Takashi Soyano, Keisuke Yokota, Hiroshi Kouchi, Atsuko Hirota, Makoto Hayashi

Plant Symbiosis Research Unit

［Abstract］

In order to confer symbiotic nitrogen fixation on non-legumes such as rice, it is crucial to identify symbiotic genes that are specific to legumes. We found that a transcription factor NIN, which is essential for induction of root nodule development, directly regulates cell division in cortical layers, where nodule primordium arises. Our findings indicate that introduction of NIN into non-legumes could facilitate induction of nodule formation.

［Keywords］

nitrogen fixation, rhizobia, root nodule development, plant-microbe interaction, transcription factor, Lotus japonicus

［Background］

Nitrogen-fixing bacteria, collectively called rhizobia, infect legume plant roots to induce formation of nodules, in which bacteria fix atmospheric nitrogen to ammonia that are supplied to host plants. This allows legume plants to survive in nitrogen-poor soil environments. On the contrary, major crops such as rice, wheat, corn etc. depend on chemical nitrogen fertilizers. It is highly demanding for agriculture to establish strategies not depending on too much chemical fertilizers because of possible depletion of underground resources, food security caused by soaring fertilizer prices, and environmental effects such as emission of greenhouse gases. In order to generate nitrogen-fixing non-legumes, we investigate molecular mechanisms in root nodule development. If we identify symbiotic genes that had been involved in legume evolution for acquiring abilities of root nodule development, then we can transfer the genes into non-legumes, to confer nodule formation on non-legumes.

［Results and Discussion］

1. By extensive researches on molecular genetics using model legumes such as Lotus japonicus, it is now generally accepted that legumes induce root nodule development through the common symbiosis pathways, which are also necessary for symbiosis with arbuscular mycorrhiza fungi. It is shown that the common symbiosis pathways are widely conserved among land plants, such as in rice, which establish symbiosis with arbuscular mycorrhizal fungi.
2. We focused on a protein that functions downstream of the common symbiosis pathways. NIN, which is required for onset of root nodule development, is legume specific (Fig. 1). This implies that the evolution of NIN might be a key factor in the acquisition of nodulation ability among legumes.
3. We identified NIN-binding cis motifs and found that they are present in the promoter region of NF-Y genes, which are important for regulation of cell division. Artificial activation of NIN directly induces the expression of NF-Y genes. Furthermore, ectopic expression of NIN induces the development of nodule-like structures that have nodule identities (Fig. 2). On the contrary, knockdown of NF-Y expression suppresses root nodule formation. These results indicate that NIN regulates the onset of root nodule development by controlling the expression of NF-Y genes.
4. Although NIN is legume specific, NF-Y genes are widely conserved among eukaryotes. Recruitment of NF-Y expression by NIN may be crucial for invention of root nodule development by legumes.

1. We identified key factors that are likely involved in evolution of nodule symbiosis.
2. Although the mechanism of conferring nodulation on non-legumes has not been clarified, we were able to show that introduction of legume-specific symbiosis genes can be a clue for that purpose.
3. It is important to clarify how NIN can be made functional in non-legumes and characterize the roles of NF-Y in non-legumes in order to confer nodulation on non-legumes.

Fig.1. Cluster alignment of NIN and its homolog NLP. NLP is conserved among land plants whereas NIN is found only in legumes. The closest NLP homolog of NIN in rice does not function in nodule formation.

Fig.2. Nodule-like structures induced by ectopic expression of NIN. NIN can induce nodule-like structures without presence of bacteria. Histological and molecular markers indicate that the structure is identical to nodules.

 

[Reference]

1. Soyano T, Kouchi H, Hirota A, Hayashi M (2013) NODULE INCEPTION directly targets NF-Y subunit genes to regulate essential processes of root nodule development in Lotus japonicus PLoS Genetics 9(3):e1003352
2. Yokota K, Hayashi M (2011) Function and evolution of nodulation genes in legumes Cellular and Molecular Life Sciences 68(8):1341-1351
3. Yokota K, Soyano T, Kouchi H, Hayashi M (2010) Function of GRAS proteins in root nodule symbiosis is retained in homologs of a non-legume, rice Plant and Cell Physiology 51(9):1436-1442