Nodulation Signaling in Legumes Requires NSP2, a Member of the GRAS Family of Transcriptional Regulators

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Science  17 Jun 2005:
Vol. 308, Issue 5729, pp. 1786-1789
DOI: 10.1126/science.1110951


Rhizobial bacteria enter a symbiotic interaction with legumes, activating diverse responses in roots through the lipochito oligosaccharide signaling molecule Nod factor. Here, we show that NSP2 from Medicago truncatula encodes a GRAS protein essential for Nod-factor signaling. NSP2 functions downstream of Nod-factor–induced calcium spiking and a calcium/calmodulin-dependent protein kinase. We show that NSP2-GFP expressed from a constitutive promoter is localized to the endoplasmic reticulum/nuclear envelope and relocalizes to the nucleus after Nod-factor elicitation. This work provides evidence that a GRAS protein transduces calcium signals in plants and provides a possible regulator of Nod-factor–inducible gene expression.

The legume/rhizobial symbiosis plays a crucial role in the introduction of fixed nitrogen into both agricultural and natural systems. Legumes form specialized organs, usually on the roots, and these “nodules” provide the low-oxygen environment required for the activity of bacterial nitrogenase. Within nodules, the bacteria reside in membrane-bound compartments within plant cells and differentiate into bacteroids, a specialized symbiotic form of the bacteria. Nod factor is central to the establishment of this symbiotic interaction. This lipochito oligosaccharide signal is produced by the bacteria in response to plant phenolics (1). Nod factor alone is sufficient to activate the majority of the early responses in the plant normally seen during the interaction with the bacteria, including the activation of cytosolic calcium spiking associated with the nucleus of epidermal root cells (2) and a wide range of plant genes (3). The recent identification of a gene (DMI3) encoding a calcium/calmodulin-dependent protein kinase (CCaMK) that functions in Nod-factor signaling (4, 5) downstream of calcium spiking (68) highlights the importance of this calcium response.

Three genes of the model legume Medicago truncatula that are central to Nod-factor signaling, including the CCaMK, are also required for the symbiosis with arbuscular mycorrizal fungi (9). This suggests that aspects of early signaling are conserved between these two symbiotic interactions. The first committed steps for nodulation signaling downstream of the conserved pathway are represented by the nodulation signaling pathway genes NSP1 and NSP2 (9, 10). Mutations in these genes reveal an essential role in Nod-factor signaling, because the majority of Nod-factor responses are absent or compromised in these mutants, whereas Nod-factor–induced calcium spiking is retained (7, 10). This shows that the NSP gene products act downstream of both calcium spiking and the conserved pathway and therefore are likely to transduce the signal downstream of CCaMK. To date, little is known about the mechanisms of signal transduction downstream of plant calcium-activated kinases.

To better understand Nod-factor signaling and the integration of calcium signals in plants, we cloned the NSP2 gene. NSP2 was mapped to a region of chromosome 3, tightly linked to marker DK201 (10). Additional markers within this region from both M. truncatula and M. sativa revealed that the marker LAX3 is 0.08 cM from NSP2. LAX3 is contained on BAC 11A20, one of a small contig of BACs defined by DNA fingerprinting and extended by BAC walking (11) (fig. S1). Genetic markers associated with the ends of two BACs within the contig defined the region containing NSP2 to 132 kb (11). Thirteen genes were predicted to be within this region, including multiple predicted transcription factors and an inositol phosphate kinase, all good candidates for playing a role in Nod-factor signaling.

We isolated the DMI3 gene (CCaMK) on the basis of decreased transcript levels in the dmi3-1 mutant, assayed with an Affymetrix microarray containing 9935 root-expressed M. truncatula genes (5). The DMI3 transcript was destabilized in the dmi3-1 mutant by a 14 base pair (bp) deletion that led to a frameshift, resulting in premature translational termination. A similar comparison of transcript levels between wild-type and nsp2-1 roots identified 17 genes that show a greater than 2-fold reduction in the mutant. Among this list was one gene that is present within the NSP2-defined region. This gene, a member of the GRAS family of transcriptional regulators, showed a 3.84-fold decrease in the mutant, ranking it seventh relative to its fold reduction.

