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DNA Research Advance Access originally published online on January 22, 2007
DNA Research 2006 13(6):255-265; doi:10.1093/dnares/dsl017
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© The Author 2006. Kazusa DNA Research Institute.
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Positional Cloning Identifies Lotus japonicus NSP2, A Putative Transcription Factor of the GRAS Family, Required for NIN and ENOD40 Gene Expression in Nodule Initiation

Yasuhiro Murakami1,3,4, Hiroki Miwa2 {dagger}, Haruko Imaizumi-Anraku1,4, Hiroshi Kouchi1, J. Allan Downie2, Masayoshi Kawaguchi3,* and Shinji Kawasaki1,4

1 National Institute of Agrobiological Sciences, 2-1-2 Kannondai Tsukuba, Ibaraki 305-8602, Japan
2 John Innes Centre Colney Lane, Norwich NR4 7UH, UK
3 Department of Biological Sciences, Graduate School of Science, The University of Tokyo 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan
4 Bio-oriented Technology Research Advancement Institution (BRAIN), Tokyo Office 3-18-19 Toranomon, Minato-ku, Tokyo 105-0001, Japan

Received 30 November 2006; revised 4 December 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgements
 REFERENCES
 
Rhizobia-secreted Nod-factors (NFs) are required for nodulation. In the early developmental process of nodulation, a large number of changes occur in gene expression. Lotus japonicus nsp2 mutants isolated from Gifu B-129 ecotype have defects in nodule initiation and display non-nodulating phenotype. Here, we describe positional cloning of LjNSP2 as a component of the nodulation-specific signaling pathway. LjNSP2 was mapped near the translocation site of chromosome 1 where the recombination is severely suppressed. To circumvent this problem, we introduced Lotus burttii as an alternative crossing partner in place of L. japonicus Miyakojima. The development of the high-resolution map using a total of 11 481 F2 plants, in combination with newly developed DNA markers and construction of BAC library, enabled us to identify the gene responsible for mutant phenotype. LjNSP2 encodes a putative transcription factor of the GRAS family that constitutes a subfamily with Medicago truncatula NSP2. LjNSP2 was expressed in roots and early nodules, but strongly suppressed in matured nodules. The expression analysis of NIN and LjENOD40-1 genes in Ljnsp2 mutants indicates that LjNSP2 functions upstream of these genes. These results suggest that LjNSP2 acts as a transcription factor to directly or indirectly switch on the NF-induced genes required for nodule initiation.

Key words: positional cloning; Lotus japonicus; transcription factor; nodule initiation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgements
 REFERENCES
 
The legume–rhizobia symbioses lead to the formation of novel organs, termed nodules, which arise from division of cortical cells in the root and the infection of these nodules by rhizobia. Rhizobia within nodule cells differentiate to bacteroids which fix atmospheric nitrogen. Lipochitin oligosaccharides, Nod-Factors (NFs) secreted by rhizobia are responsible for nodule formation and induce a variety of responses in a host-specific manner, including root hair deformation and cortical cell division, during the early steps of nodulation. Ca2+ spiking in root hair cells is one of the most early responses to NFs.1Go

In the past few years, the phenotypes of an increasing number of symbiotically defective mutants have been analyzed in the model legumes, Lotus japonicus and Medicago truncatula to dissect the NF signaling pathway.2Go–4Go Several genes required for nodulation have been identified by positional cloning. L. japonicus NFR1,5Go NFR56Go and Medicago LYK37Go encode transmembrane receptor-like serine/threonine kinases with putative extracellular regions similar to LysM domains, which are thought to be directly involved in perception of NF signal. In subsequent signal transduction, L. japonicus SYMRK,8Go CASTOR and POLLUX,9Go M. truncatula DMI1,10Go DMI211Go and DMI312Go, and L. japonicus CCaMK13Go and Nup13314Go have been identified as components required for the common symbiosis pathway shared between the fungal and bacterial endosymbiotic systems. The L. japonicus nin mutant showed normal mycorrhization and early responses following rhizobial inoculation, including root hair deformation,15Go Ca2+ influx and Ca2+ spiking.16Go NIN was cloned as a putative transcription factor gene and shown to be expressed during both early and late stages of nodule development, suggesting that NIN controls various developmental aspects of nodulation after the perception of rhizobia in root hairs. However, little is known about what signal component relays the signal from the common symbiotic pathway into the nodulation-specific program in L. japonicus. Recently, GRAS family genes, nodulation signaling pathway 1 (NSP1) and NSP2, have been identified as putative transcription factors functioning downstream of Ca2+ spiking and CCaMK in Medicago truncatula.17Go,18Go

