In the non-legume rhizobial nodulator Parasponia andersonii, the transcription factor NODULE INCEPTION (PanNIN) is required for nodule organogenesis and for early rhizobium-induced expression of PanNF-YA1, while PanNF-YA1 is specifically required for intracellular rhizobium infection — establishing NIN and NF-YA1 as a core genetic network shared with legumes (Bu et al. 2020). Three linked questions remain open: whether PanNIN directly binds and transcriptionally activates the PanNF-YA1 promoter (its putative NIN-binding sites have never been functionally tested); whether the direct NIN→NF-YA1 cis-regulatory module is conserved in non-legume nodulators and thus ancestral to the nitrogen-fixing clade rather than assembled in the legume lineage (the DNA-binding capacity itself was recently shown to predate nodulation, but the cis-regulatory wiring outside legumes is unresolved); and which direct transcriptional targets of PanNF-YA1, acting with its NF-YB/NF-YC partners, execute intracellular infection.

Background

Some flowering plants form nitrogen-fixing root nodule symbioses with soil bacteria: legumes (Fabales) and a few other lineages associate with rhizobia, while actinorhizal plants (e.g., Casuarina, Alnus, Datisca) associate with Frankia bacteria. The trait is confined to the 'nitrogen-fixing clade' (NFC) — four plant orders (Fabales, Fagales, Cucurbitales, Rosales) — and is hypothesized to have originated once, roughly 110 million years ago, followed by many independent losses; this single-origin-versus-repeated-assembly question is itself actively debated. Establishing the endosymbiosis requires two coordinated developmental programs: nodule organogenesis (formation of a new root organ) and intracellular infection (uptake of bacteria into living plant cells, in legumes typically through tubular infection-thread structures). In legumes, both programs are governed by the transcription factor NODULE INCEPTION (NIN) — a member of the RWP-RK DNA-binding family derived from nitrate-responsive NIN-LIKE PROTEIN (NLP) regulators — and by genes of the NUCLEAR FACTOR Y (NF-Y) complex, a conserved heterotrimer of NF-YA, NF-YB and NF-YC subunits that binds CCAAT-box elements to control transcription. In Lotus japonicus, NIN directly binds the promoters of the NF-Y subunit genes LjNF-YA1 and LjNF-YB1 (Soyano et al. 2013); NF-YA1 in turn directly targets SHI/STY-family transcription factors that drive local auxin biosynthesis during nodule emergence (Hossain et al. 2016; Shrestha et al. 2021); in Medicago truncatula, NF-YA1 controls rhizobial infection (Laporte et al. 2014) and specific NF-YA/NF-YB/NF-YC subunits form conserved nodulation complexes (Baudin et al. 2015).

Parasponia andersonii (Cannabaceae, Rosales) is the only non-legume genus known to nodulate with rhizobia. Because Parasponia and the legumes diverged soon after nodulation arose, Parasponia is a decisive outgroup for testing whether the legume regulatory network is an inherited ancestral program. Using CRISPR-Cas9 mutagenesis, Bu et al. (2020) showed that PanNIN is required both for nodule organogenesis and for early rhizobium-induced PanNF-YA1 expression, that PanNF-YA1 is specifically required for intracellular infection (knockout mutants form nodules but no infection threads, unlike legume nf-ya1 mutants whose organogenesis is impaired), and that PanNF-YA1, PanNF-YA3 and PanNF-YA6 act redundantly in nodule growth. Putative NIN-binding motifs were found in the PanNF-YA1 promoter, but binding was not tested; at that time, direct NIN→NF-YA1 regulation had been demonstrated only in Lotus. The paper therefore posed its central unresolved question: whether the direct NIN→NF-YA1 transcriptional module is ancestral to the nitrogen-fixing clade, or whether it evolved together with the nodulation trait — a question whose answer bears directly on how the nodulation program was assembled in the NFC ancestor. Recent work has meanwhile shown that the capacity of NIN proteins to bind and activate NF-YA1 predates nodulation (nonsymbiotic NIN orthologs from non-nodulating plants outside the NFC can do so in vitro and in vivo), which sharpens but does not settle the question: what remains untested is the cis-regulatory wiring — whether NF-YA1 promoters are under direct NIN control in non-legume nodulators — and the downstream mechanism, i.e., the set of genes that PanNF-YA1 regulates to enable bacterial entry into nodule cells.

