Comparative phylogenomics and phylotranscriptomics provide insights into the genetic complexity of nitrogen-fixing root-nodule symbiosis.

Plant root-nodule symbiosis (RNS) with mutualistic nitrogen-fixing bacteria is restricted to a single clade of angiosperms, the Nitrogen-Fixing Nodulation Clade (NFNC), and is best understood in the legume family. Nodulating species share many commonalities, explained either by divergence from a common ancestor over 100 million years ago or by convergence following independent origins over that same time period. Regardless, comparative analyses of diverse nodulation syndromes can provide insights into constraints on nodulation-what must be acquired or cannot be lost for a functional symbiosis-and the latitude for variation in the symbiosis. However, much remains to be learned about nodulation, especially outside of legumes. Here, we employed a large-scale phylogenomic analysis across 88 species, complemented by 151 RNA-seq libraries, to elucidate the evolution of RNS. Our phylogenomic analyses further emphasize the uniqueness of the transcription factor NIN as a master regulator of nodulation and identify key mutations that affect its function across the NFNC. Comparative transcriptomic assessment revealed nodule-specific upregulated genes across diverse nodulating plants, while also identifying nodule-specific and nitrogen-response genes. Approximately 70% of symbiosis-related genes are highly conserved in the four representative species, whereas defense-related and host-range restriction genes tend to be lineage specific. Our study also identified over 900 000 conserved non-coding elements (CNEs), over 300 000 of which are unique to sampled NFNC species. NFNC-specific CNEs are enriched with the active H3K9ac mark and are correlated with accessible chromatin regions, thus representing a pool of candidate regulatory elements for genes involved in RNS. Collectively, our results provide novel insights into the evolution of nodulation and lay a foundation for engineering of RNS traits in agriculturally important crops.

Primary and Secondary Metabolites in Lotus japonicus.

Lotus japonicus is a leguminous model plant used to gain insight into plant physiology, stress response, and especially symbiotic plant-microbe interactions, such as root nodule symbiosis or arbuscular mycorrhiza. Responses to changing environmental conditions, stress, microbes, or insect pests are generally accompanied by changes in primary and secondary metabolism to account for physiological needs or to produce defensive or signaling compounds. Here we provide an overview of the primary and secondary metabolites identified in L. japonicus to date. Identification of the metabolites is mainly based on mass spectral tags (MSTs) obtained by gas chromatography linked with tandem mass spectrometry (GC-MS/MS) or liquid chromatography-MS/MS (LC-MS/MS). These MSTs contain retention index and mass spectral information, which are compared to databases with MSTs of authentic standards. More than 600 metabolites are grouped into compound classes such as polyphenols, carbohydrates, organic acids and phosphates, lipids, amino acids, nitrogenous compounds, phytohormones, and additional defense compounds. Their physiological effects are briefly discussed, and the detection methods are explained. This review of the exisiting literature on L. japonicus metabolites provides a valuable basis for future metabolomics studies.

RPG interacts with E3-ligase CERBERUS to mediate rhizobial infection in Lotus japonicus.

Symbiotic interactions between rhizobia and legumes result in the formation of root nodules, which fix nitrogen that can be used for plant growth. Rhizobia usually invade legume roots through a plant-made tunnel-like structure called an infection thread (IT). RPG (Rhizobium-directed polar growth) encodes a coiled-coil protein that has been identified in Medicago truncatula as required for root nodule infection, but the function of RPG remains poorly understood. In this study, we identified and characterized RPG in Lotus japonicus and determined that it is required for IT formation. RPG was induced by Mesorhizobium loti or purified Nodulation factor and displayed an infection-specific expression pattern. Nodule inception (NIN) bound to the RPG promoter and induced its expression. We showed that RPG displayed punctate subcellular localization in L. japonicus root protoplasts and in root hairs infected by M. loti. The N-terminal predicted C2 lipid-binding domain of RPG was not required for this subcellular localization or for function. CERBERUS, a U-box E3 ligase which is also required for rhizobial infection, was found to be localized similarly in puncta. RPG co-localized and directly interacted with CERBERUS in the early endosome (TGN/EE) compartment and near the nuclei in root hairs after rhizobial inoculation. Our study sheds light on an RPG-CERBERUS protein complex that is involved in an exocytotic pathway mediating IT elongation.

