Unraveling plant-microbe symbioses using single-cell and spatial transcriptomics.

Plant-microbe symbioses require intense interaction and genetic coordination to successfully establish in specific cell types of the host and symbiont. Traditional RNA-seq methodologies lack the cellular resolution to fully capture these complexities, but single-cell and spatial transcriptomics (ST) are now allowing scientists to probe symbiotic interactions at an unprecedented level of detail. Here, we discuss the advantages that novel spatial and single-cell transcriptomic technologies provide in studying plant-microbe endosymbioses and highlight key recent studies. Finally, we consider the remaining limitations of applying these approaches to symbiosis research, which are mainly related to the simultaneous capture of both plant and microbial transcripts within the same cells.

Zinc mediates control of nitrogen fixation via transcription factor filamentation.

Plants adapt to fluctuating environmental conditions by adjusting their metabolism and gene expression to maintain fitness1. In legumes, nitrogen homeostasis is maintained by balancing nitrogen acquired from soil resources with nitrogen fixation by symbiotic bacteria in root nodules2-8. Here we show that zinc, an essential plant micronutrient, acts as an intracellular second messenger that connects environmental changes to transcription factor control of metabolic activity in root nodules. We identify a transcriptional regulator, FIXATION UNDER NITRATE (FUN), which acts as a sensor, with zinc controlling the transition between an inactive filamentous megastructure and an active transcriptional regulator. Lower zinc concentrations in the nodule, which we show occur in response to higher levels of soil nitrate, dissociates the filament and activates FUN. FUN then directly targets multiple pathways to initiate breakdown of the nodule. The zinc-dependent filamentation mechanism thus establishes a concentration readout to adapt nodule function to the environmental nitrogen conditions. In a wider perspective, these results have implications for understanding the roles of metal ions in integration of environmental signals with plant development and optimizing delivery of fixed nitrogen in legume crops.

Phenotyping revealed tolerance traits and genotypes for acidity and aluminum toxicity in European Vicia faba L.

Soil acidity is a global issue; soils with pH <4.5 are widespread in Europe. This acidity adversely affects nutrient availability to plants; pH levels <5.0 lead to aluminum (Al3+) toxicity, a significant problem that hinders root growth and nutrient uptake in faba bean (Vicia faba L.) and its symbiotic relationship with Rhizobium. However, little is known about the specific traits and tolerant genotypes among the European faba beans. This study aimed to identify response traits associated with tolerance to root zone acidity and Al3+ toxicity and potentially tolerant genotypes for future breeding efforts. Germplasm survey was conducted using 165 genotypes in a greenhouse aquaponics system. Data on the root and shoot systems were collected. Subsequently, 12 genotypes were selected for further phenotyping in peat medium, where data on physiological and morphological parameters were recorded along with biochemical responses in four selected genotypes. In the germplasm survey, about 30% of genotypes showed tolerance to acidity and approximately 10% exhibited tolerance to Al3+, while 7% showed tolerance to both. The phenotyping experiment indicated diverse morphological and physiological responses among treatments and genotypes. Acid and Al3+ increased proline concentration. Interaction between genotype and environment was observed for ascorbate peroxidase activity, malondialdehyde, and proline concentrations. Genomic markers associated with acidity and acid+Al3+-toxicity tolerances were identified using GWAS analysis. Four faba bean genotypes with varying levels of tolerance to acidity and Al3+ toxicity were identified.

Rhizobium nodule diversity and composition are influenced by clover host selection and local growth conditions.

While shaping of plant microbiome composition through 'host filtering' is well documented in legume-rhizobium symbioses, it is less clear to what extent different varieties and genotypes of the same plant species differentially influence symbiont community diversity and composition. Here, we compared how clover host varieties and genotypes affect the structure of Rhizobium populations in root nodules under conventional field and controlled greenhouse conditions. We first grew four Trifolium repens (white clover) F2 crosses and one variety in a conventional field trial and compared differences in root nodule Rhizobium leguminosarum symbiovar trifolii (Rlt) genotype diversity using high-throughput amplicon sequencing of chromosomal housekeeping (rpoB and recA) genes and auxiliary plasmid-borne symbiosis genes (nodA and nodD). We found that Rlt nodule diversities significantly differed between clover crosses, potentially due to host filtering. However, variance in Rlt diversity largely overlapped between crosses and was also explained by the spatial distribution of plants in the field, indicative of the role of local environmental conditions for nodule diversity. To test the effect of host filtering, we conducted a controlled greenhouse trial with a diverse Rlt inoculum and several host genotypes. We found that different clover varieties and genotypes of the same variety selected for significantly different Rlt nodule communities and that the strength of host filtering (deviation from the initial Rhizobium inoculant composition) was positively correlated with the efficiency of symbiosis (rate of plant greenness colouration). Together, our results suggest that selection by host genotype and local growth conditions jointly influence white clover Rlt nodule diversity and community composition.