Here, we describe the positional cloning and characterization of nodulation-specific LjNSP2 gene encoding a plant-specific GRAS protein most similar to MtNSP2, which might lead to the induction of expression of genes required for rhizobial infection and early nodule development.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgements
 REFERENCES
 
2.1. Plant materials
The mutant carrying Ljnsp2-1 was isolated from the EMS-mutagenesis experiments of L. japonicus Gifu B-129.3Go The mutant Ljsym35 was isolated from a population of Gifu derived from a transposon-/DNA-tagging trial, as a non-tagged culture mutant,19Go and was kindly provided by Prof. Jens Stougaard (Arrhus University, Denmark).

2.2. Root hair deformation and assays of Ca2+ spiking
Seeds of L. japonicus were germinated and grown on BNM agar medium essentially as described previously,9 except that the roots were grown between two filter papers (grade 0860; Schleicher and Schull, UK), one of which was on the agar surface. Root hair deformation was scored as described previously following 16 h exposure to 10–8 M NF in 1 ml BNM medium in a chamber on a microscope slide. Images were taken with a digital-camera attached to an inverted microscope. Ca2+ spiking was assayed as described previously,9Go following the addition of 10–8 M NF. Representative traces were selected from at least 10 independent cells.

2.3. Genetic mapping population and genomic DNA isolation
Genetic mapping of the LjNSP2 gene was performed with F2/F3 population derived from cross between Ljnsp2-1 (Gifu B-129) and the early flowering ecotype Miyakojima MG-2020Go or Lotus burttii B-303.21Go For AFLP analysis, genomic DNA was extracted from 0.1 g leaves using a Qiagen DNeasy plant kit according to the manufacture's protocol. For PCR markers analysis, DNA was extracted from one young leaf in 100 µl of PEB (200 mM Tris–HCl pH 7.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS), precipitated with isopropanol, washed and dissolved in 100 µl of TE (pH 8.0).

2.4. High efficiency genome scanning (HEGS)/AFLP or PCR-based screening of LjNSP2-inked markers
Genomic DNA (100 ng) was digested with EcoRI and MseI, ligated to EcoRI- and MseI-adaptors and preamplified by using EcoRI- and MseI-adaptor primers. Preamplified DNA was prepared at 0.05 mg/µl concentration before amplification with selective primers. A bulked segregant analysis was performed to identify markers linked to LjNSP2. Bulks were constructed from preamplified DNAs of 10 recessive (Nod–) or dominant (Nod+) homozygous F2 plants, 4096 selective primer combinations of EcoRI+3/MseI+3 were screened to identify markers present only in the bulk of dominant homozygotes.

Subsequently, LjNSP2-linked HEGS/AFLP markers were excised directly from polyacrylamide gels and cloned with the TOPO TA Cloning Kit (Invitrogen). The cloned markers were sequenced and primers were designed by using the software Primer 3 (Whitehead Institute, Cambridge, MA). SCAR (sequence characterized amplified region) markers, which revealed polymorphism between L. japonicus accession Gifu and Miyakojima MG-20 or L. burttii, were analyzed in 2989 or 8472 F2/3 individuals from Miyakojima MG-20 or L. burttii, respectively.

To facilitate the efficiency of electrophoresis of AFLP or PCR products, the HEGS system22Go was adapted. In this system, a set of electrophoresis apparatus is equipped with two sets of 24.5 x 26.5 cm glass plates, each accommodating a gel with 100 lanes and analysis of 400 samples is practicable in single run. After electrophoresis, the gels were stained with Vistra Green (Amersham Biosciences) and scanned by fluorescent gel scanner (FluorImager 595; Amersham Biosciences).

