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

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.

Identification of ferredoxin II as a major calcium binding protein in the nitrogen-fixing symbiotic bacterium Mesorhizobium loti

“…In rhizobium-legume symbiosis, Ca 2+ has been demonstrated to be involved in the perception of symbiotic signals not only by the plant host [ 33 ] but also by the bacterial partner. In particular, several plant-derived diffusible molecules, such as tetronic acid in M. loti [ 7 ] and the flavonoid naringenin in R. leguminosarum [ 8 , 10 ] were found to trigger transient intracellular Ca 2+ changes leading to the expression of bacterial nodulation genes. Although the molecular identity of the Ca 2+ -permeable channels involved in the observed Ca 2+ fluxes remains elusive, in this work another essential component of the Ca 2+ homeostatic machinery, i.e.…”

“…In particular, the role of calcium as critical intracellular messenger involved in the perception and transduction of symbiotic signalling molecules has become increasingly apparent. A Ca 2+ -mediated symbiotic signalling pathway has been shown to be activated by symbiosis-related signals not only in the host plant [ 6 ], but also in the rhizobial partner [ 7 - 10 ]. Measurements of intracellular Ca 2+ concentration by means of recombinant aequorin expression demonstrated a tight regulation of cytosolic Ca 2+ level in M. loti and the occurrence of Ca 2+ -based mechanisms to detect changes in the soil environment [ 7 ].…”

Multidisciplinary approaches for studying rhizobium–legume symbioses

“…Additionally, comparative proteomic studies have been used in the identification of putative symbiotic genes (Gomes et al 2012b). Several processes of more direct relevance to SNF have also been studied using proteomics (Table 3), such as the proteomic response to flavonoids (Guerreiro et al 1997;Chen et al 2000b;Hempel et al 2009;da Silva Batista and Hungria 2012;Arrigoni et al 2013;Tolin et al 2013;Gao et al 2015;Meneses et al 2017); and to micro-aerobic or anaerobic conditions (Dainese-Hatt et al 1999). The secretome of rhizobia, with a particular focus on the proteins exported by type III secretion systems, has also been investigated (Saad et al 2005;Rodrigues et al 2007;Hempel et al 2009;Okazaki et al 2009).…”

Plant–Microbe Symbiosis: What Has Proteomics Taught Us?

“…Steps in which proteomics has been widely applied to explore plant/rhizobia interaction are depicted in green circles. 1) Roots prior to inoculation, 2) free‐living Rhizobium and the perception of the plant flavonoid signal, 3) the perception of Rhizobium ‐secreted Nod factors by the host plant's root, 4) the molecular events occurring during early interaction phase, 5) mature nitrogen‐fixing bacteroids within the root nodule …”

“…Several plant‐derived diffusible molecules, for example, the flavonoid naringenin, are known to trigger transient intracellular Ca 2+ changes in the bacteria which in turn induces the expression of bacterial NFs. The elevation of cytosolic Ca 2+ levels is an early key event associated with the initiation of nodulation . The application of proteomics to Rhizobium leguminosarum cultures grown in the presence or absence of naringenin showed altered protein expression profiles.…”