The single-cell transcriptome program of nodule development cellular lineages in Medicago truncatula.

Legumes establish a symbiotic relationship with nitrogen-fixing rhizobia by developing nodules. Nodules are modified lateral roots that undergo changes in their cellular development in response to bacteria, but the transcriptional reprogramming that occurs in these root cells remains largely uncharacterized. Here, we describe the cell-type-specific transcriptome response of Medicago truncatula roots to rhizobia during early nodule development in the wild-type genotype Jemalong A17, complemented with a hypernodulating mutant (sunn-4) to expand the cell population responding to infection and subsequent biological inferences. The analysis identifies epidermal root hair and stele sub-cell types associated with a symbiotic response to infection and regulation of nodule proliferation. Trajectory inference shows cortex-derived cell lineages differentiating to form the nodule primordia and, posteriorly, its meristem, while modulating the regulation of phytohormone-related genes. Gene regulatory analysis of the cell transcriptomes identifies new regulators of nodulation, including STYLISH 4, for which the function is validated.

TML1 AND TML2 SYNERGISTICALLY REGULATE NODULATION BUT NOT ARBUSCULAR MYCORRHIZA IN MEDICAGO TRUNCATULA.

Two symbiotic processes, nodulation and arbuscular mycorrhiza, are primarily controlled by the plant's need for nitrogen (N) and phosphorus (P), respectively. Autoregulation of Nodulation (AON) and Autoregulation of Mycorrhization (AOM) share multiple components - plants that make too many nodules usually have higher arbuscule density. The protein TML (TOO MUCH LOVE) was shown to function in roots to maintain susceptibly to rhizobial infection under low N conditions and control nodule number through AON in Lotus japonicus. M. truncatula has two sequence homologs: MtTML1 and MtTML2. We report the generation of stable single and double mutants harboring multiple allelic variations in MtTML1 and MtTML2 using CRISPR-Cas9 targeted mutagenesis and screening of a transposon mutagenesis library. Plants containing single mutations in either gene produced twice the nodules of wild type plants whereas plants containing mutations in both genes displayed a synergistic effect, forming 20x more nodules and short roots compared to wild type plants. The synergistic effect on nodulation was maintained in the presence of 10mM nitrogen, but not observed in root length phenotypes. Examination of expression and heterozygote effects suggest genetic compensation may play a role in the observed synergy. However, plants with mutations in both TMLs had no detectable change in arbuscular mycorrhizal associations, suggesting that MtTMLs are specific to nodulation and nitrate signaling. The mutants created will be useful tools to dissect the mechanism of synergistic action of MtTML1 and MtTML2 in M. truncatula nodulation as well as the separation of AON from AOM.

Laser Capture Microdissection Transcriptome Reveals Spatiotemporal Tissue Gene Expression Patterns of Medicago truncatula Roots Responding to Rhizobia.

We report a public resource for examining the spatiotemporal RNA expression of 54,893 Medicago truncatula genes during the first 72 h of response to rhizobial inoculation. Using a methodology that allows synchronous inoculation and growth of more than 100 plants in a single media container, we harvested the same segment of each root responding to rhizobia in the initial inoculation over a time course, collected individual tissues from these segments with laser capture microdissection, and created and sequenced RNA libraries generated from these tissues. We demonstrate the utility of the resource by examining the expression patterns of a set of genes induced very early in nodule signaling, as well as two gene families (CLE peptides and nodule specific PLAT-domain proteins) and show that despite similar whole-root expression patterns, there are tissue differences in expression between the genes. Using a rhizobial response dataset generated from transcriptomics on intact root segments, we also examined differential temporal expression patterns and determined that, after nodule tissue, the epidermis and cortical cells contained the most temporally patterned genes. We circumscribed gene lists for each time and tissue examined and developed an expression pattern visualization tool. Finally, we explored transcriptomic differences between the inner cortical cells that become nodules and those that do not, confirming that the expression of 1-aminocyclopropane-1-carboxylate synthases distinguishes inner cortical cells that become nodules and provide and describe potential downstream genes involved in early nodule cell division. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Fixation and Laser Capture Microdissection of Plant Tissue for RNA Extraction and RNASeq Library Preparation.

