Calcium-Dependent Regulation of Genes for Plant Nodulation in Rhizobium leguminosarum Detected by iTRAQ Quantitative Proteomic Analysis

Rhizobia, the nitrogen-fixing bacterial symbionts of legumes, represent an agricultural application of primary relevance and a model of plant–microbe molecular dialogues. We recently described rhizobium proteome alterations induced by plant flavonoids using iTRAQ. Herein, we further extend that experimentation, proving that the transient elevation in cytosolic calcium is a key signaling event necessary for the expression of the nodulation (nod) genes. Ca²⁺ involvement in nodulation is a novel issue that we recently flagged with genetic and physiological approaches and that hereby we demonstrate also by proteomics. Exploiting the multiple combinations of 4-plex iTRAQ, we analyzed Rhizobium leguminosarum cultures grown with or without the <i>nod</i> gene-inducing plant flavonoid naringenin and in the presence or absence of the extracellular Ca²⁺ chelator EGTA. We quantified over a thousand proteins, 189 of which significantly altered upon naringenin and/or EGTA stimulation. The expression of NodA, highly induced by naringenin, is strongly reduced when calcium availability is limited by EGTA. This confirms, from a proteomic perspective, that a Ca²⁺ influx is a necessary early step in flavonoid-mediated legume nodulation by rhizobia. We also observed other proteins affected by the different treatments, whose identities and roles in nodulation and rhizobium physiology are likewise discussed

Location of sampling sites: latitude and longitude coordinates in decimal values.

<p>The “Car” samples were found within the car impounded from the suspect.</p

Dataset

The file contains the abundance levels of all the OTUs for each sampl

Left: bi-plot after Principal components and classification analysis based on correlation of the content of the 54 chemical elements (empty marker, not all labelled) in the 29 soil samples (full marker).

<p>The variance explained by the each principal component is shown in parentheses. Right: plot of canonical scores after Discriminant analysis with canonical analysis performed with the four chemical elements most, and significantly, correlated (absolute value) with factor 1 (Cs, Ga, Ca, S) and factor 2 (Cd, Zn, Hg, Sr). Classification of soil samples has an high probability to be correct (93%) as the predicted classification deviates only for sample “Le”, that is not distinct from “X” (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020222#pone-0020222-g001" target="_blank">fig. 1d</a>). Since these two samples are in the same group (the green one), group classification is 100% correct. For technical details of multivariate techniques see Statistica 7.0 Electronic Manual (Statsoft Inc., 2005).</p

Results of the analyses on the soil samples compared with two specimens of soil (CarR, CarL), found respectively on the right and left carpets of the suspect's car floor.

<p>a, b) ICP analysis of the content of 54 mineral elements. a): Box and whiskers synthetic representation of the variability of the 12 zones; b) Cluster analysis (single linkage, Euclidean distance) of the data; c) Amplified Ribosomal DNA Restriction Analysis (ARDRA) of the soil bacterial communities. The Neighbour Joining dendrogram resulting from Pearson correlation analysis of the combined three enzymes electrophoretic profiles is shown. The horizontal scale indicates the percent distance. In b) and c) sample replicates are included to show the degree of inter-replicate variability.</p

Fasta

OTU sequence

Air view maps of the crime scene and surroundings depicting the sites from which samples were taken, starting from the murder spot, and at increasing distances.

<p>The inset map of Italy shows the area location as well as the sites of two soils taken at far distance as outgroup references. X: spot where the corpse was found (margin of a corn field), CF (inside corn field), Le (ridge of the levee bordering the corn field). 1.7 Km, 3 Km, 18 Km, 19 Km: sites located at 1.7, 3, 18, 19 kilometers from the murder site and sharing the same crop (corn); 1.8 KmF: (fallow), site located at 1.8 km but not cropped for over 50 years and featuring natural vegetation and secondary growth. Sar: soil from an uncultivated area in Sardinia (Castelsardo); Alp: soil from an uncultivated area in the Alps (Soranzen). All samples, except the two outgroup references, were chosen in equivalent soil conditions as regards parent material, depositional basin river and soil type (Hypercalcaric-Fluvic Cambisols, WRB 1998, or Oxyaquic Eutrudept fine-silty, carbonatic, mesic, USDA 1998) to minimize the variability that would occur across different soil types. For details and exact coordinates, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020222#pone-0020222-t001" target="_blank">Tab.1</a>. Scale bars equal: 500 km (a); 5 km (b); 500 m (c); 50 m (d).</p

Symbiont phylogeny (16S rDNA) - ML analysis

Symbiont phylogeny (16S rDNA) - ML analysi

Insects host phylogeny (CO) - ML analysis

Insects host phylogeny (CO gene sequences) - ML analysi

Insects host alignment (16S rDNA + CO)

Combined data se