A collection of novel Lotus japonicus LORE1 mutants perturbed in the nodulation program induced by the Agrobacterium pusense strain IRBG74.

The Lotus japonicus population carrying new Lotus retrotransposon 1 (LORE1) insertions represents a valuable biological resource for genetic research. New insertions were generated by activation of the endogenous retroelement LORE1a in the germline of the G329-3 plant line and arranged in a 2-D system for reverse genetics. LORE1 mutants identified in this collection contributes substantially to characterize candidate genes involved in symbiotic association of L. japonicus with its cognate symbiont, the nitrogen-fixing bacteria Mesorhizobium loti that infects root nodules intracellularly. In this study we aimed to identify novel players in the poorly explored intercellular infection induced by Agrobacterium pusense IRBG74 sp. For this purpose, a forward screen of > 200,000 LORE1 seedlings, obtained from bulk propagation of G329-3 plants, inoculated with IRBG74 was performed. Plants with perturbed nodulation were scored and the offspring were further tested on plates to confirm the symbiotic phenotype. A total of 110 Lotus mutants with impaired nodulation after inoculation with IRBG74 were obtained. A comparative analysis of nodulation kinetics in a subset of 20 mutants showed that most of the lines were predominantly affected in nodulation by IRBG74. Interestingly, additional defects in the main root growth were observed in some mutant lines. Sequencing of LORE1 flanking regions in 47 mutants revealed that 92 Lotus genes were disrupted by novel LORE1 insertions in these lines. In the IM-S34 mutant, one of the insertions was located in the 5´UTR of the LotjaGi5g1v0179800 gene, which encodes the AUTOPHAGY9 protein. Additional mutant alleles, named atg9-2 and atg9-3, were obtained in the reverse genetic collection. Nodule formation was significantly reduced in these mutant alleles after M. loti and IRBG74 inoculation, confirming the effectiveness of the mutant screening. This study describes an effective forward genetic approach to obtain novel mutants in Lotus with a phenotype of interest and to identify the causative gene(s).

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.

Lotus japonicus.

Andersen and Stougaard introduce the model legume Lotus japonicus.

Major effect loci for plant size before onset of nitrogen fixation allow accurate prediction of yield in white clover.

KEY MESSAGE: Accurate genomic prediction of yield within and across generations was achieved by estimating the genetic merit of individual white clover genotypes based on extensive genetic replication using cloned material. White clover is an agriculturally important forage legume grown throughout temperate regions as a mixed clover-grass crop. It is typically cultivated with low nitrogen input, making yield dependent on nitrogen fixation by rhizobia in root nodules. Here, we investigate the effects of clover and rhizobium genetic variation by monitoring plant growth and quantifying dry matter yield of 704 combinations of 145 clover genotypes and 170 rhizobium inocula. We find no significant effect of rhizobium variation. In contrast, we can predict yield based on a few white clover markers strongly associated with plant size prior to nitrogen fixation, and the prediction accuracy for polycross offspring yield is remarkably high. Several of the markers are located near a homolog of Arabidopsis thaliana GIGANTUS 1, which regulates growth rate and biomass accumulation. Our work provides fundamental insight into the genetics of white clover yield and identifies specific candidate genes as breeding targets.

Genetic variation is associated with differences in facilitative and competitive interactions in the Rhizobium leguminosarum species complex.

Competitive and facilitative interactions influence bacterial community composition, diversity and functioning. However, the role of genetic diversity for determining interactions between coexisting strains of the same, or closely related, species remains poorly understood. Here, we investigated the type (facilitative/inhibitory) and potential underlying mechanisms of pairwise interactions between 24 genetically diverse bacterial strains belonging to three genospecies (gsA,C,E) of the Rhizobium leguminosarum species complex. Interactions were determined indirectly, based on secreted compounds in cell-free supernatants, and directly, as growth inhibition in cocultures. We found supernatants mediated both facilitative and inhibitory interactions that varied greatly between strains and genospecies. Overall, gsE strains indirectly suppressed growth of gsA strains, while their own growth was facilitated by other genospecies' supernatants. Similar genospecies-level patterns were observed in direct competition, where gsA showed the highest susceptibility and gsE the highest inhibition capacity. At the genetic level, increased gsA susceptibility was associated with a non-random distribution of quorum sensing and secondary metabolite genes across genospecies. Together, our results suggest that genetic variation is associated with facilitative and competitive interactions, which could be important ecological mechanisms explaining R. leguminosarum diversity.

Competition, Nodule Occupancy, and Persistence of Inoculant Strains: Key Factors in the Rhizobium-Legume Symbioses.

