Pharmacological Evidence That Multiple Phospholipid Signaling Pathways Link Rhizobium Nodulation Factor Perception in Medicago truncatula Root Hairs to Intracellular Responses, Including Ca2+ Spiking and Specific ENOD Gene Expression

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Abstract
Rhizobium nodulation (Nod) factors are specific lipochito-oligosaccharide signals essential for initiating in root hairs of the host legume developmental responses that are required for controlled entry of the microsymbiont. In this article, we focus on the Nod factor signal transduction pathway leading to specific and cell autonomous gene activation in Medicago truncatula cv Jemalong in a study making use of the Nod factor-inducible MtENOD11 gene. First, we show that pharmacological antagonists that interfere with intracellular ion channel and Ca2+ pump activities are efficient blockers of Nod factor-elicited pMtENOD11-β-glucuronidase (GUS) expression in root hairs of transgenic M. truncatula. These results indicate that intracellular Ca2+ release and recycling activities, essential for Ca2+ spiking, are also required for specific gene activation. Second, pharmacological effectors that inhibit phospholipase D and phosphoinositide-dependent phospholipase C activities are also able to block pMtENOD11-GUS activation, thus underlining a central role for multiple phospholipid signaling pathways in Nod factor signal transduction. Finally, pMtENOD11-GUS was introduced into all three Nod−/Myc− dmi M. truncatula mutant backgrounds, and gene expression was evaluated in response to the mastoparan peptide agonist Mas7. We found that Mas7 elicits root hair MtENOD11 expression in dmi1 and dmi2 mutants, but not in the dmi3 mutant, suggesting that the agonist acts downstream of DMI1/DMI2 and upstream of DMI3. In light of these results and the recently discovered identities of the DMI gene products, we propose an integrated cellular model for Nod factor signaling in legume root hairs in which phospholipids play a key role in linking the Nod factor perception apparatus to downstream components such as Ca2+ spiking and ENOD gene expression.