of this cellular expression pattern was further investigated in symbiotic mutants. Nod factor induction of Ern1 in epidermal cells was found to depend on Nfr1, Cyclops, and Nsp2 but was independent of Nin and Nf-ya1. These results suggest that ERN1 functions as a transcriptional regulator involved in the formation of infection threads and development of nodule primordia and may coordinate these two processes.

Sinorhizobium fredii HH103 Invades Lotus burttii by Crack Entry in a Nod Factor-and Surface Polysaccharide-Dependent Manner.

Sinorhizobium fredii HH103-Rifr, a broad host range rhizobial strain, induces nitrogen-fixing nodules in Lotus burttii but ineffective nodules in L. japonicus. Confocal microscopy studies showed that Mesorhizobium loti MAFF303099 and S. fredii HH103-Rifr invade L. burttii roots through infection threads or epidermal cracks, respectively. Infection threads in root hairs were not observed in L. burttii plants inoculated with S. fredii HH103-Rifr. A S. fredii HH103-Rifr nodA mutant failed to nodulate L. burttii, demonstrating that Nod factors are strictly necessary for this crack-entry mode, and a noeL mutant was also severely impaired in L. burttii nodulation, indicating that the presence of fucosyl residues in the Nod factor is symbiotically relevant. However, significant symbiotic impacts due to the absence of methylation or to acetylation of the fucosyl residue were not detected. In contrast S. fredii HH103-Rifr mutants showing lipopolysaccharide alterations had reduced symbiotic capacity, while mutants affected in production of either exopolysaccharides, capsular polysaccharides, or both were not impaired in nodulation. Mutants unable to produce cyclic glucans and purine or pyrimidine auxotrophic mutants formed ineffective nodules with L. burttii. Flagellin-dependent bacterial mobility was not required for crack infection, since HH103-Rifr fla mutants nodulated L. burttii. None of the S. fredii HH103-Rifr surface-polysaccharide mutants gained effective nodulation with L. japonicus.

Hemoglobin LjGlb1-1 is involved in nodulation and regulates the level of nitric oxide in the Lotus japonicus-Mesorhizobium loti symbiosis.

Leghemoglobins transport and deliver O2 to the symbiosomes inside legume nodules and are essential for nitrogen fixation. However, the roles of other hemoglobins (Hbs) in the rhizobia-legume symbiosis are unclear. Several Lotus japonicus mutants affecting LjGlb1-1, a non-symbiotic class 1 Hb, have been used to study the function of this protein in symbiosis. Two TILLING alleles with single amino acid substitutions (A102V and E127K) and a LORE1 null allele with a retrotransposon insertion in the 5'-untranslated region (96642) were selected for phenotyping nodulation. Plants of all three mutant lines showed a decrease in long infection threads and nodules, and an increase in incipient infection threads. About 4h after inoculation, the roots of mutant plants exhibited a greater transient accumulation of nitric oxide (NO) than did the wild-type roots; nevertheless, in vitro NO dioxygenase activities of the wild-type, A102V, and E127K proteins were similar, suggesting that the mutated proteins are not fully functional in vivo The expression of LjGlb1-1, but not of the other class 1 Hb of L. japonicus (LjGlb1-2), was affected during infection of wild-type roots, further supporting a specific role for LjGlb1-1. In conclusion, the LjGlb1-1 mutants reveal that this protein is required during rhizobial infection and regulates NO levels.

High-resolution genetic maps of Lotus japonicus and L. burttii based on re-sequencing of recombinant inbred lines.

Recombinant inbred lines (RILs) derived from bi-parental populations are stable genetic resources, which are widely used for constructing genetic linkage maps. These genetic maps are essential for QTL mapping and can aid contig and scaffold anchoring in the final stages of genome assembly. In this study, two Lotus sp. RIL populations, Lotus japonicus MG-20 × Gifu and Gifu × L. burttii, were characterized by Illumina re-sequencing. Genotyping of 187 MG-20 × Gifu RILs at 87,140 marker positions and 96 Gifu × L. burttii RILs at 357,973 marker positions allowed us to accurately identify 1,929 recombination breakpoints in the MG-20 × Gifu RILs and 1,044 breakpoints in the Gifu × L. burttii population. The resulting high-density genetic maps now facilitate high-accuracy QTL mapping, identification of reference genome mis-assemblies, and characterization of structural variants.

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.