Analysis of the two fast neutron-generated nsp2 mutants showed deletions within this GRAS gene (Fig. 1A), and sequencing revealed the same 435-bp deletion in both alleles (Fig. 1B). This deletion removes a major portion of the conserved GRAS domain but does not induce a frameshift. Therefore, we would not expect this deletion to destabilize the NSP2 transcript as was the case in the DMI3 transcript analysis. A comparison of the individual probes that represent this GRAS gene revealed that most of the Affymetrix oligonucleotide probes were contained within the region deleted in the nsp2-1/2 mutant (Fig. 1B). The two probes that are not contained within the deletion show equivalent hybridization levels in both the wild type and the mutant (Fig. 1C). In contrast, the six probes contained within the deleted region showed substantially reduced expression in the mutant compared with the wild type. This indicates that the apparent reduction in transcript level was due to the lack of hybridization of the nsp2-1 transcript to a subset of the probes rather than destabilization of this transcript.

Fig. 1.

(A) PCR amplification of a region of the NSP2 gene reveals a deletion in nsp2-1 and nsp2-2 but not nsp2-3. Mt A17, wild-type plant; MW, molecular size marker. (B) NSP2 encodes a GRAS protein with a conserved GRAS domain and variable N-terminal region. There are no introns contained in NSP2. The nsp2-3 and pea sym7-1 mutations are contained within the GRAS domain, as is the deletion in nsp2-1 and nsp2-2. The positions of the Affymetrix oligonucleotide probes representing NSP2 from (C) are indicated by arrows. (C) Assessment of the individual probes representing NSP2 on the Affymetrix microarray. The data presented are the average hybridization intensities ± SD from three biological replicates, after normalization in arbitrary units. Three NSP2 oligonucleotide probes that showed no alterations under any treatment were disregarded. The entire set of probes was induced in a hybridization with cRNA from wild-type roots 24 hours after inoculation with S. meliloti (intermittent dashed line), as compared with buffer-treated wild-type roots (solid line). Probes 3 to 8 (underlined) are loaded in the region deleted in the nsp2-1/2 mutant and show greatly reduced hybridization in the buffer-treated nsp2-1 roots (dashed line), whereas probes 1 and 2 that are still present in the nsp2-1/2 mutant show hybridization equivalent to buffer-treated wild type. (D) Complementation of nsp2-2 mutants by the NSP2 gene is revealed by the formation of nodules on the roots of nsp2-2 plants transformed with A. rhizogenes carrying 35S-NSP2. Scale bar, 2 mm.

An ethyl methane sulfonate mutant allele, nsp2-3 (12), revealed a mutation that causes the nonconservative change E232K in the same GRAS gene (Fig. 1B). In contrast to nsp2-1/2, this allele shows a few small white nodules after rhizobial inoculation, which indicates that this mutation results in a weak mutant allele as compared with the presumed null of nsp2-1/2. We verified the identity of NSP2 by complementation of nsp2-2 with the NSP2 cDNA under the regulation of the 35S promoter. This construct was introduced into the roots of nsp2-2 mutant plants with Agrobacterium rhizogenes. This procedure results in the formation of a chimeric plant with transformed roots attached to an untransformed shoot. The transformed roots produced nodules (Fig. 1D), and no nodules were seen on roots of nsp2 mutants transformed with A. rhizogenes alone or with CCaMK in the equivalent binary vector. This complementation validates the genetic identity of NSP2.

Pea sym7-1 has a mutant phenotype similar to nsp2-1/2, and SYM7 resides in an approximately syntenic position in the pea genome (13). Therefore, SYM7 is a possible ortholog of NSP2. We isolated the homologous cDNA from pea using primers generated against the M. truncatula gene. The protein product shows 89.5% identity to the M. truncatula protein (Fig. 2). Analysis of the sym7-1 sequence revealed a translation stop at the position equivalent to R239 in M. truncatula NSP2 (Fig. 1B). Thus, null mutations of NSP2 show consistent phenotypes across two related species of legumes.