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.

A CCaMK/Cyclops response element in the promoter of Lotus japonicus calcium-binding protein 1 (CBP1) mediates transcriptional activation in root symbioses.

Early gene expression in arbuscular mycorrhiza (AM) and the nitrogen-fixing root nodule symbiosis (RNS) is governed by a shared regulatory complex. Yet many symbiosis-induced genes are specifically activated in only one of the two symbioses. The Lotus japonicus T-DNA insertion line T90, carrying a promoterless uidA (GUS) gene in the promoter of Calcium Binding Protein 1 (CBP1) is exceptional as it exhibits GUS activity in both root endosymbioses. To identify the responsible cis- and trans-acting factors, we subjected deletion/modification series of CBP1 promoter : reporter fusions to transactivation and spatio-temporal expression analysis and screened ethyl methanesulphonate (EMS)-mutagenized T90 populations for aberrant GUS expression. We identified one cis-regulatory element required for GUS expression in the epidermis and a second element, necessary and sufficient for transactivation by the calcium and calmodulin-dependent protein kinase (CCaMK) in combination with the transcription factor Cyclops and conferring gene expression during both AM and RNS. Lack of GUS expression in T90 white mutants could be traced to DNA hypermethylation detected in and around this element. We concluded that the CCaMK/Cyclops complex can contribute to at least three distinct gene expression patterns on its direct target promoters NIN (RNS), RAM1 (AM), and CBP1 (AM and RNS), calling for yet-to-be identified specificity-conferring factors.

A strawberry accession with elevated methyl anthranilate fruit concentration is naturally resistant to the pest fly Drosophila suzukii.

During the past decade, Drosophila suzukii has established itself as a global invasive fruit pest, enabled by its ability to lay eggs into fresh, ripening fruit. In a previous study, we investigated the impact of different strawberry accessions on the development of D. suzukii eggs, in the search of natural resistance. We identified several accessions that significantly reduced adult fly emergence from infested fruit. In the present study, we aimed at understanding the chemical basis of this effect. We first noted that one of the more resistant accessions showed an unusual enrichment of methyl anthranilate within its fruit, prompting us to investigate this fruit compound as a possible cause limiting fly development. We found that methyl anthranilate alone triggers embryo lethality in a concentration-dependent manner, unlike another comparable organic fruit compound. We also showed that a chemical fraction of the resistant strawberry accession that contains methyl anthranilate carries some activity toward the egg hatching rate. Surprisingly, in spite of the lethal effect of this compound to their eggs, adult females are not only attracted to methyl anthranilate at certain concentrations, but they also display a concentration-dependent preference to lay on substrates enriched in methyl anthranilate. This study demonstrates that methyl anthranilate is a potent agonist molecule against D. suzukii egg development. Its elevated concentration in a specific strawberry accession proven to reduce the fly development may explain, at least in part the fruit resistance. It further illustrates how a single, natural compound, non-toxic to humans could be exploited for biological control of a pest species.

Maintenance and Quantitative Phenotyping of the Oomycete-plant Model Pathosystem Hyaloperonospora arabidopsidis-Arabidopsis.

