Direct coupling of Nod factor signaling to legume vesicular trafficking: A key event in nodulation initiation

Legume–rhizobium symbiosis relies on the precise integration of receptor-mediated signaling with dynamic membrane trafficking to initiate infection threads (ITs) and accommodate bacterial infection via root hairs. Here, we identify the soybean Qa-SNARE GmSYNTAXIN111a (GmSYP111a), a close paralog of the cytokinesis-specific protein KNOLLE, as a critical regulator in symbiotic infection. A kinase client (KiC) assay revealed that GmSYP111a is a direct phosphorylation substrate of the receptor kinase GmSymRKβ (also known as DMI2 or NORK in other legumes), which modifies Ser-8 at its N-terminus. BiFC, co-immunoprecipitation, and in vitro kinase assays confirmed that GmSymRKβ phosphorylates GmSYP111a without disrupting their basal association, but phosphorylation alters the subcellular distribution of the complex. In soybean root hairs, GmSYP111a and GmSymRKβ co-localize to the plasma membrane, and Nod factor perception induces clathrin-mediated endocytosis accompanied by GmSYP111a re-localization into intracellular vesicles. A non-phosphorylatable mutant ( GmSYP111a ^S8A ) showed markedly reduced internalization and a corresponding decrease in infection events, resulting in impaired IT formation and a phenotype resembling GmSymRKβ -RNAi roots. In contrast, a phosphomimetic variant ( GmSYP111a ^S8D ) displayed partial vesicular recruitment upon infection. Functional analyses further demonstrated that GmSYP111a , but not its paralog SYP111b , is indispensable for nodule initiation in soybean, with this role conserved in Lotus japonicus . Collectively, our results show that GmSymRKβ-mediated phosphorylation of GmSYP111a functions as a molecular switch that links symbiotic signaling to clathrin-dependent endocytosis. This mechanism coordinates localized membrane remodeling at infection sites, parallels the role of KNOLLE in cytokinesis, and highlights how duplication and neo-functionalization of GmSYP111a contributed to the evolution of legume-specific symbiotic pathways.