Intercellular communication in the fern endosymbiotic cyanobacterium Nostoc azollae

The water fern Azolla spp. harbors as an endobiont the N 2 -fixing, filamentous, heterocyst-forming cyanobacterium Nostoc azollae. N. azollae provide the fern with fixed nitrogen permitting its growth in nitrogen-poor environments. In the filaments of heterocyst-forming cyanobacteria, an intercellular exchange of regulators and metabolites occur in which heterocysts provide vegetative cells with fixed nitrogen and vegetative cells provide heterocysts with reduced carbon. Intercellular molecular exchange takes place by diffusion through septal junctions and can be probed by fluorescence recovery after photobleaching (FRAP) analysis with fluorescent markers such as calcein and 5-carboxyfluorescein. The septal junctions traverse the septal peptidoglycan through nanopores that can be visualized in isolated septal peptidoglycan disks by electron microscopy. Here we obtained N. azollae material from Azolla plants, which contains the symbiotic cyanobacterium in a viable state and with different morphologies, including heterocyst-containing filaments. FRAP analysis showed effective transfer of the fluorescent markers between vegetative cells as well as from vegetative cells to heterocysts. Interestingly, communicating and noncommunicating vegetative cells and heterocysts could be distinguished showing conservation in the endobiont of a regulatory mechanism capable of opening and closing septal junctions. Peptidoglycan sacculi were also isolated and showed septal disks with arrays of nanopores that conform to those visualized in other heterocyst-forming cyanobacteria. However, a wider range of septal disk size was observed in N. azollae . In spite of its eroded genome, N. azollae maintains the intercellular communication system that is key for its growth as a multicellular organism. Importance The water fern Azolla constitutes a unique symbiotic system in which cyanobacterial endobionts capable of fixation of atmospheric nitrogen provide the plant with the nitrogen needed for growth. This symbiosis is an important fertilizer for rice crops worldwide, thereby reducing the reliance on fossil fuel-derived nitrogen fertilizers. The symbiotic cyanobacterium, Nostoc azollae , is a heterocyst-forming strain in which a filament of cells is the organismic unit of growth. Here we show that the intercellular molecular exchange function necessary for the multicellular behavior of the organism is conserved in the endobiotic Nostoc azollae .