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

Summary ● 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.


Table S1
Sequences of sgRNAs used for creating single, double and triple knockout mutants.

Table S2
Primers used in this work.

Table S3
Putative promoter sequences used for promoter-reporter GUS assays.

Table S4
Gene identifiers for NF-YA proteins used to build the phylogenetic tree depicted in   Figure   1A. Plants were grown in vitro (a-f), or in a perlite potting system (g). Data shown are obtained using transgenic PanNF-YA1pro:GUS line 1E5. ep: epidermis; pc: pericycle; px: protoxylem; arrowhead indicates lateral root primordia.      Orthogroups are indicated by coloured circles. Node labels indicate posterior probability, Node labels with a value above 0.9 are not shown. Pannin mutant (line B3) roots detected by qRT-PCR. Data were generated from the RNA samples used in Figure 2. RNA was isolated from root segments encompassing the elongation and part of the differentiation zone at 1 DPI with R. tropici CIAT899.pMP604. Data represent means of 2 independent experiments with a total of 5 biological replicates each ± SE. Data were normalized against the mock-treated CTR44 sample. np: nodule primordium; nodule: mature nodule.    Figure   6B were included in this analysis. Data represent means (n = 4-5 plants) ± SE. Different letters indicate statistical significance (Student's t-test, p < 0.05).    S14 Parasponia andersonii nf-ya1, nf-ya3, nf-ya6 and Pannf-ya1;Pannf-ya3;Pannf-ya6 mutants can form arbuscular mycorrhiza.

Fig. S15: Putative NIN Binding sites in the PanNF-YA1 promoter region.
Shown is the annotated PanNF-YA1 gene, including the putative promoter region (2,823 bp) and 5'UTR (977 bp containing an intron of 643 bp) that was used for PanNF-YA1pro:GUS reporter studies.