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

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出典： Nature 462, 514-517 (26 November 2009)
DOI： 10.1038/nature08594
リンク先： http://dx.doi.org/10.1038/nature08594

Host plant genome overcomes the lack of a bacterial
gene for symbiotic nitrogen fixation
Tsuneo Hakoyama1,2, Kaori Niimi2, Hirokazu Watanabe2, Ryohei Tabata2, Junichi
Matsubara2, Shusei Sato3, Yasukazu Nakamura3, Satoshi Tabata3, Li Jichun4, Tsuyoshi
Matsumoto4, Kazuyuki Tatsumi4, Mika Nomura5, Shigeyuki Tajima5, Masumi Ishizaka6,
Koji Yano1, Haruko Imaizumi-Anraku1, Masayoshi Kawaguchi7, Hiroshi Kouchi1 &
Norio Suganuma2
1National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
2Department of Life Science, Aichi University of Education, Kariya, Aichi 448-8542,
Japan. 3Kazusa DNA Research Institute, Kisarazu, Chiba 292-0812, Japan. 4Research
Center for Materials Science and Department of Chemistry, Graduate School of Science,
Nagoya University, Nagoya, Aichi 464-8602, Japan. 5Faculty of Agriculture, Kagawa
University, Kita, Kagawa 761-0795, Japan. 6National Institute of Agro-Environmental
Sciences, Tsukuba, Ibaraki 305-8604, Japan. 7National Institute for Basic Biology,
Okazaki, Aichi 444-8585, Japan.
Homocitrate is a component of the iron-molybdenum cofactor (FeMo-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 of rhizobium species that exert 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– plant mutant, fen1, forms morphologically normal but ineffective
nodules4,5. The causal gene, FEN1, was shown to encode HCS by its ability to

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complement a Saccharomyces cerevisiae HCS-defective mutant. Homocitrate was
present abundantly in wild-type nodules but was absent in 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 bacteroids. These results indicate that
homocitrate derived from the host plant cells is essential for the efficient and
continuing synthesis of nitrogenase system in endosymbionts, and thus provides a
molecular basis for the complementary and indispensable partnership between
legumes and rhizobia in symbiotic nitrogen fixation.
The major source of nitrogen for all living organisms is atmospheric dinitrogen,
which is mainly fixed by microorganisms that have an ability to reduce dinitrogen to
ammonium by a nitrogenase system. In legume plants, soil bacteria of the family
Rhizobiaceae (rhizobia) are hosted within a symbiotic organ, the root nodule, in which
the endosymbiotic rhizobia are able to fix dinitrogen. This enables the host legumes to
grow without an exogenous nitrogen source. Unlike many free-living diazotrophs,
rhizobia are able to exhibit highly efficient nitrogen fixation only when they are in the
host nodule cells as an endosymbiotic form, the bacteroid. This indicates that rhizobial
nitrogen fixation is controlled by the host plant. Fix– mutants of the host legumes that
form ineffective nodules are key tools to identify the host genes essential for
establishment of symbiotic nitrogen fixation.
A L. japonicus Fix– mutant, fen14,5, forms small, pale-pink nodules and
displays nitrogen deficiency symptoms under symbiotic conditions (Supplementary Fig.
1a-c and f-h). In the fen1 nodules, rhizobial invasion of the nodule cells appeared to be
comparable to wild-type Gifu (Supplementary Fig. 1d and e), but the nitrogenase
activity remained at very low levels (Supplementary Fig. 1i and j).

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We identified the responsible gene, FEN1, for the fen1 mutant through map-
based cloning, and confirmed the complementation of the mutant phenotypes by
Agrobacterium rhizogenes-mediated hairy root transformation (Supplementary Results
and Supplementary Fig. 2). Transcripts of FEN1 were detected only in nodules,
indicating that expression of the FEN1 gene was regulated in a nodule specific manner
(Fig. 1a). When the FEN1 promoter-ß-glucuronidase (GUS) fusion was introduced,
GUS activity was detected only in infected cells of nodules (Fig. 1b and c). By
searching L. japonicus EST database6, we found a paralogous clone to the FEN1 gene,
MWM049f12, of which predicted amino-acid sequence had 91% identity to that of the
FEN1 gene. However, expression of MWM049f12 was detected in all organs of L.
japonicus at low levels, and was not enhanced in nodules. These results indicate that
FEN1 is closely associated with nitrogen-fixing activity of the nodules.
The predicted FEN1 protein consisted of 540 amino acids with a molecular
mass of 58,600. Any signal peptide sequences were not found, suggesting that it is a
cytosolic protein. Deduced amino-acid sequence of FEN1 had 71% identity to that for
the Glycine max nodule-specific gene, GmN56. The introduction of GmN56 cDNA into
the fen1 mutant recovered growth and nitrogenase activity of the mutant
(Supplementary Fig. 3a and b), indicating that GmN56 is an ortholog of the FEN1 gene.
GmN56 has been shown to be induced with the onset of nitrogen fixation and the
transcripts are localized in the bacterial infected cells of mature nodules of soybean7,
consistent with the expression pattern of the FEN1 gene in L. japonicus nodules. The
predicted GmN56 protein showed homology to 2-isopropylmalate synthase (IPMS) and
homocitrate synthase (HCS), though the exact function of the GmN56 protein has not
been confirmed. Besides GmN56, several genes encoding IPMS isolated from plants
such as Brassica atlantica, Arabidopsis thaliana, and Lycopersicon pennellii were
found to show high similarity (around 66% amino acid sequence identity) with FEN1.