Fig. 2.

Alignment of NSP2 with pea SYM7, At4g08250 (the closest Arabidopsis homolog), and NSP1. NSP2 and SYM7 show high similarity across the whole protein, including the variable N-terminal region, whereas the Arabidopsis protein shows homology only in the GRAS domain, indicating that although SYM7 is a true ortholog, the Arabidopsis protein is not. The GRAS domain contains two leucine-rich regions (LHRI and LHRII) and three separate conserved motifs: VHIID, PFYRE, and SAW. The conserved motifs (double underlined) are within regions (underlined) that contain additional conserved residues (not indicated).

Analysis of NSP2 expression levels on the Affymetrix microarrays revealed a 1.95 ± 0.05–fold induction of this gene 24 hours after treatment with the M. truncatula symbiont Sinorhizobium meliloti strain 1021 and a similar induction following Nod-factor treatment (Fig. 3A). This induction did not occur with SL44, a mutant strain of S. meliloti unable to generate Nod factor, or in mutants of plant genes known to be required for Nod-factor signal transduction (Fig. 3A). To verify these microarray data, we analyzed expression of NSP2 by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and found a similar induction by both Nod factor and S. meliloti (Fig. 3B). Furthermore, quantitative real-time PCR analysis revealed a 1.6 ± 0.19–fold induction of NSP2 4 days after S. meliloti inoculation and a 3.2 ± 0.55–fold induction 7 days after inoculation. The RT-PCR data also revealed expression of NSP2 in shoots and leaves (Fig. 3B). This shoot expression is surprising because the mutant phenotypes are restricted to the root, and complementation with A. rhizogenes indicates that root expression of NSP2 is sufficient for nodulation. It is possible that NSP2 has additional functions in shoots, but these must be either limited or redundant.

Fig. 3.

(A) Expression of NSP2 assayed on the Affymetrix microarray in roots from wild-type and Nod-factor signaling mutants. Red indicates higher levels than the median signal; blue indicates lower levels than the median, assessed as an average from three biological replicates. WT, wild type; NF, Nod factor; Sm, S. meliloti; SL44, S. meliloti mutant defective in Nod-factor production; exoA, S. meliloti mutant defective in exopolysaccharide production; dmi and nsp, plant mutants defective in Nod-factor signaling. (B) NSP2 expression assayed by RT-PCR. NSP2 is expressed in shoots, leaves, and roots and induced in roots treated with either S. meliloti or Nod factor. ENOD11 is a gene induced during nodulation that acts as a positive control for S. meliloti inoculation. Overexposure of the blot reveals low levels of ENOD11 after Nod-factor treatment. 1D to 14D, days after inoculation with S. meliloti; 2H to 24H, hours after treatment with Nod factor.

NSP2 encodes a gene with similarity to members of the GRAS family of putative transcriptional regulators. This gene family is found throughout the plant kingdom (Arabidopsis contains 33 members) and contains genes involved in gibberellin and phytochrome signaling, root development, axillary shoot development, and maintenance of the shoot apical meristem (14). NSP2 contains a conserved GRAS domain and a variable N-terminal region, as observed in other family members. It shows closest similarity to At4 g08250 (Fig. 2), an Arabidopsis GRAS protein of unknown function. The GRAS domain consists of two leucine-rich regions that may indicate protein-protein interactions and some small invariantly conserved motifs whose functions are unknown (Fig. 2). Most GRAS domain proteins, including NSP2, show homopolymeric stretches of amino acids in the N-terminal region, and these are also found in the activation domain of transcription factors. This, coupled with the leucine-rich regions and the fact that many of these proteins show nuclear localization (14), suggests a possible role in transcriptional regulation. Smit et al. (15) reveal that NSP1 also encodes a GRAS family member. However, the homology between NSP1 and NSP2 is mostly limited to the residues that are conserved throughout this family of proteins (Fig. 2). This is in contrast to the DELLA proteins, a group of closely related GRAS proteins that function in gibberellin signaling (13). It is likely that NSP1 and NSP2 fulfill similar, but nonredundant, functions.