To study the transcriptome of individual plant cells at specific points in time, we developed protocols for fixation, embedding, and sectioning of plant tissue followed by laser capture microdissection (LCM) and processing for RNA recovery. LCM allows the isolation of individual cell types from heterogeneous tissue sections and is particularly suited to plant processing because it does not require the breakdown of cell walls. This approach allows accurate separation of a small volume of cells that can be used to study gene expression profiles in different tissues or cell layers. The technique requires neither separation of cells by enzymatic digestion of any kind nor cell-specific reporter genes, and it allows storage of fixed and embedded tissue for months before capture. The methods for fixation, embedding, sectioning, and capturing of plant cells that we describe yield high-quality RNA suitable for making libraries for RNASeq. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Tissue Preparation for Laser Capture Microdissection Basic Protocol 2: Tissue Sectioning Basic Protocol 3: Laser Capture Microdissection of Embedded Tissue Basic Protocol 4: RNA Extraction from Laser Capture Microdissection Samples.

A Medicago truncatula Autoregulation of Nodulation Mutant Transcriptome Analysis Reveals Disruption of the SUNN Pathway Causes Constitutive Expression Changes in Some Genes, but Overall Response to Rhizobia Resembles Wild-Type, Including Induction of TML1 and TML2.

Nodule number regulation in legumes is controlled by a feedback loop that integrates nutrient and rhizobia symbiont status signals to regulate nodule development. Signals from the roots are perceived by shoot receptors, including a CLV1-like receptor-like kinase known as SUNN in Medicago truncatula. In the absence of functional SUNN, the autoregulation feedback loop is disrupted, resulting in hypernodulation. To elucidate early autoregulation mechanisms disrupted in SUNN mutants, we searched for genes with altered expression in the loss-of-function sunn-4 mutant and included the rdn1-2 autoregulation mutant for comparison. We identified constitutively altered expression of small groups of genes in sunn-4 roots and in sunn-4 shoots. All genes with verified roles in nodulation that were induced in wild-type roots during the establishment of nodules were also induced in sunn-4, including autoregulation genes TML2 and TML1. Only an isoflavone-7-O-methyltransferase gene was induced in response to rhizobia in wild-type roots but not induced in sunn-4. In shoot tissues of wild-type, eight rhizobia-responsive genes were identified, including a MYB family transcription factor gene that remained at a baseline level in sunn-4; three genes were induced by rhizobia in shoots of sunn-4 but not wild-type. We cataloged the temporal induction profiles of many small secreted peptide (MtSSP) genes in nodulating root tissues, encompassing members of twenty-four peptide families, including the CLE and IRON MAN families. The discovery that expression of TML2 in roots, a key factor in inhibiting nodulation in response to autoregulation signals, is also triggered in sunn-4 in the section of roots analyzed, suggests that the mechanism of TML regulation of nodulation in M. truncatula may be more complex than published models.

A modified aeroponic system for growing small-seeded legumes and other plants to study root systems.