Problem Statement

Determine the direct regulatory wiring, evolutionary origin, and mechanistic output of the NIN→NF-YA1 transcriptional module in nitrogen-fixing nodule symbiosis, as posed by Bu et al. (2020) for the non-legume nodulator Parasponia andersonii. Three linked questions define the problem, with the module's assembly history as the overarching objective: (i) Does PanNIN directly bind and transcriptionally activate PanNF-YA1 in P. andersonii — are the putative NIN-binding sites in the PanNF-YA1 promoter functional in vivo, established for example by chromatin immunoprecipitation, electrophoretic mobility shift assays, or promoter deletion/site-directed mutagenesis linked to loss of NIN-dependent PanNF-YA1 induction? (ii) Is the direct NIN→NF-YA1 cis-regulatory link conserved outside the legumes — in Parasponia and, in addition, other non-legume nodulating lineages such as actinorhizal Fagales — so that the module can be inferred to be ancestral to the nitrogen-fixing clade rather than having been assembled within the legume lineage after the split? (Note: the DNA-binding capacity of NIN orthologs for NF-YA1-type sites has been shown to predate nodulation; what remains unresolved is the cis-regulatory deployment of that capacity in non-legume nodulators.) (iii) Which direct transcriptional targets of PanNF-YA1 — acting with its NF-YB/NF-YC partners in the NF-Y complex, and in relation to the redundant PanNF-YA3/PanNF-YA6 — execute intracellular rhizobium infection in P. andersonii, i.e., what is the PanNF-YA1 regulon (the set of genes directly controlled by PanNF-YA1) for infection-thread initiation and bacterial uptake into nodule cells? All three linked questions are required to resolve the task: question (i) is the Parasponia instance of the conservation question in (ii), so a positive answer to (i) settles the non-legume case, while the wider survey of other non-legume nodulating lineages is a strengthening of the ancestral inference rather than an independent requirement.