Biological nitrogen fixation by Rhizobium-legume symbioses represents an environmentally friendly and inexpensive alternative to the use of chemical nitrogen fertilizers in legume crops. Rhizobial inoculants, applied frequently as biofertilizers, play an important role in sustainable agriculture. However, inoculants often fail to compete for nodule occupancy against native rhizobia with inferior nitrogen-fixing abilities, resulting in low yields. Strains with excellent performance under controlled conditions are typically selected as inoculants, but the rates of nodule occupancy compared to native strains are rarely investigated. Lack of persistence in the field after agricultural cycles, usually due to the transfer of symbiotic genes from the inoculant strain to naturalized populations, also limits the suitability of commercial inoculants. When rhizobial inoculants are based on native strains with a high nitrogen fixation ability, they often have superior performance in the field due to their genetic adaptations to the local environment. Therefore, knowledge from laboratory studies assessing competition and understanding how diverse strains of rhizobia behave, together with assays done under field conditions, may allow us to exploit the effectiveness of native populations selected as elite strains and to breed specific host cultivar-rhizobial strain combinations. Here, we review current knowledge at the molecular level on competition for nodulation and the advances in molecular tools for assessing competitiveness. We then describe ongoing approaches for inoculant development based on native strains and emphasize future perspectives and applications using a multidisciplinary approach to ensure optimal performance of both symbiotic partners.

Recombination Facilitates Adaptive Evolution in Rhizobial Soil Bacteria.

Homologous recombination is expected to increase natural selection efficacy by decoupling the fate of beneficial and deleterious mutations and by readily creating new combinations of beneficial alleles. Here, we investigate how the proportion of amino acid substitutions fixed by adaptive evolution (α) depends on the recombination rate in bacteria. We analyze 3,086 core protein-coding sequences from 196 genomes belonging to five closely related species of the genus Rhizobium. These genes are found in all species and do not display any signs of introgression between species. We estimate α using the site frequency spectrum (SFS) and divergence data for all pairs of species. We evaluate the impact of recombination within each species by dividing genes into three equally sized recombination classes based on their average level of intragenic linkage disequilibrium. We find that α varies from 0.07 to 0.39 across species and is positively correlated with the level of recombination. This is both due to a higher estimated rate of adaptive evolution and a lower estimated rate of nonadaptive evolution, suggesting that recombination both increases the fixation probability of advantageous variants and decreases the probability of fixation of deleterious variants. Our results demonstrate that homologous recombination facilitates adaptive evolution measured by α in the core genome of prokaryote species in agreement with studies in eukaryotes.

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.

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.

Recent advances in faba bean genetic and genomic tools for crop improvement.

Faba bean (Vicia faba L.), a member of the Fabaceae family, is one of the important food legumes cultivated in cool temperate regions. It holds great importance for human consumption and livestock feed because of its high protein content, dietary fibre, and nutritional value. Major faba bean breeding challenges include its mixed breeding system, unknown wild progenitor, and genome size of ~13 Gb, which is the largest among diploid field crops. The key breeding objectives in faba bean include improved resistance to biotic and abiotic stress and enhanced seed quality traits. Regarding quality traits, major progress on reduction of vicine-convicine and seed coat tannins, the main anti-nutritional factors limiting faba bean seed usage, have been recently achieved through gene discovery. Genomic resources are relatively less advanced compared with other grain legume species, but significant improvements are underway due to a recent increase in research activities. A number of bi-parental populations have been constructed and mapped for targeted traits in the last decade. Faba bean now benefits from saturated synteny-based genetic maps, along with next-generation sequencing and high-throughput genotyping technologies that are paving the way for marker-assisted selection. Developing a reference genome, and ultimately a pan-genome, will provide a foundational resource for molecular breeding. In this review, we cover the recent development and deployment of genomic tools for faba bean breeding.

Lotus japonicus Nuclear Factor YA1, a nodule emergence stage-specific regulator of auxin signalling.

Organogenesis of legume root nodules begins with the nodulation factor-dependent stimulation of compatible root cells to initiate divisions, signifying an early nodule primordium formation event. This is followed by cellular differentiation, including cell expansion and vascular bundle formation, and we previously showed that Lotus japonicus NF-YA1 is essential for this process, presumably by regulating three members of the SHORT INTERNODES/STYLISH (STY) transcription factor gene family. In this study, we used combined genetics, genomics and cell biology approaches to characterize the role of STY genes during root nodule formation and to test a hypothesis that they mediate nodule development by stimulating auxin signalling. We show here that L. japonicus STYs are required for nodule emergence. This is attributed to the NF-YA1-dependent regulatory cascade, comprising STY genes and their downstream targets, YUCCA1 and YUCCA11, involved in a local auxin biosynthesis at the post-initial cell division stage. An analogous NF-YA1/STY regulatory module seems to operate in Medicago truncatula in association with the indeterminate nodule patterning. Our data define L. japonicus and M. truncatula NF-YA1 genes as important nodule emergence stage-specific regulators of auxin signalling while indicating that the inductive stage and subsequent formation of early nodule primordia are mediated through an independent mechanism(s).