BACKGROUND: Various growth systems are available for studying plant root growth and plant-microbe interactions including hydroponics and aeroponics. Although some of these systems work well with Arabidopsis thaliana and smaller cereal model plants, they may not scale up as well for use with hundreds of plants at a time from a larger plant species. The aim of this study is to present step-by-step instructions for fabricating an aeroponic system, also called a "caisson," that has been in use in several legume research labs studying the development of symbiotic nitrogen fixing nodules, but for which detailed directions are not currently available. The aeroponic system is reusable and is adaptable for many other types of investigations besides root nodulation. RESULTS: An aeroponic system that is affordable and reusable was adapted from a design invented by French engineer René Odorico. It consists of two main components: a modified trash can with a lid of holes and a commercially available industrial humidifier that is waterproofed with silicon sealant. The humidifier generates a mist in which plant roots grow, suspended from holes in trash can lid. Results from use of the aeroponic system have been available in the scientific community for decades; it has a record as a workhorse in the lab. CONCLUSIONS: Aeroponic systems present a convenient way for researchers to grow plants for studying root systems and plant-microbe interactions in root systems. They are particularly attractive for phenotyping roots and following the progress of nodule development in legumes. Advantages include the ability to precisely control the growth medium in which the plants grow and easy observations of roots during growth. In this system, mechanical shear potentially killing microbes found in some other types of aeroponic devices is not an issue. Disadvantages of aeroponic systems include the likelihood of altered root physiology compared to root growth on soil and other solid substrates and the need to have separate aeroponic systems for comparing plant responses to different microbial strains.

FKF1 Interacts with CHUP1 and Regulates Chloroplast Movement in Arabidopsis.

Plants have mechanisms to relocate chloroplasts based on light intensities in order to maximize photosynthesis and reduce photodamage. Under low light, chloroplasts move to the periclinal walls to increase photosynthesis (accumulation) and move to the anticlinal walls under high light to avoid photodamage, and even cell death (avoidance). Arabidopsis blue light receptors phot1 and phot2 (phototropins) have been reported to regulate chloroplast movement. This study discovered that another blue light receptor, FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1), regulates chloroplast photorelocation by physically interacting with chloroplast unusual positioning protein 1 (CHUP1), a critical component of the chloroplast motility system. Leaf cross-sectioning and red-light transmittance results showed that overexpression of FKF1 compromised the avoidance response, while the absence of FKF1 enhanced chloroplast movements under high light. Western blot analysis showed that CHUP1 protein abundance is altered in FKF1 mutants and overexpression lines, indicating a potential regulation of CHUP1 by FKF1. qPCR results showed that two photorelocation pathway genes, JAC1 and THRUMIN1, were upregulated in FKF1-OE lines, and overexpression of FKF1 in the THRUMIN1 mutant weakened its accumulation and avoidance responses, indicating that JAC1 and THRUMIN1 may play a role in the FKF1-mediated chloroplast avoidance response. However, the precise functional roles of JAC1 and THRUMIN1 in this process are not known.

Mutation of BAM2 rescues the sunn hypernodulation phenotype in Medicago truncatula, suggesting that a signaling pathway like CLV1/BAM in Arabidopsis affects nodule number.

The unique evolutionary adaptation of legumes for nitrogen-fixing symbiosis leading to nodulation is tightly regulated by the host plant. The autoregulation of nodulation (AON) pathway negatively regulates the number of nodules formed in response to the carbon/nitrogen metabolic status of the shoot and root by long-distance signaling to and from the shoot and root. Central to AON signaling in the shoots of Medicago truncatula is SUNN, a leucine-rich repeat receptor-like kinase with high sequence similarity with CLAVATA1 (CLV1), part of a class of receptors in Arabidopsis involved in regulating stem cell populations in the root and shoot. This class of receptors in Arabidopsis includes the BARELY ANY MERISTEM family, which, like CLV1, binds to CLE peptides and interacts with CLV1 to regulate meristem development. M. truncatula contains five members of the BAM family, but only MtBAM1 and MtBAM2 are highly expressed in the nodules 48 hours after inoculation. Plants carry mutations in individual MtBAMs, and several double BAM mutant combinations all displayed wild-type nodule number phenotypes. However, Mtbam2 suppressed the sunn-5 hypernodulation phenotype and partially rescued the short root length phenotype of sunn-5 when present in a sunn-5 background. Grafting determined that bam2 suppresses supernodulation from the roots, regardless of the SUNN status of the root. Overexpression of MtBAM2 in wild-type plants increases nodule numbers, while overexpression of MtBAM2 in some sunn mutants rescues the hypernodulation phenotype, but not the hypernodulation phenotypes of AON mutant rdn1-2 or crn. Relative expression measurements of the nodule transcription factor MtWOX5 downstream of the putative bam2 sunn-5 complex revealed disruption of meristem signaling; while both bam2 and bam2 sunn-5 influence MtWOX5 expression, the expression changes are in different directions. We propose a genetic model wherein the specific root interactions of BAM2/SUNN are critical for signaling in nodule meristem cell homeostasis in M. truncatula.