The interaction between the host plant Arabidopsis thaliana (Arabidopsis) and the oomycete Hyaloperonospora arabidopsidis (Hpa) is an established model system for the study of an obligate biotrophic downy mildew interaction. The evaluation of the developmental success of Hpa is often based on the quantification of reproductive structures that are formed on the surface of leaves, such as the sporangiophores or the conidiospores they carry. However, the structural basis of this interaction lies within the plant tissue and, in particular, the haustoria that form inside plant cells. Therefore, valuable additional information about the performance and compatibility of the downy mildew interaction can be gained by light microscopical inspection of the hyphal and haustorial shape inside the plant tissue and within plant cells respectively. Here we describe a protocol for the visualization and quantification of morphological phenotypes inside the plant. While we focus specifically on the quantification of haustorial shape variants, the protocol can easily be adapted for the quantification of other morphological features such as hyphal deformations, or oogonia frequency. By including and refining already existing protocols from a variety of sources, we assembled the entire experimental pipeline for the Arabidopsis Hpa bioassay to provide a practical guide for the initial setup of this system in the laboratory. This pipeline includes the following steps: A) growing Arabidopsis, B) Hpa propagation and strain maintainance C) Hpa inoculation and incubation D) staining of plant tissues for visualization of the pathogen and E) an introduction of the Keyence VHX microscope and Fiji plugin of ImageJ for the quantification of structures of interest. While described here for Arabidopsis and Hpa, the protocol steps B-E should be easily adjustable for the study of other plant-oomycete pathosystems.

LCO Receptors Involved in Arbuscular Mycorrhiza Are Functional for Rhizobia Perception in Legumes.

Bacterial lipo-chitooligosaccharides (LCOs) are key mediators of the nitrogen-fixing root nodule symbiosis (RNS) in legumes. The isolation of LCOs from arbuscular mycorrhizal fungi suggested that LCOs are also signaling molecules in arbuscular mycorrhiza (AM). However, the corresponding plant receptors have remained uncharacterized. Here we show that petunia and tomato mutants in the LysM receptor-like kinases LYK10 are impaired in AM formation. Petunia and tomato LYK10 proteins have a high affinity for LCOs (Kd in the nM range) comparable to that previously reported for a legume LCO receptor essential for the RNS. Interestingly, the tomato and petunia LYK10 promoters, when introduced into a legume, were active in nodules similarly to the promoter of the legume orthologous gene. Moreover, tomato and petunia LYK10 coding sequences restored nodulation in legumes mutated in their orthologs. This combination of genetic and biochemical data clearly pinpoints Solanaceous LYK10 as part of an ancestral LCO perception system involved in AM establishment, which has been directly recruited during evolution of the RNS in legumes.

A unified multi-kingdom Golden Gate cloning platform.

Assembling composite DNA modules from custom DNA parts has become routine due to recent technological breakthroughs such as Golden Gate modular cloning. Using Golden Gate, one can efficiently assemble custom transcription units and piece units together to generate higher-order assemblies. Although Golden Gate cloning systems have been developed to assemble DNA plasmids required for experimental work in model species, they are not typically applicable to organisms from other kingdoms. Consequently, a typical molecular biology laboratory working across kingdoms must use multiple cloning strategies to assemble DNA constructs for experimental assays. To simplify the DNA assembly process, we developed a multi-kingdom (MK) Golden Gate assembly platform for experimental work in species from the kingdoms Fungi, Eubacteria, Protista, Plantae, and Animalia. Plasmid backbone and part overhangs are consistent across the platform, saving both time and resources in the laboratory. We demonstrate the functionality of the system by performing a variety of experiments across kingdoms including genome editing, fluorescence microscopy, and protein interaction assays. The versatile MK system therefore streamlines the assembly of modular DNA constructs for biological assays across a range of model organisms.

A set of Arabidopsis genes involved in the accommodation of the downy mildew pathogen Hyaloperonospora arabidopsidis.

The intracellular accommodation structures formed by plant cells to host arbuscular mycorrhiza fungi and biotrophic hyphal pathogens are cytologically similar. Therefore we investigated whether these interactions build on an overlapping genetic framework. In legumes, the malectin-like domain leucine-rich repeat receptor kinase SYMRK, the cation channel POLLUX and members of the nuclear pore NUP107-160 subcomplex are essential for symbiotic signal transduction and arbuscular mycorrhiza development. We identified members of these three groups in Arabidopsis thaliana and explored their impact on the interaction with the oomycete downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa). We report that mutations in the corresponding genes reduced the reproductive success of Hpa as determined by sporangiophore and spore counts. We discovered that a developmental transition of haustorial shape occurred significantly earlier and at higher frequency in the mutants. Analysis of the multiplication of extracellular bacterial pathogens, Hpa-induced cell death or callose accumulation, as well as Hpa- or flg22-induced defence marker gene expression, did not reveal any traces of constitutive or exacerbated defence responses. These findings point towards an overlap between the plant genetic toolboxes involved in the interaction with biotrophic intracellular hyphal symbionts and pathogens in terms of the gene families involved.

