Calcium-Dependent Regulation of Genes for Plant Nodulation in Rhizobium leguminosarum Detected by iTRAQ Quantitative Proteomic Analysis Giorgio Arrigoni, †,§ Serena Tolin, †,‡ Roberto Moscatiello, ⊥ Antonio Masi, ‡ Lorella Navazio, ⊥ and Andrea Squartini* ,‡ † Proteomics Center of Padova University, Via G. Orus 2b, 35129 Padova, Italy ‡ Department of Agronomy, Food, Natural Resources, Animals and Environment, DAFNAE, Viale dell’Universita ̀ 16, 35020 Legnaro, Padova, Italy § Department of Biomedical Sciences and ⊥ Department of Biology, University of Padova, Via U. Bassi 58/B, 35131 Padova, Italy * S Supporting Information ABSTRACT: 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. Ca 2+ 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 nod gene-inducing plant flavonoid naringenin and in the presence or absence of the extracellular Ca 2+ 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 Ca 2+ 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. KEYWORDS: calcium signaling, plant-microbe interactions, nitrogen fixation, Rhizobium leguminosarum, nod genes, NodA, naringenin, iTRAQ ■ INTRODUCTION The interaction between leguminous plants and symbiotic nitrogen-fixing bacteria entails a subject of paramount importance both in natural ecosystems’ productivity and in agricultural applications. The controlled invasion of host plants by specific bacteria is also a favorite theme for model-testing studies aiming at unravelling the stepwise molecular dialogue displayed by the two partners. 1,2 The signal exchange starts with flavonoids, 3 the secondary plant metabolites which trigger gene expression in the symbiotic bacteria. 4,5 While initially acting as chemoattractants and stimulators of bacterial multiplication in the rhizosphere, flavonoids are also recognized as specific inducers of the common nodulation (nod) genes in rhizobia. Proteins codified by these genes lead to the synthesis of the chito-lipo oligosaccharide signal (Nod factor) that, in turn, triggers the nodule formation in plants. 6,7 The long-standing model on nod gene induction sees the rhizobial NodD transcriptional activator protein as a flavonoid- perceiving unit that is localized at the cytoplasmic bacterial membrane 8 where it is postulated to bind with the incoming plant flavonoids. 9-11 While the binding of NodD to the nod box promoter of the common nod genes is an ascertained event, the binding of NodD to flavonoids has not yet been demonstrated in vivo. Our recent work has put in evidence a role for calcium in the early signaling events on the bacterial partner side. By using the recombinant expression of the bioluminescent Ca 2+ reporter aequorin, we showed that Mesorhizobium loti, which nodulates Lotus japonicus, senses host plant root exudates via transient intracellular Ca 2+ elevations. 12 Subsequently, in Rhizobium leguminosarum bv. viciae, the transient intracellular Ca 2+ increase induced by the flavonoid naringenin was proven to be essential for the activation of nod gene expression, thus Special Issue: Agricultural and Environmental Proteomics Received: June 30, 2013 Published: September 16, 2013 Article pubs.acs.org/jpr © 2013 American Chemical Society 5323 dx.doi.org/10.1021/pr400656g | J. Proteome Res. 2013, 12, 5323-5330