The Defective in Autoregulation (DAR) gene of Medicago truncatula encodes a protein involved in regulating nodulation and arbuscular mycorrhiza.

BACKGROUND: Legumes utilize a long-distance signaling feedback pathway, termed Autoregulation of Nodulation (AON), to regulate the establishment and maintenance of their symbiosis with rhizobia. Several proteins key to this pathway have been discovered, but the AON pathway is not completely understood. RESULTS: We report a new hypernodulating mutant, defective in autoregulation, with disruption of a gene, DAR (Medtr2g450550/MtrunA17_Chr2g0304631), previously unknown to play a role in AON. The dar-1 mutant produces ten-fold more nodules than wild type, similar to AON mutants with disrupted SUNN gene function. As in sunn mutants, suppression of nodulation by CLE peptides MtCLE12 and MtCLE13 is abolished in dar. Furthermore, dar-1 also shows increased root length colonization by an arbuscular mycorrhizal fungus, suggesting a role for DAR in autoregulation of mycorrhizal symbiosis (AOM). However, unlike SUNN which functions in the shoot to control nodulation, DAR functions in the root. CONCLUSIONS: DAR encodes a membrane protein that is a member of a small protein family in M. truncatula. Our results suggest that DAR could be involved in the subcellular transport of signals involved in symbiosis regulation, but it is not upregulated during symbiosis. DAR gene family members are also present in Arabidopsis, lycophytes, mosses, and microalgae, suggesting the AON and AOM may use pathway components common to other plants, even those that do not undergo either symbiosis.

Detection and Characterization of Xylella fastidiosa subsp. fastidiosa in Rabbiteye Blueberry in South Carolina.

Xylella fastidiosa causes bacterial leaf scorch in southern highbush (Vaccinium corymbosum interspecific hybrids) and is also associated with a distinct disease phenotype in rabbiteye blueberry (V. virgatum) cultivars in the southeastern United States. Both X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. multiplex have been reported to cause problems in southern highbush blueberry, but so far only X. fastidiosa subsp. multiplex has been reported in rabbiteye cultivars in Louisiana. In this study, we report detection of X. fastidiosa in rabbiteye blueberry plants in association with symptoms of foliar reddening and shoot dieback. High throughput sequencing of an X. fastidiosa-positive plant sample and comparative analyses identified the strain in one of these plants as being X. fastidiosa subsp. fastidiosa. We briefly discuss the implications of these findings, which may spur research into blueberry as a potential inoculum source that could enable spread to other susceptible fruit crops in South Carolina.

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.

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.

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.

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.

The systemic nodule number regulation kinase SUNN in Medicago truncatula interacts with MtCLV2 and MtCRN.

Autoregulation of nodulation (AON), a systemic signaling pathway in legumes, limits the number of nodules formed by the legume in its symbiosis with rhizobia. Recent research suggests a model for the systemic regulation in Medicago truncatula in which root signaling peptides are translocated to the shoot where they bind to a shoot receptor complex containing the leucine-rich repeat receptor-like kinase SUNN, triggering signal transduction which terminates nodule formation in roots. Here we show that a tagged SUNN protein capable of rescuing the sunn-4 phenotype is localized to the plasma membrane and is associated with the plasmodesmata. Using bimolecular fluorescence complementation analysis we show that, like its sequence ortholog Arabidopsis CLV1, SUNN interacts with homologous CLV1-interacting proteins MtCLAVATA2 and MtCORYNE. All three proteins were also able to form homomers and MtCRN and MtCLV2 also interact with each other. A crn Tnt1 insertion mutant of M. truncatula displayed a shoot controlled increased nodulation phenotype, similar to the clv2 mutants of pea and Lotus japonicus. Together these data suggest that legume AON signaling could occur through a multi-protein complex and that both MtCRN and MtCLV2 may play roles in AON together with SUNN.

The M. truncatula SUNN gene is expressed in vascular tissue, similarly to RDN1, consistent with the role of these nodulation regulation genes in long distance signaling.

