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

Abstract

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.

Transcriptome analysis ofMedicago truncatulaAutoregulation of Nodulation mutants reveals that disruption of the SUNN pathway causes constitutive expression changes in a small group of genes, but the overall response to rhizobia resembles wild type, including induction ofTML1andTML2

“…In this work, we used a harvesting procedure described in (Poehlman et al, 2019) incorporating simultaneous inoculation of all plants in an aeroponic system and harvest of the zone of developing nodules tracked via root growth (summarized in Supplemental Fig 1). In our laboratory experience, nodulation progresses more uniformly and more rapidly in this system than on plates or in pots, and we presume this is due to the apparatus simultaneously spraying the aerosolized solution of rhizobia on all plants.…”

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

“…Medicago truncatula A17 plants were grown, inoculated and root sections harvested as in Poehlman et al ( 2019 ). Specifically, M.truncatula A17 seeds were scarified for 5–8 min in sulfuric acid, rinsed 5 times with distilled water, washed with 3% bleach, rinsed another 5 times in distilled water, and imbibed in water with gentle rocking for 2 h at room temperature.…”

“…RNA libraries were made and sequenced by Novogene Co., 740 Ltd. (Beijing) from 100 to 1,000 ng of total RNA prepared by a stranded kit (Illumina TruSeq Stranded 741 Total RNA Kit or NEB Next UltraTM II Directional RNA Library Prep Kit for Illumina). These libraries were included in a final dataset consisting of 60 libraries, including 30 libraries from this study (three replicates of five time points each for inoculated and uninoculated wild type (A17) shoot segments) and 30 libraries previously reported from inoculated and uninoculated wild type (A17) root segments (Poehlman et al, 2019 ). The PBS-GEM workflow ( https://github.com/wpoehlm/PBS-GEM ) was used to process RNA sequencing reads on Clemson University's Palmetto Cluster (Pertea et al, 2016 ).…”

Bacterial bioinoculants adapted for sustainable plant health and soil fertility enhancement in Namibia

“…Root nodule formation is triggered by simultaneous correlations between plants and their soil environment. The release of Nod factors into the soil by rhizobia temporarily activates plant genes that code for specific hormones (Spaink, 2000;Poehlman et al, 2019). Peptide hormones, for example, together with signal receptors and low levels of nitrogen in soil induce nodule formation with close association with nitrogen fixing bacteria (Taleski et al, 2018).…”