Intercellular communication in the fern endosymbiotic cyanobacterium <i>Nostoc azollae</i>

The water fern <i>Azolla</i> spp. harbors as an endobiont the N₂-fixing, filamentous, heterocyst-forming cyanobacterium <i>Nostoc azollae. N. azollae</i> provides the fern with fixed nitrogen permitting its growth in nitrogen-poor environments. In the diazotrophic filaments of heterocyst-forming cyanobacteria, intercellular molecular exchange occurs 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 (PG) through nanopores that can be visualized in isolated septal PG disks by electron microscopy. Here, we obtained from <i>Azolla</i> plants material containing 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 mechanism regulating the septal junctions. PG 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 <i>N. azollae</i>. In spite of its eroded genome, <i>N. azollae</i> maintains the intercellular communication system that is key for its growth as a multicellular organism. Additionally, labeling with the fluorescent sucrose analog esculin suggests sucrose as a source of reduced carbon for the endobiont.IMPORTANCEThe water fern <i>Azolla</i> constitutes a unique symbiotic system in which cyanobacterial endobionts capable of fixing 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, <i>Nostoc azollae</i>, 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 <i>N. azollae</i>.