Mutant analysis in the nonlegume Parasponia andersonii identifies NIN and NF-YA1 transcription factors as a core genetic network in nitrogen-fixing nodule symbioses

Nitrogen-fixing nodulation occurs in 10 taxonomic lineages, with either rhizobia or Frankia bacteria. To establish such an endosymbiosis, two processes are essential: nodule organogenesis and intracellular bacterial infection. In the legume-rhizobium endosymbiosis, both processes are guarded by the transcription factor NODULE INCEPTION (NIN) and its downstream target genes of the NUCLEAR FACTOR Y (NF-Y) complex. It is hypothesized that nodulation has a single evolutionary origin c. 110 Ma, followed by many independent losses. Despite a significant body of knowledge of the legume-rhizobium symbiosis, it remains elusive which signalling modules are shared between nodulating species in different taxonomic clades. We used Parasponia andersonii to investigate the role of NIN and NF-YA genes in rhizobium nodulation in a nonlegume system. Consistent with legumes, P. andersonii PanNIN and PanNF-YA1 are coexpressed in nodules. By analyzing single, double and higher-order CRISPR-Cas9 knockout mutants, we show that nodule organogenesis and early symbiotic expression of PanNF-YA1 are PanNIN-dependent and that PanNF-YA1 is specifically required for intracellular rhizobium infection. This demonstrates that NIN and NF-YA1 have conserved symbiotic functions. As Parasponia and legumes diverged soon after the birth of the nodulation trait, we argue that NIN and NF-YA1 represent core transcriptional regulators in this symbiosis. BLACK CIRCLEBLACK CIRCLEBLACK CIRCLEBLACK CIRCLE