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.

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 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.

Relatedness of chromosomal and plasmid DNAs of Erwinia pyrifoliae and Erwinia amylovora.

The plant pathogen Erwinia pyrifoliae has been classified as a separate species from Erwinia amylovora based in part on differences in molecular properties. In this study, these and other molecular properties were examined for E. pyrifoliae and for additional strains of E. amylovora, including strains from brambles (Rubus spp.). The nucleotide composition of the internal transcribed spacer (ITS) region was determined for six of the seven 16S-23S rRNA operons detected in these species with a 16S rRNA gene probe. Each species contained four operons with a tRNA(Glu) gene and two with tRNA(Ile) and tRNA(Ala) genes, and analysis of the operons from five strains of E. amylovora indicated a high degree of ITS variability among them. One tRNA(Glu)-containing operon from E. pyrifoliae Ep1/96 was identical to one in E. amylovora Ea110, but three tRNA(Glu) operons and two tRNA(Ile) and tRNA(Ala) operons from E. pyrifoliae contained unique nucleotide changes. When groEL sequences were used for species-specific identification, E. pyrifoliae and E. amylovora were the closest phylogenetic relatives among a set of 12 bacterial species. The placement of E. pyrifoliae distinct from E. amylovora corroborated molecular hybridization data indicating low DNA-DNA similarity between them. Determination of the nucleotide sequence of plasmid pEP36 from E. pyrifoliae Ep1/96 revealed a number of presumptive genes that matched genes previously found in pEA29 from E. amylovora and similar organization for the genes and origins of replication. Also, pEP36 and pEA29 were incompatible with clones containing the reciprocal origin regions. Finally, the ColE1-like plasmid pEP2.6 from strain Ep1/96 contained sequences found in small plasmids in E. amylovora strains IL-5 and IH3-1.