Encoding a conserved protein of unknown function, the Medicago truncatula RDN1 gene is involved in autoregulation of nodulation through signaling in the root.  In contrast, the SUNN kinase in M. truncatula has been shown by grafting of mutant scions to control nodule number in the root by communication of a signal from the shoot to the root.  GUS staining patterns resulting from expression of the SUNN promoter fused to uidA showed expression of SUNN in most parts of plant including the root, but confined to the vascular tissue, a pattern that overlaps with that published for RDN1.  Real Time qRT-PCR analysis showed levels of both SUNN RNA and RDN1 RNA did not change significantly during early nodulation signaling (0-72 hours after inoculation).  The similarity in expression in cell types strongly suggests vascular signaling for nodule number regulation, while the lack of changes over early nodule development suggest post transcriptional mechanisms such as protein association or phosphorylation transmit the signal.  

The ROOT DETERMINED NODULATION1 gene regulates nodule number in roots of Medicago truncatula and defines a highly conserved, uncharacterized plant gene family.

The formation of nitrogen-fixing nodules in legumes is tightly controlled by a long-distance signaling system in which nodulating roots signal to shoot tissues to suppress further nodulation. A screen for supernodulating Medicago truncatula mutants defective in this regulatory behavior yielded loss-of-function alleles of a gene designated ROOT DETERMINED NODULATION1 (RDN1). Grafting experiments demonstrated that RDN1 regulatory function occurs in the roots, not the shoots, and is essential for normal nodule number regulation. The RDN1 gene, Medtr5g089520, was identified by genetic mapping, transcript profiling, and phenotypic rescue by expression of the wild-type gene in rdn1 mutants. A mutation in a putative RDN1 ortholog was also identified in the supernodulating nod3 mutant of pea (Pisum sativum). RDN1 is predicted to encode a 357-amino acid protein of unknown function. The RDN1 promoter drives expression in the vascular cylinder, suggesting RDN1 may be involved in initiating, responding to, or transporting vascular signals. RDN1 is a member of a small, uncharacterized, highly conserved gene family unique to green plants, including algae, that we have named the RDN family.

Transcript profiling in M. truncatula lss and sunn-1 mutants reveals different expression profiles despite disrupted SUNN gene function in both mutants.

A novel autoregulation of nodulation locus in Medicago truncatula, lss, silences the SUNN gene thorough a cis-acting mechanism. Microarray analysis was performed on the Affymetrix Gene Chip® Medicago Genome Array with cDNA isolated from seven-day-old seedlings of wild type, sunn-1 and lss plants. The results suggest that in lss plants expression of only a few dozen genes differs significantly from wild type while in sunn-1 plants expression of several hundred genes represented by over 800 probe sets is altered. These results suggest that the kinase domain modification caused by the sunn-1 mutation alters the receptor's influence on gene expression and that these differences are present even in the absence of nodulation.

The lss supernodulation mutant of Medicago truncatula reduces expression of the SUNN gene.

The number of nodules that form in a legume when interacting with compatible rhizobia is regulated by the plant. We report the identification of a mutant in nodule regulation in Medicago truncatula, like sunn supernodulator (lss), which displays shoot-controlled supernodulation and short roots, similar to sunn mutants. In contrast with the sunn-1 mutant, nodulation in the lss mutant is more extensive and is less sensitive to nitrate and ethylene, resembling the sunn-4 presumed null allele phenotype. Although the lss locus maps to the SUNN region of linkage group 4 and sunn and lss do not complement each other, there is no mutation in the genomic copy of the SUNN gene or in the 15-kb surrounding region in the lss mutant. However, expression of the SUNN gene in the shoots of lss plants is greatly reduced compared with wild-type plants. Analysis of cDNA from plants heterozygous for lss indicates that lss is a cis-acting factor affecting the expression of SUNN, and documented reversion events show it to be unstable, suggesting a possible reversible DNA rearrangement or an epigenetic change in the lss mutant. Assessment of the SUNN promoter revealed low levels of cytosine methylation in the 700-bp region proximal to the predicted transcription start site in both wild-type and lss plants, indicating that promoter hypermethylation is not responsible for the suppression of SUNN expression in lss. Thus, lss represents either a distal novel locus within the mapped region affecting SUNN expression or an uncharacterized epigenetic modification at the SUNN locus.