2.5. BAC contig development
Three-dimensional BAC DNA pools prepared from our BAC library23Go were screened with LjNSP2-linked HEGS/AFLP or SCAR markers as described above. HindIII-digested DNA from positive BACs was fractionated on an agarose gel for fingerprinting and determination of overlaps. BAC ends were sequenced using M13 reverse or forward primer and non-repetitive sequences in BAC were used for chromosome walking to screen 3-D BAC pools with PCR or AFLP. The process was repeated as needed to complete the BAC contig. Polymorphic PCR fragments in BAC sequences were analyzed to directly score recombinants in F2/F3 populations.

BAC clone 188C5 containing the LjNSP2 gene was shotgun-sequenced and then annotated by Rice-GAAS (Rice Genome Automated Annotation System) (http://ricegaas.dna.affrc.go.jp/).

2.6. Complementation experiments
For complementation, a 6.9 kb PstI fragment, carrying only the wild-type LjNSP2 1500 bp ORF and 4486 and 899 bp of upstream and downstream sequence, respectively, was cloned into the hairy root transformation vector, which was made from pCAMBIA1300 by replacing the hygromycin-resistant gene with sGFP(S65T).24Go The resulting LjNSP2 recombinant plasmid was introduced into A. rhizogenes LBA133425Go by electroporation. Hairy root transformation of Ljnsp2-1 mutant was performed as described.26Go The plants with transgenic hairy roots were grown in vermiculite pots and inoculated with Mesorhizobium loti TONO. GFP fluorescence and nodule formations were confirmed 4 weeks after inoculation.

2.7. Southern and northern hybridizations
Genomic DNA was extracted from leaves using CTAB method27Go from L. japonicus, and 2.5 µg DNA were digested by EcoRI, electrophoresed on 0.8% agarose gel and blotted to nylon membrane (Biodyne A, Pall).

Twelve-day-old plants were inoculated with M. loti TONO. Infected roots at 4 days post inoculation (dpi) and nodules at 8–32 dpi were immediately frozen in liquid nitrogen. Total RNA was isolated from flower, shoot, root and nodule tissue, and 5 µg aliquots were electrophoresed by denaturing agarose gel and blotted as above.

LjNSP2 probe (1322 bp) was amplified from 188C5 BAC DNA with primers 5'-ACTTCCACCACCTCATCGAC-3' and 5'-ACAAGTCCAAAGGGATGCAG-3', and labeled with 32P using a random primer labeling kit (Takara). Hybridization was done at 63°C in Church buffer [0.5 M sodium phosphate, pH 7.2, 7% (w/v) SDS, 1 mM EDTA]28Go and the filters were washed once in 2x SSC containing 0.5% SDS at room temperature for 10 min, and twice in 0.2x SSC, 0.1% SDS at 63°C for 15 min. The hybridized membranes were then exposed for 3 days to phosphor imaging plates (Fuji, Tokyo, Japan), which were then scanned by a phosphor imaging scanner (Storm840, Amersham Bioscience). After stripping, the same filters were reprobed with the 400 bp fragment from L. japonicus ubiquitin cDNA as a loading control.28Go

2.8. Transient expression of the LjNSP2-GFP fusion protein in onion epidermal cells
The 1.5 kb ORF of LjNSP2 was amplified from 188C5 BAC DNA using the primers: [5'-ACGCGTCGACATGGAAATGGATATAGATTGCATCC-3' (SalI-site underlined), 5'-CATGTCATGAATGCACAATCTGATTCTGAAGAAC-3' (BspHI-site underlined)], digested with SalI and BspHI, and cloned into pUC18-CaMV35S{Omega}-sGFP(s65T)-NOS plasmid29Go at the SalI/NcoI sites just upstream of GFP gene. Onion epidermal cells were bombarded with DNA-coated particles using a Biolistic PDS-1000/He Particle Delivery System (Bio-Rad).9Go About 18–24 h after bombardment, the cells were observed using a Bio-Rad Radiance2000 confocal laser scanning microscope.

