Conserved symbiosis-associated genes in the cycad Encephalartos natalensis suggest co-option for cyanobacterial symbiosis.

Plant-cyanobacterial symbioses have evolved independently at least four times across land plants, yet their underlying molecular mechanisms remain largely elusive. Here, we elucidate the pathways involved in this specialised symbiosis and nutrient exchange within coralloid roots (CRs) of Encephalartos natalensis. Using anatomical analysis and RNA sequencing, we characterise the structural and transcriptional features of CRs harbouring active, heterocyst-rich, nitrogen-fixing Nostoc cyanobacteria. Notably, no fungal hyphae or arbuscular structures were observed under the sampled conditions. CR-associated upregulation of core common symbiosis signalling pathway (CSSP) genes was evident, genes shared across multiple nodulating symbioses. Transcriptome-wide analysis further revealed elevated expression of citrulline and ornithine biosynthesis genes, indicating host assimilation of Nostoc-fixed ammonia. Together, these findings demonstrate that cycads retain and transcriptionally upregulate conserved symbiosis signalling genes during cyanobacterial associations. Building on the evolutionary link between CSSP genes and the ancient arbuscular mycorrhizal (AM) signalling toolkit, our results support differential retention of these genes across plant lineages. Specifically, in E. natalensis, CSSP gene expression in CRs suggests transcriptional co-option for cyanobacterial symbiosis in CRs. This study provides a framework for understanding the role of ancient molecular pathways in driving plant-microbe symbiosis evolution and diversification.