Nod factors integrate spontaneously in biomembranes and transfer rapidly between membranes and to root hairs, but transbilayer flip-flop does not occur

Three novel nodulation (Nod) factors were synthesized from chitotetraose an d three structurally different fluorescent BODIPY-tagged fatty acids. With fluorescence spectroscopic and microscopic techniques, the following aspect s were studied: whether these amphiphilic molecules insert in membranes, wh ether they transfer between different membranes, and whether they are able to transfer from a membrane to a legume root hair. Fluorescence correlation spectroscopy showed that fluorescent Nod factors are present as monomers i n PBS buffer at a concentration of 10 nM, but that when either Triton X-100 micelles or dioleoylphosphatidylcholine (DOPC) vesicles are present, the N od factors are associated with these particles. With time-correlated single -photon counting fluorescence spectroscopy, it was shown that upon Nod fact or insertion in the membrane, the rotation of the fluorescent acyl chain wa s markedly reduced. A fluorescence resonance energy transfer assay was used to study the transfer of Nod factors from one membrane to the other, or fr om vesicles to root hairs. Nod factors transfer rapidly between membranes o r from vesicles to root hair cell walls. However, they do not flip-flop bet ween membrane leaflets. The results provide novel insights for the mode of secretion and transfer of Nod factors during the early steps of the Rhizobi um-legume interaction.