Phosphoproteome Analysis of Lotus japonicus Roots Reveals Shared and Distinct Components of Symbiosis and Defense

Plant roots form an intracellular symbiosis with nitrogen-fixing bacteria while maintaining the capacity for defending themselves against bacterial pathogens. To investigate the molecular relationship between these opposing cellular responses, we compared changes in the root phosphoproteome of the legume Lotus japonicus occurring within minutes after perception of nodulation factor (NF), a symbiotic signaling molecule, to those elicited by flagellin peptide (flg22), a conserved pathogen-associated peptide motif present in flagellar protein of a wide range of bacteria. Phosphoproteins were visualized by autoradiography of two-dimensional polyacrylamide gels after in vivo labeling with P-33-orthophosphate. Comparisons of NF- and flg22-induced phosphoprotein patterns revealed signal-specific responses but also a surprisingly large overlap. Specificity of the responses was observed because the NF receptor kinases NFR1 and NFR5 were both required for NF- but not for flg22-mediated changes in the phosphoproteome. Moreover. NF did not stimulate an oxidative burst or activation of mitogen-activated protein kinases, two common markers for early defense responses that were induced by flg22. Inhibitor studies revealed that phosphorylation of at least some of the proteins in response to NF requires phospholipase I) (PIA)) whereas regulation of the flg22 phosphoproteome is PLD-independent. Although plant signal transduction during symbiosis and defense utilizes distinct components, phosphorylation of overlapping sets of proteins is achieved.