Dryas as a Model for Studying the Root Symbioses of the Rosaceae.

The nitrogen-fixing root nodule symbiosis is restricted to four plant orders: Fabales (legumes), Fagales, Cucurbitales and Rosales (Elaeagnaceae, Rhamnaceae, and Rosaceae). Interestingly all of the Rosaceae genera confirmed to contain nodulating species (i.e., Cercocarpus, Chamaebatia, Dryas, and Purshia) belong to a single subfamily, the Dryadoideae. The Dryas genus is particularly interesting from an evolutionary perspective because it contains closely related nodulating (Dryas drummondii) and non-nodulating species (Dryas octopetala). The close phylogenetic relationship between these two species makes Dryas an ideal model genus to study the genetic basis of nodulation by whole genome comparison and classical genetics. Therefore, we established methods for plant cultivation, transformation and DNA extraction for these species. We optimized seed surface sterilization and germination methods and tested growth protocols ranging from pots and Petri dishes to a hydroponic system. Transgenic hairy roots were obtained by adapting Agrobacterium rhizogenes-based transformation protocols for Dryas species. We compared several DNA extraction protocols for their suitability for subsequent molecular biological analysis. Using CTAB extraction, reproducible PCRs could be performed, but CsCl gradient purification was essential to obtain DNA in sufficient purity for high quality de novo genome sequencing of both Dryas species. Altogether, we established a basic toolkit for the culture, transient transformation and genetic analysis of Dryas sp.

Uptake of bacteria into living plant cells, the unifying and distinct feature of the nitrogen-fixing root nodule symbiosis.

Despite the presence of complex microbiota on the surfaces of all plants, the uptake of bacteria into plant cells and the subsequent accommodation in a membrane-enclosed compartment is restricted to the nitrogen-fixing root nodule and the Gunnera-Nostoc symbiosis. The plant cell wall and the outward-directed turgor pressure are major constraints for bacterial uptake because localised lysis of the cell wall endangers the integrity of the protoplast. Host cell integrity is consistently maintained by turgescent neighbours, connected via apoplastic polymers that seal a bacteria-containing extracellular compartment prior to localized cell wall lysis. Its unifying and almost exclusive phylogenetic distribution pinpoints the ability to take up bacteria into living plant cells as a key step during the evolution of the nitrogen-fixing root nodule symbiosis.

Phylogenomics reveals multiple losses of nitrogen-fixing root nodule symbiosis.

The root nodule symbiosis of plants with nitrogen-fixing bacteria affects global nitrogen cycles and food production but is restricted to a subset of genera within a single clade of flowering plants. To explore the genetic basis for this scattered occurrence, we sequenced the genomes of 10 plant species covering the diversity of nodule morphotypes, bacterial symbionts, and infection strategies. In a genome-wide comparative analysis of a total of 37 plant species, we discovered signatures of multiple independent loss-of-function events in the indispensable symbiotic regulator NODULE INCEPTION in 10 of 13 genomes of nonnodulating species within this clade. The discovery that multiple independent losses shaped the present-day distribution of nitrogen-fixing root nodule symbiosis in plants reveals a phylogenetically wider distribution in evolutionary history and a so-far-underestimated selection pressure against this symbiosis.

Advances and current challenges in calcium signaling.

