Project description:The soybean–Bradyrhizobium symbiosis enables symbiotic nitrogen fixation (SNF) within root nodules, reducing reliance on synthetic N fertilizers. However, nitrogen fixation is transient, peaking several weeks after Bradyrhizobium colonization and declining as nodules senesce in coordination with host development. To investigate the regulatory mechanisms governing SNF and senescence, we conducted a temporal transcriptomic analysis of soybean nodules colonized with Bradyrhizobium diazoefficiens USDA110. Weekly nodule samples (2 to 10 weeks postinoculation, wpi) were analyzed using RNA and small RNA sequencing, and acetylene reduction assays assessed nitrogenase activity from 4 to 7 wpi. We identified three major nodule developmental phases: early development (2 to 3 wpi), nitrogen fixation (3 to 8 wpi), and senescence (8 to 10 wpi). Soybean showed extensive transcriptional reprogramming during senescence, whereas Bradyrhizobium underwent major transcriptional shifts early in development before stabilizing during nitrogen fixation. We identified seven soybean genes and several microRNAs as candidate biomarkers of nitrogen fixation, including lipoxygenases (Lox), suggesting roles for oxylipin metabolism. Soy hemoglobin-2 (Hb2), previously classified as nonsymbiotic, was upregulated during senescence, implicating oxidative stress responses within aging nodules. Upregulation of the Bradyrhizobium paa operon and rpoH during senescence suggesting metabolic adaptation for survival beyond symbiosis. Additionally, Bradyrhizobium nif gene expression showed stage-specific regulation, with nifK peaking at 2 wpi, nifD and nifA at 2 and 10 wpi, and nifH, nifW, and nifS at 10 wpi. These findings provide insights into SNF regulation and nodule aging, revealing temporal gene expression patterns that could inform breeding or genetic engineering strategies to enhance nitrogen fixation in soybeans and other legume crops.
Project description:Metabolomics and transcriptomics of Bradyrhizobium diazoefficiens-induced root nodules Bradyrhizobium diazoefficiens is a nitrogen-fixing endosymbiont, which can grow inside root-nodule cells of the agriculturally important soybean and other host plants. Our previous studies described B. diazoefficiens host-specific global expression changes occurring during legume infection at the transcript and protein level. In order to further characterize nodule metabolism, we here determine by flow injection -time of flight mass spectrometry analysis the metabolome of i) nodules and roots from four different B. diazoefficiens host plants, ii) soybean nodules harvested at different time points during nodule development, and iii) soybean nodules infected by two strains mutated in key genes for nitrogen fixation, respectively. Ribose (soybean), tartaric acid (mungbean), hydroxybutanoyloxybutanoate (siratro) and catechol (cowpea) were among the metabolites found to be specifically elevated in one of the respective host plants. While the level of C4-dicarboxylic acids decreased during soybean nodule development, we observed an accumulation of trehalose-phosphate at 21 days post infection (dpi). Moreover, nodules from non-nitrogen-fixing bacteroids (nifA and nifH mutants) showed specific metabolic alterations; these were also supported by transcriptomics data that was generated for the two mutant strains and were helpful to separate for some examples the respective bacterial and plant contributions to the metabolic profile. The alterations included signs of nitrogen limitation in both mutants, and an increased level of a phytoalexin in nodules induced by the nifA mutant, suggesting that the tissue of these nodules exhibits defense and stress reactions.
Project description:Legumes and rhizobia establish a nitrogen-fixing symbiosis that involves the formation of a lateral root organ, the nodule, and the infection process that allows intracellular accommodation of rhizobia within nodule cells. This process involves significant gene expression changes regulated at the transcriptional and post-transcriptional levels. We have previously shown that a transcript encoding the subunit 3 of the Superkiller Complex (SKI), which guides mRNAs to the exosome for 3´-to-5´ degradation, is required for nodule formation and bacterial persistence within the nodule, as well as the induction of early nodulation genes (e.g., MtENOD40) during the Medicago truncatula-Sinorhizobium meliloti symbiosis. Here, we reveal through transcript degradome and small RNA sequencing analysis that knockdown of MtSKI3 impairs the miR172-directed endonucleolytic cleavage of the mRNA encoding Nodule Number Control 1 (MtNNC1), an APETALA2 transcription factor that negatively modulates nodulation. Knockdown of MtNNC1 enhances nodule number, bacterial infection, and the induction of MtENOD40 upon inoculation with S. meliloti whereas overexpression of a miR172-resistant form of MtNNC1 significantly reduces nodule formation. This work identifies miR172 cleavage of MtNNC1 and its control by MtSKI3, a component of the 3´-to- 5´mRNA degradation pathway, as a new regulatory hub controlling indeterminate nodulation.
Project description:Global bottom-up proteomics analysis of proteins purified from soybean root nodules infected with either WT or nifH- mutant Bradyrhizobium japonicum. Nine glycoproteins containing Lewis-a N-glycans, with 3 distinct Lewis-a epitopes (Hex:5 HexNAc:4 dHex:3 Pent:1, Hex:4 HexNAc:4 dHex:2 Pent:1, and Hex:4 HexNAc:3 dHex:2 Pent:1) were observed. Proteins purified from WT and nifH- infected soybean root nodules (five biological replicates each) were reduced using dithiothreitol, alkylated with iodoacetamide and trypsin digested followed by C18 SPE clean-up and LC-MS/MS analysis. Raw data files were processed using FragPipe v17.1, then output files 'combined_modified_peptide.tsv' and 'combined_protein.tsv' were used to identify glycopeptides and for global protein quantitation. These files, along with Excel files containing global quantitation analysis files for soybean nodule (Glycine max) and bacterial (Bradyrhizobium), are available in directory 'Quantification/MSFragger_results'. Glycopeptide data were also processed with PMI Byonic, and Excel file results are available in directory 'Quantification/PMI_Byonic_results'.