Establishment of a functional symbiosis between the cyanobacterium Nostoc punctiforme and the bryophyte Anthoceros punctatus requires genes involved in nitrogen control and initiation of heterocyst differentiation.

Three mutant strains (ntcA, hetR, hetF) of the cyanobacterium Nostoc punctiforme unable to differentiate heterocysts were characterized and examined for their ability to form a symbiotic association with the bryophyte Anthoceros punctatus. Previously unknown characteristics of the N. punctiforme hetR mutant include differentiation of chilling-resistant akinetes, while vegetative cells of the ntcA mutant randomly lysed, yielding short filaments, following ammonium deprivation. Strains with mutations in hetF and hetR infected A. punctatus with similar frequency to that of wild-type N. punctiforme but did not support growth of the plant partner. These results confirm that the infection of A. punctatus by hormogonia leading to the establishment of an association is physiologically uncoupled from the development of a functional diazotrophic association. They also indicate that heterocyst regulatory elements downstream from HetR and HetF are required in both free-living and symbiotic heterocyst differentiation and nitrogenase expression. A strain with a mutation in the global nitrogen regulator ntcA did not infect A. punctatus despite its ability to differentiate hormogonia at a low frequency. When complemented with one or more copies of ntcA, the mutant strain infected A. punctatus at a similar frequency as the wild-type and supported growth of the plant partner in the absence of combined nitrogen. These results established a connection between the presence of a functional copy of ntcA and the magnitude of hormogonium differentiation, and the behaviour of the formed hormogonia.