Content Summary 414 I. Introduction 415 II. Ca2+ importer and exporter in plants 415 III. The Ca2+ decoding toolkit in plants 415 IV. Mechanisms of Ca2+ signal decoding 417 V. Immediate Ca2+ signaling in the regulation of ion transport 418 VI. Ca2+ signal integration into long-term ABA responses 419 VII Integration of Ca2+ and hormone signaling through dynamic complex modulation of the CCaMK/CYCLOPS complex 420 VIII Ca2+ signaling in mitochondria and chloroplasts 422 IX A view beyond recent advances in Ca2+ imaging 423 X Modeling approaches in Ca2+ signaling 424 XI Conclusions: Ca2+ signaling a still young blooming field of plant research 424 Acknowledgements 425 ORCID 425 References 425 SUMMARY: Temporally and spatially defined changes in Ca2+ concentration in distinct compartments of cells represent a universal information code in plants. Recently, it has become evident that Ca2+ signals not only govern intracellular regulation but also appear to contribute to long distance or even organismic signal propagation and physiological response regulation. Ca2+ signals are shaped by an intimate interplay of channels and transporters, and during past years important contributing individual components have been identified and characterized. Ca2+ signals are translated by an elaborate toolkit of Ca2+ -binding proteins, many of which function as Ca2+ sensors, into defined downstream responses. Intriguing progress has been achieved in identifying specific modules that interconnect Ca2+ decoding proteins and protein kinases with downstream target effectors, and in characterizing molecular details of these processes. In this review, we reflect on recent major advances in our understanding of Ca2+ signaling and cover emerging concepts and existing open questions that should be informative also for scientists that are currently entering this field of ever-increasing breath and impact.

A quantitative hypermorphic CNGC allele confers ectopic calcium flux and impairs cellular development.

The coordinated control of Ca2+ signaling is essential for development in eukaryotes. Cyclic nucleotide-gated channel (CNGC) family members mediate Ca2+ influx from cellular stores in plants (Charpentier et al., 2016; Gao et al., 2016; Frietsch et al., 2007; Urquhart et al., 2007). Here, we report the unusual genetic behavior of a quantitative gain-of-function CNGC mutation (brush) in Lotus japonicus resulting in a leaky tetrameric channel. brush resides in a cluster of redundant CNGCs encoding subunits which resemble metazoan voltage-gated potassium (Kv1-Kv4) channels in assembly and gating properties. The recessive mongenic brush mutation impaired root development and infection by nitrogen-fixing rhizobia. The brush allele exhibited quantitative behavior since overexpression of the cluster subunits was required to suppress the brush phenotype. The results reveal a mechanism by which quantitative competition between channel subunits for tetramer assembly can impact the phenotype of the mutation carrier.

Lipid transfer from plants to arbuscular mycorrhiza fungi.

Arbuscular mycorrhiza (AM) symbioses contribute to global carbon cycles as plant hosts divert up to 20% of photosynthate to the obligate biotrophic fungi. Previous studies suggested carbohydrates as the only form of carbon transferred to the fungi. However, de novo fatty acid (FA) synthesis has not been observed in AM fungi in absence of the plant. In a forward genetic approach, we identified two Lotus japonicus mutants defective in AM-specific paralogs of lipid biosynthesis genes (KASI and GPAT6). These mutants perturb fungal development and accumulation of emblematic fungal 16:1ω5 FAs. Using isotopolog profiling we demonstrate that 13C patterns of fungal FAs recapitulate those of wild-type hosts, indicating cross-kingdom lipid transfer from plants to fungi. This transfer of labelled FAs was not observed for the AM-specific lipid biosynthesis mutants. Thus, growth and development of beneficial AM fungi is not only fueled by sugars but depends on lipid transfer from plant hosts.

Cell Biology: Control of Partner Lifetime in a Plant-Fungus Relationship.

Arbuscules are tree-shaped fungal structures inside plant root cells that facilitate the exchange of nutrients delivered by the fungus with carbon sources from the host. To maintain symbiotic efficiency, plant cells can trigger degeneration of underperforming arbuscules. A recent study reveals the first transcription factor, which induces genes encoding hydrolytic enzymes, to mediate arbuscule degeneration.

