title: Interface Symbiotic Membrane Formation in Root Nodules ofMedicago truncatula: the Role of SynaptotagminsMtSyt1,MtSyt2andMtSyt3.
abstract: Symbiotic bacteria (rhizobia) are maintained and conditioned to fix atmospheric nitrogen in infected cells of legume root nodules. Rhizobia are confined to the asymmetrical protrusions of plasma membrane (PM): infection threads (IT), cell wall-free unwalled droplets and symbiosomes. These compartments rapidly increase in surface and volume due to the microsymbiont expansion, and remarkably, the membrane resources of the host cells are targeted to interface membrane quite precisely. We hypothesized that the change in the membrane tension around the expanding microsymbionts creates a vector for membrane traffic toward the symbiotic interface. To test this hypothesis, we selected calcium sensors from the group of synaptotagmins:MtSyt1, Medicago truncatulahomolog ofAtSYT1fromArabidopsis thalianaknown to be involved in membrane repair, and two other homologs expressed in root nodules:MtSyt2andMtSyt3.Here we show thatMtSyt1, MtSyt2, andMtSyt3are expressed in the expanding cells of the meristem, zone of infection and proximal cell layers of zone of nitrogen fixation (MtSyt1, MtSyt3). All three GFP-tagged proteins delineate the interface membrane of IT and unwalled droplets and create a subcompartments of PM surrounding these structures. The localization of MtSyt1 by EM immunogold labeling has shown the signal on symbiosome membrane and endoplasmic reticulum (ER). To specify the role of synaptotagmins in interface membrane formation, we compared the localization of MtSyt1, MtSyt3 and exocyst subunit EXO70i, involved in the tethering of post-Golgi secretory vesicles and operational in tip growth. The localization of EXO70i in root nodules and arbusculated roots was strictly associated with the tips of IT and the tips of arbuscular fine branches, but the distribution of synaptotagmins on membrane subcompartments was broader and includes lateral parts of IT, the membrane of unwalled droplets as well as the symbiosomes. The double silencing of synaptotagmins caused a delay in rhizobia release and blocks symbiosome maturation confirming the functional role of synaptotagmins.
  
  
  CONCLUSIONS
  synaptotagmin-dependent membrane fusion along with tip-targeted exocytosis is operational in the formation of symbiotic interface.
authors.affiliation: Laboratory of Molecular Biology, Department of Plant Sciences, Graduate School Experimental Plant Sciences, Wageningen UniversityWageningen, Netherlands; Sainsbury Laboratory, University of CambridgeCambridge, UK.
authors.forename: Aleksandr
authors.initials: A
authors.lastname: Gavrin
authors.affiliation: Laboratory of Molecular Biology, Department of Plant Sciences, Graduate School Experimental Plant Sciences, Wageningen University Wageningen, Netherlands.
authors.forename: Olga
authors.initials: O
authors.lastname: Kulikova
authors.affiliation: Laboratory of Molecular Biology, Department of Plant Sciences, Graduate School Experimental Plant Sciences, Wageningen University Wageningen, Netherlands.
authors.forename: Ton
authors.initials: T
authors.lastname: Bisseling
authors.affiliation: Laboratory of Molecular Biology, Department of Plant Sciences, Graduate School Experimental Plant Sciences, Wageningen University Wageningen, Netherlands.
authors.forename: Elena E
authors.initials: EE
authors.lastname: Fedorova
keywords: Medicago truncatula; arbuscular mycorrhiza; interface membrane; membrane tension/repair; root nodule; symbiosis; synaptotagmin1