The sequence data of LjNSP2 and genomic sequences (B-129 Gifu) of LjNSP2 have been deposited with the DDBJ data library under accession numbers AB241456 [GenBank] and AB241457 [GenBank] , respectively.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgements
 REFERENCES
 
3.1. Early infection phenotypes of a Ljnsp2 mutant
The Ljnsp2-1 mutant of L. japonicus grew normally in nitrogen-rich compost, and established a normal symbiosis with the mycorrhizal fungus,30Go but did not form nodules under nitrogen limitation when inoculated with M. loti. The addition of M. loti to Ljnsp2-1 seedlings induced no phenotypes, such as root hair curling, infection thread formation and cortical cell division, typically seen in the wild-type (data not shown). However, NF did induce swelling and branching in root hair tips, although at a reduced level compared with the wild-type (Fig. 1A and H). Cytological staining of M. loti expressing lacZ revealed no infection foci and no infection thread formation (data not shown). Intracellular Ca2+ spiking, which is induced by NF and has been proposed to be integrated by a Ca2+-calmodulin-dependent protein kinase required for activation of early nodulation gene expression and mycorrhization1Go, was indistinguishable in the mutant from the wild-type (Fig. 1I). The observed induction of Ca2+ spiking and normal mycorrhization of the mutant indicates that the mutation in Ljnsp2 affects a nodulation-specific signaling component that is downstream of the common pathway for mycorrhizal and rhizobial symbioses.


Figure 1
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  		Figure 1. L. japonicus root hair responses induced by NF. (A–D) Root hair deformation induced with 10–8 M NF. Slight swelling and branching were observed also in Ljnsp2-1 mutant (C and D), although to a lesser extent than the wild-type (Gifu B-129; A and B). (E–H) Non-treated root hairs look similar between the mutant (G and H) and wild-type (E and F). Scale bar: 50 µm. (I) Analysis of Ca2+ spiking in root hairs. Each trace is from a single root hair using seedlings of wild-type (Gifu B-129), or the Ljnsp2 mutant. After about 20 min from injection of the Ca2+-sensitive dye Oregon green-dextran to the root hairs, NF was added to the medium at 10–8 M. Both wild-type (upper) and the mutant (lower) responded positively for the addition of NF in 10–20 min and similar effects were observed with at least 10 independent seedlings.

 
3.2. High-resolution genetic mapping of LjNSP2
The mutant allele Ljnsp2-1 (Gifu B-129) was crossed to the early flowering ecotype Miyakojima MG-20. The LjNSP2 locus was mapped near translocation site of the short arm of chromosome 1.31Go Since the recombination is significantly suppressed in the chromosomal segment, total of 4096 EcoRI/MseI primer combinations were examined for bulked segregant analysis on bulks 10 Nod– recessive and Nod+ dominant homozygous F2 plants in the cross of Ljnsp2-1 and Miyakojima MG-20. LjNSP2-linked AFLP markers from this screen were further analyzed in additional 2989 F2 plants with HEGS/AFLP system. Nineteen markers are located on the southern side of LjNSP2 locus at distances of 0.07 cM and seven markers on the northern side of LjNSP2 locus at distances of 0.45 cM, respectively, while nine markers cosegregated with LjNSP2 locus (Table 1, Fig. 2A). The screened markers were converted into the 11 polymorphic SCAR markers (Table 2).


Figure 2
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  		Figure 2. Positional cloning of the LjNSP2 gene (A) Genetic map of LjNSP2 region. This map is based on the analysis of 3 009 F2 plant of a cross between Ljnsp2-1 and Miyakojima MG-20. These HEGS/AFLP makers were selected from 4 096 of AFLP primer combinations with HEGS system. The markers conversed into polymorphic SCAR markers are underlined. (B) About 2 Mb of BAC contig around LjNSP2 genomic region. The numbers below the markers indicates recombination events detected in 16 944 chromosomes from 8 472 siblings of the heterozygotes from crosses of Ljnsp2-1 x L. burttii21. Only overlapping BAC clones spanning LjNSP2 are displayed in this figure. Finally, LjNSP2 region was delimited within a 130 kb region on a 211 kb BAC clone (188C5). (C) Putative genes in LjNSP2 region. ORFs of putative genes (closed arrows) and retro-elements (RE; open arrows) are marked in this 130 kb region. Predicted genes are GTK, glutamine transferase K; P4P5K, phosphatidilinositol-4-phosphate-5-kinase; UP, unknown proteins. There were no introns in the 1 500 bp of the predicted LjNSP2 ORF.