Time Series Transcriptome Analysis in Medicago truncatula Shoot and Root Tissue During Early Nodulation.

In response to colonization by rhizobia bacteria, legumes are able to form nitrogen-fixing nodules in their roots, allowing the plants to grow efficiently in nitrogen-depleted environments. Legumes utilize a complex, long-distance signaling pathway to regulate nodulation that involves signals in both roots and shoots. We measured the transcriptional response to treatment with rhizobia in both the shoots and roots of Medicago truncatula over a 72-h time course. To detect temporal shifts in gene expression, we developed GeneShift, a novel computational statistics and machine learning workflow that addresses the time series replicate the averaging issue for detecting gene expression pattern shifts under different conditions. We identified both known and novel genes that are regulated dynamically in both tissues during early nodulation including leginsulin, defensins, root transporters, nodulin-related, and circadian clock genes. We validated over 70% of the expression patterns that GeneShift discovered using an independent M. truncatula RNA-Seq study. GeneShift facilitated the discovery of condition-specific temporally differentially expressed genes in the symbiotic nodulation biological system. In principle, GeneShift should work for time-series gene expression profiling studies from other systems.

The Regulation of Nodule Number in Legumes Is a Balance of Three Signal Transduction Pathways.

Nitrogen is a major determinant of plant growth and productivity and the ability of legumes to form a symbiotic relationship with nitrogen-fixing rhizobia bacteria allows legumes to exploit nitrogen-poor niches in the biosphere. But hosting nitrogen-fixing bacteria comes with a metabolic cost, and the process requires regulation. The symbiosis is regulated through three signal transduction pathways: in response to available nitrogen, at the initiation of contact between the organisms, and during the development of the nodules that will host the rhizobia. Here we provide an overview of our knowledge of how the three signaling pathways operate in space and time, and what we know about the cross-talk between symbiotic signaling for nodule initiation and organogenesis, nitrate dependent signaling, and autoregulation of nodulation. Identification of common components and points of intersection suggest directions for research on the fine-tuning of the plant's response to rhizobia.

Whole genome bisulfite sequencing of Medicago truncatula A17 wild type and lss mutants.

OBJECTIVES: Earlier work in our lab identified a spontaneous mutant (likesunnsupernodulator-lss) in Medicago truncatula, resulting in increased nodulation. Molecular genetic evidence indicated the phenotype was due to an unknown lesion resulting in cis-silencing of the SUNN gene. Altered methylation of the promoter was suspected, but analysis of the SUNN promoter by bisulfite sequencing at the time of publication revealed no significant methylation differences between the SUNN promoter in wild type and lss plants. Using advances in methylome generation we compared the methylome of wild type and the lss mutant in the larger 810 kB area of the genome where lss maps. DATA DESCRIPTION: The data show the distribution of types of methylation across the entire genome between A17 wild type and lss mutants, the number of differentially methylated cytosines between genotypes, and the overall pattern of gene methylation between genotypes. We expect the wild type data will be especially useful as a reference for other investigations of methylation using M. truncatula.

Celebrating 20 Years of Genetic Discoveries in Legume Nodulation and Symbiotic Nitrogen Fixation.

