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Host plant genome overcomes the lack of a bacterial gene for symbiotic nitrogen fixation


Homocitrate is a component of the iron–molybdenum cofactor in nitrogenase, where nitrogen fixation occurs1,2. NifV, which encodes homocitrate synthase (HCS)3, has been identified from various diazotrophs but is not present in most rhizobial species that perform efficient nitrogen fixation only in symbiotic association with legumes. Here we show that the FEN1 gene of a model legume, Lotus japonicus, overcomes the lack of NifV in rhizobia for symbiotic nitrogen fixation. A Fix- (non-fixing) plant mutant, fen1, forms morphologically normal but ineffective nodules4,5. The causal gene, FEN1, was shown to encode HCS by its ability to complement a HCS-defective mutant of Saccharomyces cerevisiae. Homocitrate was present abundantly in wild-type nodules but was absent from ineffective fen1 nodules. Inoculation with Mesorhizobium loti carrying FEN1 or Azotobacter vinelandii NifV rescued the defect in nitrogen-fixing activity of the fen1 nodules. Exogenous supply of homocitrate also recovered the nitrogen-fixing activity of the fen1 nodules through de novo nitrogenase synthesis in the rhizobial bacteroids. These results indicate that homocitrate derived from the host plant cells is essential for the efficient and continuing synthesis of the nitrogenase system in endosymbionts, and thus provide a molecular basis for the complementary and indispensable partnership between legumes and rhizobia in symbiotic nitrogen fixation.

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Figure 1: Expression analyses of FEN1 from Lotus japonicus.
Figure 2: Functional complementation of a Saccharomyces cerevisiae homocitrate-synthase defective mutant, and homocitrate and 2-oxoglutarate content in nodules.
Figure 3: Complementation of fen1 mutants by inoculation with Mesorhizobium loti carrying FEN1 and Azotobacter vinelandii NifV.
Figure 4: Effect of supplying homocitrate to the fen1 mutant.

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Data deposits

The sequences have been deposited at the DNA Data Bank of Japan with the accession numbers AP004466 (LjT09C23), AP010267 (LjT02F04), AP010268 (LjT28C03) and AB494481 (mRNA sequence (Gifu B-129) of FEN1).


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We thank E. Casalone and E. Dubois for providing Saccharomyces cerevisiae mutants; T. Bisseling for providing nitrogenase antibodies; Y. Kawaharada and H. Mitsui for technical help; and R. W. Ridge for critical reading of the manuscript. This work was supported by the Special Coordination Funds for Promoting Science and Technology of the Japanese Ministry of Education, Culture, Sports, Science and Technology.

Author Contributions All authors contributed extensively to the experimental work. The manuscript was written by T.H., H.K. and N.S.

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Correspondence to Norio Suganuma.

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Hakoyama, T., Niimi, K., Watanabe, H. et al. Host plant genome overcomes the lack of a bacterial gene for symbiotic nitrogen fixation. Nature 462, 514–517 (2009).

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