 


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  		Table 1. AFLP markers linked to LjNSP2 locus

 


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  		Table 2. PCR-based markers linked to LjNSP2 locus

 
3.3. Construction of a 2 Mb physical BAC contig spanning LjNSP2
At the first step, all SCAR markers were used to screen 3-D BAC DNA pools prepared from our L. japonicus Gifu BAC library. The contigs containing these SCAR markers were extended by the chromosome walking with the screening of 3-D BAC DNA pool using PCR primer combinations based on end sequences of BAC clones. In several cases, however, we could not obtain non-repetitive PCR fragments from BAC end sequences and screen BAC library. As an alternative strategy, AFLP fragments from BAC with EcoRI+1 selective and MseI+1 selective primers were searched for non-repetitive sequences of BAC inner. Subsequently, the corresponding EcoRI+3 selective and MseI+3 selective primers were used to screen 3-D BAC DNA pools with AFLP system. Finally, we constructed a ~2 Mb physical BAC contig that spans the LjNSP2 locus (Fig. 2B).

3.4. The narrowing of LjNSP2 genomic region using L. burttii as an alternative crossing partner and identification of LjNSP2 gene
As no more recombination was found from the population of this cross of Ljnsp2-1 x Miyakojima MG-20, even with new markers developed from the BAC clones in the contig, we made another cross with L. burttii.21Go Among 8 472 F2 progenies from this cross, six recombination events were found between the flanking markers S26d and 183R derived from the BAC clone 188C5; this located the mutation within a 130 kb region (Fig. 2B). Among the 5 ORFs (Fig. 2C) predicted from the sequence in this region (excluding transposable elements), only one was identified as having a mutation in the mutant. A 6.9 kb fragment including this ORF complemented the mutant for nodulation in hairy roots transformed by the Agrobacterium rhizogenes carrying the cloned region (Fig. 3). No nodules were formed using empty vector, confirming that mutation of this gene caused the mutant phenotype.


Figure 3
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  		Figure 3. Complementation of Ljnsp2-1 mutant. Roots of the Ljnsp2 mutant were transformed by Agrobacterium rhizogenes strain LBA1334 carrying appropriate plasmids carrying the 1 500 bp LjNSP2 ORF and 4 486 and 899 bp of upstream and downstream sequences and a transformation indicator 35S-GFP. On inoculation with Mesorhizobium loti, the GFP-marked transformants formed nodules complementing the mutant phenotype. Scale bar: 1 mm.

 
The ORF in this region corresponding to LjNSP2 encodes a protein belonging to the plant GRAS family of putative transcription factors32Go–34Go and analysis of the major plant GRAS family protein sequences indicated that the closest in sequence to LjNSP2 were the M. truncatula and Pisum sativum NSP2 proteins.18Go AtSCL26 of Arabidopsis was about twice distant from them, and together these proteins made an apparent subfamily of GRAS proteins (Fig. 4). However, genome sequences around the LjNSP2 and M. truncatula NSP2 revealed no clear co-linearity based on the available data.


Figure 4
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  		Figure 4. Phylogenetic tree of plant GRAS family proteins. Members of the family of plant GRAS proteins aligned using Clustal W are shown as a neighbor-joining dendrogram with 1000 bootstrap replicates. At, Arabidopsis thaliana; Mt, Medicago truncatula; Ph, Petunia hybrida; Ps, Pisum sativum; Le, Lycopersicum esculentum; NSP, nodulation signaling pathway; SCR, SCARECROW; SCL, SCARECROW-like; SHR, short-root; HAM, hairy meristem; Ls, lateral suppressor; RGA, repressor of ga1-3; PAT, phytochrome A signal transduction.