Since 1999, various forward- and reverse-genetic approaches have uncovered nearly 200 genes required for symbiotic nitrogen fixation (SNF) in legumes. These discoveries advanced our understanding of the evolution of SNF in plants and its relationship to other beneficial endosymbioses, signaling between plants and microbes, the control of microbial infection of plant cells, the control of plant cell division leading to nodule development, autoregulation of nodulation, intracellular accommodation of bacteria, nodule oxygen homeostasis, the control of bacteroid differentiation, metabolism and transport supporting symbiosis, and the control of nodule senescence. This review catalogs and contextualizes all of the plant genes currently known to be required for SNF in two model legume species, Medicago truncatula and Lotus japonicus, and two crop species, Glycine max (soybean) and Phaseolus vulgaris (common bean). We also briefly consider the future of SNF genetics in the era of pan-genomics and genome editing.

Identifying Temporally Regulated Root Nodulation Biomarkers Using Time Series Gene Co-Expression Network Analysis.

Root nodulation results from a symbiotic relationship between a plant host and Rhizobium bacteria. Synchronized gene expression patterns over the course of rhizobial infection result in activation of pathways that are unique but overlapping with the highly conserved pathways that enable mycorrhizal symbiosis. We performed RNA sequencing of 30 Medicago truncatula root maturation zone samples at five distinct time points. These samples included plants inoculated with Sinorhizobium medicae and control plants that did not receive any Rhizobium. Following gene expression quantification, we identified 1,758 differentially expressed genes at various time points. We constructed a gene co-expression network (GCN) from the same data and identified link community modules (LCMs) that were comprised entirely of differentially expressed genes at specific time points post-inoculation. One LCM included genes that were up-regulated at 24 h following inoculation, suggesting an activation of allergen family genes and carbohydrate-binding gene products in response to Rhizobium. We also identified two LCMs that were comprised entirely of genes that were down regulated at 24 and 48 h post-inoculation. The identity of the genes in these modules suggest that down-regulating specific genes at 24 h may result in decreased jasmonic acid production with an increase in cytokinin production. At 48 h, coordinated down-regulation of a specific set of genes involved in lipid biosynthesis may play a role in nodulation. We show that GCN-LCM analysis is an effective method to preliminarily identify polygenic candidate biomarkers of root nodulation and develop hypotheses for future discovery.

A CLE-SUNN module regulates strigolactone content and fungal colonization in arbuscular mycorrhiza.

During arbuscular mycorrhizal symbiosis, colonization of the root is modulated in response to the physiological status of the plant, with regulation occurring locally and systemically. Here, we identify differentially expressed genes encoding CLAVATA3/ESR-related (CLE) peptides that negatively regulate colonization levels by modulating root strigolactone content. CLE function requires a receptor-like kinase, SUNN; thus, a CLE-SUNN-strigolactone feedback loop is one avenue through which the plant modulates colonization levels.

Correction to: Comparison of efficiency and time to regeneration of Agrobacterium-mediated transformation methods in Medicago truncatula.

[This corrects the article DOI: 10.1186/s13007-019-0404-1.].

The Medicago truncatula CLAVATA3-LIKE CLE12/13 signaling peptides regulate nodule number depending on the CORYNE but not the COMPACT ROOT ARCHITECTURE2 receptor.

We previously showed that the rdn1 and sunn supernodulation mutants of Medicago truncatula respond differentially to overexpression of the rhizobial CLAVAT3/EMBRYO SURROUNDING REGION (CLE) signaling peptides MtCLE12p and MtCLE13p, allowing the order of action of the genes to be determined in the autoregulation of nodulation (AON) signal transduction pathway. We tested the same gene constructs that lead to the production of proteolytically processed peptides (indicated by a p after the name) in plants mutant for two other proteins that control nodule number (CRN and CRA2) and were able to determine that CRN is involved in the same signaling pathway as MtCLE12p and MtCLE13p, while regulation in CRA2 mutants responds normally to the peptides, suggesting CRA2 likely signals separately from SUNN, RDN1, and CRN. Based on the analysis of the double mutant of cra2-2 and sunn-4, we also confirm recent findings that CRA2 acts independently of SUNN in nodule number regulation.