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To explore the function of FEN1, we first introduced the FEN1 gene into a S.
cerevisiae IPMS defective mutant8. FEN1 failed to complement leucine auxotrophy of
the S. cerevisiae mutant, and the cell extract exhibited no IPMS activity (Supplementary
Fig. 4a and b). By contrast, we detected substantial activity of IPMS in the extract of S.
cerevisiae transformed with the Arabidopsis IPMS2 (At1g74040) gene9 and the
complementation of leucine auxotrophy, even though in part (see the legend for
Supplementary Fig. 4). In addition, the Fix– phenotype of fen1 was not recovered by
introduction of the AtIPMS2 gene (Supplementary Fig. 3a and b). From these results,
we concluded that the FEN1 gene does not code for IPMS.
We next focused on HCS, which catalyzes the synthesis of homocitrate from 2-
oxoglutarate and acetyl-CoA. IPMS and HCS are different enzymes, but they have
some structural similarity7. They both catalyze similar reactions; the transfer of an acyl-
group from acetyl-CoA to 2-oxo acid to generate the alkyl-group in 2-oxo acid. The
FEN1 protein has 36% identity to HCS (NIFV) of the nitrogen-fixing aerobic bacteria
Azotobacter vinelandii3. We introduced the FEN1 gene into a S. cerevisiae mutant
which shows lysine auxotrophy caused by the lack of HCS10. The introduction of the
FEN1 gene, but not Arabidopsis IPMS2 and mutated FEN1 gene, complemented lysine
auxotrophy of the mutant (Fig. 2a). Furthermore, significant accumulation of
homocitrate was found in the transformed S. cerevisiae mutant when expression of the
FEN1 gene was induced (Supplementary Fig. 5). These results demonstrated that the
recombinant FEN1 protein confers HCS activity. In the present study, we were unable
to detect HCS activity in vitro in cell-free extracts of Lotus nodules. We thus
investigated the presence of homocitrate in various tissues of L. japonicus to confirm
HCS activity in vivo. LC/MS/MS analysis showed that, in wild-type Gifu plants,
homocitrate was detected abundantly in nodules, but neither in roots nor shoots (Fig.
2b). By contrast, it was barely detectable (less than 1% of wild-type nodules) in
ineffective nodules formed on the fen1 mutant (Fig. 2c). The ineffective nodules formed

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by the NifH defective mutant of M. loti contained homocitrate at the level comparable to
that in the wild-type nodules, indicating that accumulation of homocitrate in nodules is
not the result of active nitrogen fixation. In addition, the level of 2-oxoglutarate was
found to be higher in the fen1 nodules than the wild-type nodules and ineffective
nodules formed by the NifH defective mutant of M. loti (Fig. 2c). These results indicate
that FEN1 encodes HCS and the activity is lost in nodules of the fen1 mutant.
In higher plants, a metabolic pathway leading to synthesis of lysine through
homocitrate as an intermediate has not been identified. Here we took notice of the fact
that homocitrate is a component of the FeMo-cofactor of nitrogenase complex in
nitrogen-fixing bacteria2. Therefore, homocitrate synthesized in host plant cells was
expected to be transported to bacteroids and utilized for biosynthesis of the nitrogenase
complex. We examined this hypothesis by introducing the FEN1 gene into M. loti under
the control of the rhizobial NifH promoter. Inoculation with M. loti carrying the FEN1
gene to the fen1 mutant rescued either the defect in nodule nitrogenase activity or the
plant growth (Fig. 3a and b). Expression of FEN1 in the bacteroids of nodules formed
by transformed M. loti was confirmed by immuno-detection of FEN1-myc fusion
protein (Fig. 3c). In a similar way, we tested inoculation with M. loti carrying the A.
vinelandii NifV gene, which has been well demonstrated to encode HCS and to be
essential for nitrogenase activity3. M. loti with expression of A. vinelandii NifV could
also rescue the fen1 mutant phenotypes (Fig. 3d-f). Furthermore, we found that addition
of homocitrate into the culture solution restored in part the effectiveness of the nodules
formed on the fen1 mutant (Fig. 4a and b). In the fen1 nodules supplied with
homocitrate, amounts of nitrogenase proteins were recovered to the level nearly
comparable to those in the wild-type nodules (Fig. 4c), indicating that the restoration of
nitrogenase activity by the supply of homocitrate was due to de novo nitrogenase
biosynthesis. Taken together, these results indicate that rhizobial nitrogen-fixing activity
depends on the homocitrate derived from the host plant, which could be utilized for