 
The LjNSP2 gene consists of a 1500 bp exon with no introns, encoding a predicted 499 amino acids protein of 55 kDa, containing the following GRAS family-specific domains: homopolymeric stretches (HPS) of polyE and polyT; first leucine heptad repeat (LHRI); a VHIID DNA-binding sequence, second leucine heptad repeat (LHRII); and a Src-homology 2 (SH2)-like domain35Go (Fig. 5A). About 15% of the N-terminal region is divergent among most GRAS family members but that of LjNSP2 showed strong homology with the M. truncatula and Pisum sativam NSP2 proteins18Go suggesting functional equivalence and that they are probably orthologs. Furthermore, LjNSP2 has a well-conserved SH2-like domain among GRAS proteins (Fig. 5B). The Ljnsp2-1 mutation causes a substitution of a conserved valine (V) to glutamate (E) in this SH2-like domain (Fig. 5C).


Figure 5
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  		Figure 5. Domain structure of the LjNSP2 protein. (A) Amino acid sequence of 499 residues and predicted functional domains of LjNSP2. HPS; homopolymeric stretches characteristic for GRAS protein near to the N-terminal, LHR (leucine heptad repeat) 1 and 2; putative leucine zipper, VHIID; putative DNA-binding sites, SH2-like region, GY; GY, Y of which is phosphorylated in STATs, is conserved about 100 residues downstream from SH2-like domain. (B) Comparison of GRAS and STAT family proteins with LjNSP2 in their SH2(-like) domains. STAT2: human STAT2; P52630, Ce-STAT: Caenorhabditis elegans STAT; Z70754, D-STAT: Drosophila stat; Q24151, Dd-STAT: Dictyostelium discoideum STAT; Y13097, GAI: Arabidopsis GAI (gibberellin insensitive); At1g14920, SCR: Arabidopsis SCR (SCARECROW); At3g54220. GAI and SCR are the representatives of plant GRAS family proteins. Conserved amino acids are indicated by red boxes. A missense mutation in Ljnsp2-1 is indicated by arrowhead.

 
Complementation tests with the various L. japonicus nodulation mutants carrying mutations mapped to linkage group 1 revealed that Ljsym35 carried a mutation allelic to Ljnsp2. DNA hybridizations detected no signal in Ljsym35 (Fig. 6A) and PCR analyses indicated that it has a deletion of >100 kb around the LjNSP2 gene; this allele was renamed Ljnsp2-2.


Figure 6
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  		Figure 6. DNA, RNA hybridization and RT–PCR analyses of LjNSP2. (A) Genomic hybridization with the full-length LjNSP2 ORF probe to the wild-type (WT; Gifu B-129) and a null deletion mutant Ljnsp2-2. Genomic DNA was digested with EcoRI. The LjNSP2 homolog seems to be unique in WT, but cannot be detected in Ljnsp2-2. (B and C) Northern analysis of LjNSP2 expression in various organs (B), and infected roots at 4 dpi of M. loti TONO and nodules at 8–32 dpi of M. loti TONO, respectively (C). DPI: days post inoculation.

 
3.5. Expression of LjNSP2 during nodulation and in different organs
RNA hybridization showed that LjNSP2 expression was detectable in roots but not in shoots and flowers (Fig. 6B), in contrast to the ubiquitously expressed Medicago NSP2.18Go LjNSP2 was also expressed in infected roots at 4 dpi and early nodules but strongly suppressed in matured nodules (Fig. 6C). These expression patterns of LjNSP2 are similar to that of the putative Nod factor receptor kinase genes, NFR15Go and NFR5,6Go and the signal transduction components CASTOR, although the decrease in expression of the latter was slight.9Go This contrasts with the expression patterns of the genes encoding the nodulation signaling pathway components M. truncatula NSP1,17Go M. truncatula NSP218Go and L. japonicus NIN15Go(Fig. 6A) whose expression increased following inoculation.

3.6. Expression of NIN and LjENOD40-1 genes in Ljnsp2-2 mutant
In the Ljnsp2-2 null mutant, RT–PCR revealed that even before inoculation with M. loti the expression levels of early nodulation genes, NIN and LjENOD40-1, were both <40% of the wild-type (Fig. 7A and B). Upon inoculation of the wild-type with M. loti, NIN and LjENOD40-1 increased by up to 65- or 3.5-fold, respectively. In contrast, the levels of NIN and LjENOD40-1 transcripts in the Ljnsp2-2 mutant remained low after inoculation reaching only about 5–20% of wild-type levels (Fig. 7A and B). This demonstrates that LjNSP2 function is required, directly or indirectly, for either the expression and/or the induction of these early nodulins.