References

  1. Bu F, Rutten L, Roswanjaya YP, Kulikova O, Rodriguez-Franco M, Ott T, Bisseling T, van Zeijl A, Geurts R. Mutant analysis in the nonlegume Parasponia andersonii identifies NIN and NF-YA1 transcription factors as a core genetic network in nitrogen-fixing nodule symbioses. New Phytologist 226(2):541–554 (2020). DOI 10.1111/nph.16386. https://doi.org/10.1111/nph.16386
  2. Soyano T, Kouchi H, Hirota A, Hayashi M. NODULE INCEPTION directly targets NF-Y subunit genes to regulate essential processes of root nodule development in Lotus japonicus. PLoS Genetics 9(3):e1003352 (2013). DOI 10.1371/journal.pgen.1003352. https://doi.org/10.1371/journal.pgen.1003352
  3. Soyano T, Shimoda Y, Kawaguchi M, Hayashi M. A shared gene drives lateral root development and root nodule symbiosis pathways in Lotus. Science 366(6468):1021–1023 (2019). DOI 10.1126/science.aax2153. https://doi.org/10.1126/science.aax2153
  4. Laporte P, Lepage A, Fournier J, Catrice O, Moreau S, Jardinaud M-F, Mun J-H, Larrainzar E, Cook DR, Gamas P, Niebel A. The CCAAT box-binding transcription factor NF-YA1 controls rhizobial infection. Journal of Experimental Botany 65(2):481–494 (2014). DOI 10.1093/jxb/ert392. https://doi.org/10.1093/jxb/ert392
  5. Baudin M, Laloum T, Lepage A, Rípodas C, Ariel F, Frances L, Crespi M, Gamas P, Blanco FA, Zanetti ME, de Carvalho-Niebel F, Niebel A. A phylogenetically conserved group of nuclear factor-Y transcription factors interact to control nodulation in legumes. Plant Physiology 169(4):2761–2773 (2015). DOI 10.1104/pp.15.01144. https://doi.org/10.1104/pp.15.01144
  6. Hossain MS, Shrestha A, Zhong S, Miri M, Austin RS, Sato S, Ross L, Huebert T, Tromas A, Torres-Jerez I, Tang Y, Udvardi M, Murray JD, Szczyglowski K. Lotus japonicus NF-YA1 plays an essential role during nodule differentiation and targets members of the SHI/STY gene family. Molecular Plant-Microbe Interactions 29(12):950–964 (2016). DOI 10.1094/mpmi-10-16-0206-r. https://doi.org/10.1094/mpmi-10-16-0206-r
  7. Shrestha A, Zhong S, Therrien J, Huebert T, Sato S, Mun T, Andersen SU, Stougaard J, Lepage A, Niebel A, Ross L, Szczyglowski K. Lotus japonicus Nuclear Factor YA1, a nodule emergence stage-specific regulator of auxin signalling. New Phytologist 229(3):1535–1552 (2021). DOI 10.1111/nph.16950. https://doi.org/10.1111/nph.16950
  8. Clavijo F, Diedhiou I, Vaissayre V, Brottier L, Acolatse J, Moukouanga D, Crabos A, Auguy F, Franche C, Gherbi H, Champion A, Hocher V, Barker D, Bogusz D, Tisa LS, Svistoonoff S. The Casuarina NIN gene is transcriptionally activated throughout Frankia root infection as well as in response to bacterial diffusible signals. New Phytologist 208(3):887–903 (2015). DOI 10.1111/nph.13506. https://doi.org/10.1111/nph.13506
  9. Liu J, Yan S, Li M, Shen D, Tichá M, Bærentsen R, Andersen KR, Verbeek F, Kulikova O, Geurts R, Bisseling T, Huisman R. Ancestral functionality and symbiotic refinement of NIN in root nodule symbiosis. Nature Communications 17:4907 (2026). DOI 10.1038/s41467-026-71330-1. https://doi.org/10.1038/s41467-026-71330-1
  10. Nosaki S, Noda M, Onoda H, Ito M, Suzaki T. The root nodule symbiosis regulator NIN exhibits broad DNA binding specificity conferred by an NLP-inherited motif. Science Advances 12(5):eaeb8825 (2026). DOI 10.1126/sciadv.aeb8825. https://doi.org/10.1126/sciadv.aeb8825
  11. Noda M, Ito M, Miyata K, Suzaki T. Nonnodulating Fagales retain the functional NODULE INCEPTION gene. New Phytologist 251(6):3080–3087 (2026). DOI 10.1111/nph.71337. https://doi.org/10.1111/nph.71337
  12. Cathebras C, Gong X, Andrade RE, Vondenhoff K, Keller J, Delaux PM, Hayashi M, Griesmann M, Parniske M. A novel cis-element enabled bacterial uptake by plant cells. Nature Plants 12(1):140–151 (2026). DOI 10.1038/s41477-025-02161-z. https://doi.org/10.1038/s41477-025-02161-z
  13. Yu H, Xiao A, Zou Z, Zou Z, Wu Q, Chen L, Zhang D, Sun Y, Wang C, Cao J, Zhu H, Zhang Z, Cao Y. Conserved cis-elements enable NODULES WITH ACTIVATED DEFENSE1 regulation by NODULE INCEPTION during nodulation. The Plant Cell 36(10):4622–4636 (2024). DOI 10.1093/plcell/koae229. https://doi.org/10.1093/plcell/koae229
  14. Doyle JJ, Ren J, Pawlowski K, James EK, Gao Y. One versus many independent assemblies of symbiotic nitrogen fixation in flowering plants. Nature Communications 16:5345 (2025). DOI 10.1038/s41467-025-60433-w. https://doi.org/10.1038/s41467-025-60433-w
  15. Kates HR, O'Meara BC, LaFrance R, Stull GW, James EK, Liu S-Y, Tian Q, Yi T-S, Conde D, Kirst M, Ané J-M, Soltis DE, Guralnick RP, Soltis PS, Folk RA. Shifts in evolutionary lability underlie independent gains and losses of root-nodule symbiosis in a single clade of plants. Nature Communications 15:4262 (2024). DOI 10.1038/s41467-024-48036-3. https://doi.org/10.1038/s41467-024-48036-3
  16. Shen L, Feng J. NIN—at the heart of nitrogen-fixing nodule symbiosis. Frontiers in Plant Science 14:1284720 (2024). DOI 10.3389/fpls.2023.1284720. https://doi.org/10.3389/fpls.2023.1284720