The ERN1 transcription factor gene is a target of the CCaMK/CYCLOPS complex and controls rhizobial infection in Lotus japonicus.

Bacterial accommodation inside living plant cells is restricted to the nitrogen-fixing root nodule symbiosis. In many legumes, bacterial uptake is mediated via tubular structures called infection threads (ITs). To identify plant genes required for successful symbiotic infection, we screened an ethyl methanesulfonate mutagenized population of Lotus japonicus for mutants defective in IT formation and cloned the responsible gene, ERN1, encoding an AP2/ERF transcription factor. We performed phenotypic analysis of two independent L. japonicus mutant alleles and investigated the regulation of ERN1 via transactivation and DNA-protein interaction assays. In ern1 mutant roots, nodule primordia formed, but most remained uninfected and bacterial entry via ITs into the root epidermis was abolished. Infected cortical nodule cells contained bacteroids, but transcellular ITs were rarely observed. A subset exhibited localized cell wall degradation and loss of cell integrity associated with bacteroid spread into neighbouring cells and the apoplast. Functional promoter studies revealed that CYCLOPS binds in a sequence-specific manner to a motif within the ERN1 promoter and in combination with CCaMK positively regulates ERN1 transcription. We conclude that the activation of ERN1 by CCaMK/CYCLOPS complex is an important step controlling IT-mediated bacterial progression into plant cells.

The relationship between thiamine and two symbioses: Root nodule symbiosis and arbuscular mycorrhiza.

Lotus japonicus THIC is expressed in all organs, and the encoded protein catalyzes thiamine biosynthesis. Loss of function produces chlorosis, a typical thiamine-deficiency phenotype, and mortality. To investigate thiamine's role in symbiosis, we focused on THI1, a thiamine-biosynthesis gene expressed in roots, nodules, and seeds. The thi1 mutant had green leaves, but formed small nodules and immature seeds. These phenotypes were rescued by THI1 complementation and by exogenous thiamine. Thus, THI1 is required for nodule enlargement and seed maturation. On the other hand, colonization by arbuscular mycorrhiza (AM) fungus Rhizophagus irregularis was not affected in the thi1 mutant or by exogenous thiamine. However, spores of R. irregularis stored more thiamine than the source (host plants), despite lacking thiamine biosynthesis genes. Therefore, disturbance of the thiamine supply would affect progeny phenotypes such as spore formation and hyphal growth. Further investigation will be required to elucidate thiamine's effect on AM.

The Thiamine Biosynthesis Gene THI1 Promotes Nodule Growth and Seed Maturation.

Thiamine (vitamin B1) is essential for living organisms. Unlike animals, plants can synthesize thiamine. In Lotus japonicus, the expression of two thiamine biosynthesis genes, THI1 and THIC, was enhanced by inoculation with rhizobia but not by inoculation with arbuscular mycorrhizal fungi. THIC and THI2 (a THI1 paralog) were expressed in uninoculated leaves. THI2-knockdown plants and the transposon insertion mutant thiC had chlorotic leaves. This typical phenotype of thiamine deficiency was rescued by an exogenous supply of thiamine. In wild-type plants, THI1 was expressed mainly in roots and nodules, and the thi1 mutant had green leaves even in the absence of exogenous thiamine. THI1 was highly expressed in actively dividing cells of nodule primordia. The thi1 mutant had small nodules, and this phenotype was rescued by exogenous thiamine and by THI1 complementation. Exogenous thiamine increased nodule diameter, but the level of arbuscular mycorrhizal colonization was unaffected in the thi1 mutant or by exogenous thiamine. Expression of symbiotic marker genes was induced normally, implying that mainly nodule growth was delayed in the thi1 mutant. Furthermore, this mutant formed many immature seeds with reduced seed weight. These results indicate that thiamine biosynthesis mediated by THI1 enhances nodule enlargement and is required for seed development in L. japonicus.