Figure 7
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  		Figure 7. RT–PCR analyses of expression of LjNSP2 and the early nodulins. (A and B) RT–PCR analysis of the change of expression of NIN (A) and LjENOD40-1 (B) after M. loti inoculation, in wild-type and Ljnsp2. Suppression of LjNSP2 expression after inoculation was confirmed, and almost complete loss of induction in NIN and LjENOD40-1 were apparent. Ubiquitin is as a loading control. Relative expression levels were normalized against the amount of ubiquitin (set as 105). Standard deviations of three independent experiments are indicated by error bars. DPI: days post inoculation.

 
3.7. Nuclear localization of LjNSP2 in onion cells
Although most GRAS family proteins have a putative nuclear localization sequence (NLS)34Go, PSORT II analysis did not identify an NLS-like sequence in LjNSP2. However, LjNSP2-GFP fusion delivered into onion (Allium cepa) epidermal cells by particle bombardment revealed it to be exclusively localized in nuclei, although not in nucleoli (Fig. 8A). The mutation in the SH-2-like domain reduced nuclear localization of Ljnsp2-1-GFP and fluorescence in the cytoplasm became noticeable in at least 30 GFP-expressed cells (Fig. 8B), but not as strong as seen with GFP alone (Fig. 8C). These observations are consistent with LjNSP2 acting as a transcription factor, and the SH2-like domain facilitating its nuclear localization.


Figure 8
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  		Figure 8. Nuclear localization of the LjNSP2-GFP. LjNSP2-GFP (A), Ljnsp2-1-GFP (B) fusion genes and control GFP gene (C), delivered by particle bombardment, were transiently expressed in onion epidermal cells and observed with a laser confocal microscope 24 h after bombardment. The nuclear localization of the Ljnsp2-1 mutant gene product is significantly suppressed compared with LjNSP2. Scale: 10 µm.

 
The above LjNSP2-GFP fusion under the control of the CaMV 35S promoter or the LjNSP2 promoter containing a 5.1 kb fragment of the 5' flanking sequence was constructed and introduced into Ljnsp2-2 mutants. These constructs complemented the Ljnsp2-2 mutant phenotype, indicating that this fusion protein retained the LjNSP2 activity for nodulation. However, the transgenic roots showed no detectable GFP fluorescence (data not shown).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgements
 REFERENCES
 
So far the only described nodulation-specific mutants of L. japonicus, which completely lack nodules but have normal mycorrhization, are Ljnsp2, nfr1,5Go nfr56Go and nin.15Go One key difference among these mutants is the induction of Ca2+ spiking, which might be required for the early common response in activation of the symbioses with both rhizobia and mycorrhizal fungi. The Ljnsp2 mutants are normal for Ca2+ spiking but the nfr1 and nfr5 mutants are blocked. Recently, NFR1 and NFR5 were shown to encode LysM-receptor-like-kinases, that were predicted to function in NF perception.5,6 NIN functions downstream of LjNSP2 as indicated in this study. It is possible that LjNSP2 is earliest known protein executing nodulation-specific gene expression from Ca2+ spiking induced through NFR1 and NFR5 in L. japonicus.

We have established a ~2 Mb physical BAC contig that spans the LjNSP2 locus (Fig. 2B). However, LjNSP2 region was only closed to the minimum of 14 BAC clones even in the population of 3 009 F2/F3 plants of the cross with Miyakojima MG-20. This corresponds to 24 Mb/cM, ~78-fold greater than the average physical to genetic distance found in our high-density map of the L. japonicus (Wang et al., manuscript in preparation). This indicates that recombination in the chromosomal segment near the translocation site is highly suppressed in the cross combination of Gifu B-129 x Miyakojima MG-20. Therefore, we changed the crossing partner from Miyakojima MG-20 to L. burttii.21Go As a result, LjNSP2 region narrowed up to 130 kb in one BAC clone (188C5) using the population of 8 472 F2 plants of the cross with L. burttii, indicating that L. burttii is significantly useful as alternative crossing partner of Gifu especially near the translocation site of chromosome 1.