MAUI-seq: Metabarcoding using amplicons with unique molecular identifiers to improve error correction.

Sequencing and PCR errors are a major challenge when characterizing genetic diversity using high-throughput amplicon sequencing (HTAS). We have developed a multiplexed HTAS method, MAUI-seq, which uses unique molecular identifiers (UMIs) to improve error correction by exploiting variation among sequences associated with a single UMI. Erroneous sequences are recognized because, across the data set, they are over-represented among the minor sequences associated with UMIs. We show that two main advantages of this approach are efficient elimination of chimeric and other erroneous reads, outperforming dada2 and unoise3, and the ability to confidently recognize genuine alleles that are present at low abundance or resemble chimeras. The method provides sensitive and flexible profiling of diversity and is readily adaptable to most HTAS applications, including microbial 16S rRNA profiling and metabarcoding of environmental DNA.

Symbiosis genes show a unique pattern of introgression and selection within a Rhizobium leguminosarum species complex.

Rhizobia supply legumes with fixed nitrogen using a set of symbiosis genes. These can cross rhizobium species boundaries, but it is unclear how many other genes show similar mobility. Here, we investigate inter-species introgression using de novo assembly of 196 Rhizobium leguminosarum sv. trifolii genomes. The 196 strains constituted a five-species complex, and we calculated introgression scores based on gene-tree traversal to identify 171 genes that frequently cross species boundaries. Rather than relying on the gene order of a single reference strain, we clustered the introgressing genes into four blocks based on population structure-corrected linkage disequilibrium patterns. The two largest blocks comprised 125 genes and included the symbiosis genes, a smaller block contained 43 mainly chromosomal genes, and the last block consisted of three genes with variable genomic location. All introgression events were likely mediated by conjugation, but only the genes in the symbiosis linkage blocks displayed overrepresentation of distinct, high-frequency haplotypes. The three genes in the last block were core genes essential for symbiosis that had, in some cases, been mobilized on symbiosis plasmids. Inter-species introgression is thus not limited to symbiosis genes and plasmids, but other cases are infrequent and show distinct selection signatures.

Identification of novel genes involved in phosphate accumulation in Lotus japonicus through Genome Wide Association mapping of root system architecture and anion content.

Phosphate represents a major limiting factor for plant productivity. Plants have evolved different solutions to adapt to phosphate limitation ranging from a profound tuning of their root system architecture and metabolic profile to the evolution of widespread mutualistic interactions. Here we elucidated plant responses and their genetic basis to different phosphate levels in a plant species that is widely used as a model for AM symbiosis: Lotus japonicus. Rather than focussing on a single model strain, we measured root growth and anion content in response to different levels of phosphate in 130 Lotus natural accessions. This allowed us not only to uncover common as well as divergent responses within this species, but also enabled Genome Wide Association Studies by which we identified new genes regulating phosphate homeostasis in Lotus. Among them, we showed that insertional mutants of a cytochrome B5 reductase and a Leucine-Rich-Repeat receptor showed different phosphate concentration in plants grown under phosphate sufficient condition. Under low phosphate conditions, we found a correlation between plant biomass and the decrease of plant phosphate concentration in plant tissues, representing a dilution effect. Altogether our data of the genetic and phenotypic variation within a species capable of AM complements studies that have been conducted in Arabidopsis, and advances our understanding of the continuum of genotype by phosphate level interaction existing throughout dicot plants.

Atypical Receptor Kinase RINRK1 Required for Rhizobial Infection But Not Nodule Development in Lotus japonicus.

During the legume-rhizobium symbiotic interaction, rhizobial invasion of legumes is primarily mediated by a plant-made tubular invagination called an infection thread (IT). Here, we identify a gene in Lotus japonicus encoding a Leu-rich repeat receptor-like kinase (LRR-RLK), RINRK1 (Rhizobial Infection Receptor-like Kinase1), that is induced by Nod factors (NFs) and is involved in IT formation but not nodule organogenesis. A paralog, RINRK2, plays a relatively minor role in infection. RINRK1 is required for full induction of early infection genes, including Nodule Inception (NIN), encoding an essential nodulation transcription factor. RINRK1 displayed an infection-specific expression pattern, and NIN bound to the RINRK1 promoter, inducing its expression. RINRK1 was found to be an atypical kinase localized to the plasma membrane and did not require kinase activity for rhizobial infection. We propose RINRK1 is an infection-specific RLK, which may specifically coordinate output from NF signaling or perceive an unknown signal required for rhizobial infection.

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.