Comparison of efficiency and time to regeneration of Agrobacterium-mediated transformation methods in Medicago truncatula.

BACKGROUND: Tissue culture transformation of plants has an element of art to it, with protocols passed on between labs but often not directly compared. As Medicago truncatula has become popular as a model system for legumes, rapid transformation is critical, and many protocols exist, with varying results. RESULTS: The M. truncatula ecotypes, R108 and A17, were utilized to compare the effect of a modification to a previously used protocol based on shoot explants on the percentage of transformed plants produced from calli. This percentage was then compared to that of two additional transformation protocols based on root explants in the R108 ecotype. Variations in embryonic tissue sources, media components, time for transformation, and vectors were analyzed. CONCLUSIONS: While no A17 transgenic plants were obtained, transgenic plantlets from the R108 ecotype were produced in as little as 4 months with a comparison of the two widely studied ecotypes under a single set of conditions. While the protocols tested gave similar results in percentage of transformed plants produced, considerations of labor and time to transgenics that vary between the root explant protocols tested were discovered. These considerations may influence which protocol to choose for introducing a single transgene versus creating lines with multiple mutations utilizing a CRISPR/Cas9 construct.

Fixation and Laser Capture Microdissection of Plant Tissue for RNA Extraction and RNASeq Library Preparation.

In order to study the transcriptome of individual plant cells at specific points in time, we developed protocols for fixation, embedding, and sectioning of plant tissue followed by laser capture microdissection (LCM) and processing for RNA recovery. LCM allows the isolation of individual cell types from heterogeneous tissue sections and is particularly suited to plant processing because it does not require the breakdown of cell walls. This approach allows accurate separation of a small volume of cells that can be used to study gene expression profiles in different tissues or cell layers. The technique does not require separation of cells by enzymatic digestion of any kind, does not require cell-specific reporter genes, and allows storage of fixed and embedded tissue for months before capture. The methods for fixation, embedding, sectioning, and capture of plant cells that we describe yield high-quality RNA suitable for making libraries for RNASeq. © 2018 by John Wiley & Sons, Inc.

ROOT DETERMINED NODULATION1 Is Required for M. truncatula CLE12, But Not CLE13, Peptide Signaling through the SUNN Receptor Kinase.

The combinatorial interaction of a receptor kinase and a modified CLE peptide is involved in several developmental processes in plants, including autoregulation of nodulation (AON), which allows legumes to limit the number of root nodules formed based on available nitrogen and previous rhizobial colonization. Evidence supports the modification of CLE peptides by enzymes of the hydroxyproline O-arabinosyltransferase (HPAT/RDN) family. Here, we show by grafting and genetic analysis in Medicago truncatula that, in the AON pathway, RDN1, functioning in the root, acts upstream of the receptor kinase SUNN, functioning in the shoot. As expected for a glycosyltransferase, we found that RDN1 and RDN2 proteins are localized to the Golgi, as was shown previously for AtHPAT1. Using composite plants with transgenic hairy roots, we show that RDN1 and RDN2 orthologs from dicots as well as a related RDN gene from rice (Oryza sativa) can rescue the phenotype of rdn1-2 when expressed constitutively, but the less related MtRDN3 cannot. The timing of the induction of MtCLE12 and MtCLE13 peptide genes (negative regulators of AON) in nodulating roots is not altered by the mutation of RDN1 or SUNN, although expression levels are higher. Plants with transgenic roots constitutively expressing MtCLE12 require both RDN1 and SUNN to prevent nodule formation, while plants constitutively expressing MtCLE13 require only SUNN, suggesting that the two CLEs have different requirements for function. Combined with previous work, these data support a model in which RDN1 arabinosylates MtCLE12, and this modification is necessary for the transport and/or reception of the AON signal by the SUNN kinase.