LjNSP2 encodes a GRAS family protein of a putative transcription factor. The phylogenetic analysis revealed that LjNSP2 is most closely related to M. truncatula NSP2 and P. sativum NSP2.18Go The comparison of the mutant phenotypes suggests that the LjNSP2, MtNSP2 and PsNSP2 genes function at similar or parallel positions in the nodulation signal transduction. However, the expression pattern is different between LjNSP2 and M. truncatula NSP2. LjNSP2 is predominantly expressed in roots and its expression decreases in developed nodules. The expression of M. truncatula NSP2 is observed in shoots as well as roots and is induced after rhizobial inoculation. These results suggest that, in contrast with a possible role for M. truncatula NSP2 in another organ development in addition to nodulation, LjNSP2 seems to be specialized in nodule initiation.

The expression of NIN and ENOD40 is induced rapidly after rhizobial inoculation and NF-treatment. The NIN transcripts are detected in different tissues during various nodule stages, such as the dividing cells of the nodule primordia and the nodule vascular bundles. The Ljnin mutants are nodulation-minus and blocked in both the infection thread formation and the cortical cell division.15Go ENOD40 is induced in the root pericycle a few hours after rhizobial inoculation, and subsequently in the dividing cortical cells of the root and nodule primordia.36Go–38Go ENOD40 RNAi knock-down lines39Go or the possible co-suppression40Go suppressed nodule primordium formation, resulting in very poor nodulation. Despite of their function in nodule initiation, however, little is known about what kinds of transcription factors in the root activate NIN and ENOD40 gene expression in response to rhizobia. In this study, we demonstrate that the induction of NIN and LjENOD40-1 gene expression is clearly cancelled by the Ljnsp2 mutation (Fig. 7). On the basis of these findings, we speculate that LjNSP2 may function as a transcriptional activator to directly or indirectly switch on the NIN and LjENOD40-1 gene expression in nodule initiation. In order to address this issue, identification of the promoter region and subsequent binding assay using LjNSP2 protein would be of great importance in future.

Kaló et al.18Go reported that M. truncatula NSP2-GFP localizes in the endoplasmic reticulum and nuclear envelope and re-localizes into the nucleus rapidly after NF-treatment. In this case, they made a functional C-terminal GFP fusion under control of the constitutive CaMV 35S promoter that was introduced in Medicago nsp2 mutant plants. Here, we attempted to detect LjNSP2-GFP, -YFP or -DsRED2 fusion in L. japonicus hairy roots. These fusions could complement the mutant phenotype but no fluorescence was detected even under the control of CaMV35S promoter. In place of L. japonicus we delivered LjNSP2-GFP fusion into onion epidermal cells by particle bombardment. The fusion exclusively localizes in the nucleus but not in the nuclear envelope. Although there is no way to explain the peculiar difference of subcellular localization of NSP2 between Medicago and onion, putative nucleoporins such as NUP13314Go and NUP85 (Saito et al. unpublished data) required for rhizobial and arbuscular mycorrhizal symbioses may retain the NSP2 putative transcription factor in the nuclear envelope of M. truncatula. Most recently, Heckmann et al. reported that non-nodulating mutant, SL781-3, carrying an allele of LjNSP2 was found by TILLING.41Go


   Acknowledgements
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgements
REFERENCES
 
We thank Prof. Jens Stougaard for kind providing the mutant seeds of Ljsym35. We wish to thank Krzyztof Szczyglowski and Martin Parniske for valuable comments on the manuscript and Anne Heckman for helpful discussion. This work was supported by the fund of Promotion of Basic Research Activities for Innovative Biosciences, (BRAIN), a grant in aid from the BBSRC (to J.A.D.), and a John Innes Foundation studentship and an Overseas Student Award (to H.M.). This work was also supported in part by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
*To whom correspondence should be addressed. Tel./Fax: +81-3-5841-4458, E-mail: masayosi{at}biol.s.u-tokyo.ac.jp

{dagger}Present address: Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo, Tokyo 113-8657, Japan Back

Communicated by Satoshi Tabata


   REFERENCES
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgements
 REFERENCES
 

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