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

Nitrogen‐fixing nodulation occurs in ten taxonomic lineages, either with 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 ~ 110 million years ago, 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 non‐legume system.Consistent with legumes, P. andersonii PanNIN and PanNF‐YA1 are co‐expressed 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 commit 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.