We analyzed the localization of NSP2 with a C-terminal GFP fusion driven by the 35S promoter; this complements nsp2-2, indicating that this fusion protein is active. NSP2-GFP showed strong localization to the nuclear envelope and weaker localization to the ER (Fig. 4A), despite the fact that NSP2 does not contain a signal peptide or ER retention signal. After application of Nod factor, we saw a shift in NSP2-GFP with a specific loss in the nuclear envelope, but little change in the ER localization, and the gain of diffuse staining throughout the nucleus (Fig. 4B). Overexpression of proteins can cause spurious localization. However, we do not believe that the Nod-factor–induced relocalization is likely to be caused by overexpression. We used the nuclear envelope localization as a marker to assess the frequency of this relocalization event. We found that 85% of cells lacked the nuclear envelope localization 24 hours after 10–8 M Nod-factor treatment (n = 87, on three roots), as compared with only 3.7% of cells that lacked the nuclear envelope localization before Nod-factor treatment (n = 106, on three roots). However, this relocalization occurs much earlier, as 39% of cells lacked the nuclear envelope localization 4 hours after 10–9 M Nod-factor treatment (n = 111, on three roots). At this stage, we cannot differentiate between the retargeting of newly synthesized NSP2 after Nod-factor application and a shift of nuclear envelope–localized NSP2 to the nucleus. If NSP2 functions as a transcriptional regulator, which is proposed for GRAS family proteins, then its relocalization to the nucleus may be central to the regulation of its activity.

Fig. 4.

NSP2 and CCaMK (DMI3) localization. Confocal sections through epidermal root cells expressing the NSP2-GFP and CCaMK-GFP fusions. (A) NSP2-GFP shows strong localization to the nuclear envelope and weaker localization in the endoplasmic reticulum, seen as a filamentous network emanating from the nuclear envelope. (B) Upon Nod-factor application, the nuclear envelope localization disappears and is replaced by diffuse fluorescence in the nucleus. (C) CCaMK shows strong nuclear localization that is unaffected by Nod-factor treatment. Scale bars in (A) and (B), 10 μM; scale bar in (C), 40 μM.

Because NSP2 appears to function downstream of CCaMK (DMI3), we analyzed the localization of CCaMK with both C- and N-terminal GFP fusions. These constructs complement the dmi3 mutant, indicating appropriate localization. Both GFP CCaMK fusions show strong nuclear localization (Fig. 4C), and a similar localization was observed when this fusion was driven by the DMI3 promoter (15). There was no change observed in the localization of CCaMK after Nod-factor treatment.

The phenotype of nsp2 mutants indicates that NSP2 is essential for Nod-factor–induced gene expression and that NSP2 acts downstream of both calcium spiking (10) and CCaMK. A distinct possibility is the direct phosphorylation of NSP2 by CCaMK after the relocalization of NSP2 to the nucleus, but such an interaction between CCaMK and NSP2 still needs to be assessed. CCaMK is a common feature of both nodulation and mycorrhizal signaling, and specificity of these signaling pathways is most likely a function of differential regulation of this protein. NSP1 and NSP2 are the earliest known nodulation-specific proteins downstream of CCaMK, and their activation may be central to the maintenance of specificity in the Nod-factor signaling pathway. The mechanisms of transduction immediately downstream of CCaMK in both the nodulation and mycorrhizal pathways are likely to be similar, and there may be analogous mycorrhizal-specific GRAS proteins. The isolation of NSP1 and NSP2 provides the first insights into the nodulation-specific components of this signaling pathway downstream of CCaMK, and deciphering their mechanism of action will provide insights into calcium signaling in plants and the mechanisms of specificity in this multifunctional signaling pathway.

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Materials and Methods

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