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 nod 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.