NSP1 of the GRAS Protein Family Is Essential for Rhizobial Nod Factor-Induced Transcription

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Science  17 Jun 2005:
Vol. 308, Issue 5729, pp. 1789-1791
DOI: 10.1126/science.1111025


Rhizobial Nod factors induce in their legume hosts the expression of many genes and set in motion developmental processes leading to root nodule formation. Here we report the identification of the Medicago GRAS-type protein Nodulation signaling pathway 1 (NSP1), which is essential for all known Nod factor–induced changes in gene expression. NSP1 is constitutively expressed, and so it acts as a primary transcriptional regulator mediating all known Nod factor–induced transcriptional responses, and therefore, we named it a Nod factor response factor.

Nod factors are lipo-chitooligosaccharide–based signal molecules secreted by rhizobia that set in motion Rhizobium–legume root nodule symbiosis (1). Nod factors are essential for rhizobial infection and induction of cortical cell divisions, leading to nodule primordium formation. These processes are preceded by induction of at least 40 genes (2). Nod factors are most likely perceived by LysM domain–containing receptor kinases identified both in Lotus japonicus (lotus) (NFR1, NFR5) and Medicago truncatula (medicago) (LYK3, LYK4) (35). Subsequent Nod factor signal transduction requires another receptor kinase (DMI2 in medicago, SymRK in lotus) and putative cation channel(s) (DMI1 in medicago, Pollux and Castor in lotus) (69). In medicago, these proteins are essential to induce Ca2+ spiking in the perinuclear region of root hairs within a few minutes after Nod factor application (10). These calcium spikes probably activate a calcium- and calmodulin-dependent protein kinase (DMI3 in medicago) that is also necessary for Nod factor signaling (2, 11, 12).

We fused the gene for green fluorescent protein (GFP) with DMI3 to make GFP-DMI3, which is driven by the DMI3 promoter (DMI3::GFP-DMI3) (13). This construct complements the dmi3 mutant TRV25, which shows that the GFP-DMI3 fusion is biologically active (fig. S1). GFP-tagged DMI3 is located in the nucleus of epidermal root cells in both uninoculated roots and roots inoculated with Sinorhizobium meliloti Sm2011 [expressing monomeric red fluorescent protein (mRFP)] (13, 14) (Fig. 1A). This suggests that activation of DMI3 and subsequent downstream Nod factor signal transduction occur in the nucleus. Putative targets of DMI3 are, therefore, primary transcription factors mediating Nod factor–induced gene expression. These transcription factors should be constitutively present in an inactive form, should be activated by Nod factor signaling, and, collectively, should be essential for all Nod factor–induced changes in gene expression. Such primary transcription factors have been identified, for example, in auxin signaling, and are called auxin response factors (ARFs) (15). Because Nod factor–activated primary transcription factors are analogous to ARFs, we named them Nod factor response factors (NRFs).

Fig. 1.

Subcellular localization of DMI3 and NSP1 by using confocal microscopy. (A) TRV25 (dmi3) mutant transformed with DMI3::GFP-DMI3. Left, GFP-DMI3 localizes in the nucleus of a root hair cell. Right, Confocal sections of both GFP-DMI3 and DsRED (used as selection marker) signals are merged with a bright-field image. (B) C54 (nsp1-2) mutant transformed with NSP1::GFP-NSP1. Left, Localization of GFP-NSP1 in the nucleus of a root hair cell. Right, Confocal sections of both GFP-DMI3 and DsRED (selection marker) signals are merged with a bright-field image. (C) C54 (nsp1-2) mutant transformed with NSP1::GFP-NSP1 2 days after inoculation with S. meliolti Sm2011. Expression of GFP-NSP1 can be detected in all epidermal and cortical root cells. Green signal in cell walls of epidermal cells and cytoplasm of root cap cells is due to autofluorescence in the green channel. The nucleolus is devoid of GFP signal, which indicates that GFP-NSP1 and GFP-DMI3 are present in the nucleoplasm.

In addition to the aforementioned Nod factor–signaling genes, medicago NODULATION SIGNALING PATHWAY 1 (NSP1) and NSP2 are the only loci identified that are also essential for all known Nod factor–induced changes in gene expression (2, 16, 17). Genetic analyses showed that NSP1 and NSP2 gene products act directly downstream of DMI3 (10, 16, 17). Therefore, we postulate that at least one of these genes encodes an NRF. This report and the accompanying report of Kaló et al. describe the positional cloning of NSP1 and NSP2, respectively, and show that both are excellent candidates for such NRFs (14).

We located NSP1 on the short arm of chromosome 8 between markers 32C19F and 76L14R on the bacterial artificial chromosome (BAC) clone MtH2-37J10 (13). This region encompasses 50 kb and contains six putative genes (fig. S2). One of these encodes a putative transcription factor having all the motifs characteristic of proteins belonging to the GRAS transcription factor family (18). This putative transcription factor was mutated in all available nsp1 mutants (B85, C54, C103, and C108) (16). The B85 mutant allele (nsp1-1) contains a premature stop codon and encodes a truncated protein of 239, instead of 554, amino acids. C54, C103, and C108 mutant alleles all have the same mutation, and therefore, we assume that they are siblings. This allele (nsp1-2) encodes a truncated protein of 487 amino acids, and it lacks the C-terminal SAW motif present in all GRAS proteins, which indicates that the SAW motif is essential for NSP1 functioning (Fig. 2). Introduction of the wild-type allele in B85 and C54, using Agrobacterium rhizogenes–mediated root transformation, resulted in infected root nodules on inoculation with S. meliloti Sm2011 (expressing GFP) (13) (fig. S3). This shows that NSP1 encodes a GRAS-type protein essential for all known Nod factor–induced changes in gene expression.

Fig. 2.

Alignment of medicago NSP1 and NSP2, poplar PtHNO1 and PtHMO2, Arabidopsis SCL29, and rice OsHNO. Five conserved motifs specific for GRAS proteins are annotated (leucine heptad I, VHIID, leucine heptad II, PFYRE, and SAW motifs). Conserved amino acids in NSP1-type proteins are highlighted in shades of black and gray. Putative nuclear localization signals present in NSP1, PtHNO1, and OsHNO are underlined (22). Arrows indicate the premature translational stop in nsp1-1 (B85) and nsp1-2 (C54). Alignment was made using CLUSTALW (23). In order of homology to NSP1: PtHNO1 (66% identity, 77% similarity); PtHNO2 (63% identity, 74% similarity); AtSCL29 (48% identity, 63% similarity); OsHNO (36% identity, 50% similarity); and NSP2 (17% identity, 32% similarity).

GRAS-type transcription factors are in general involved in plant developmental processes (19). The involvement of NSP1 in Nod factor–induced nodule development is in line with such function. Also, NSP2 encodes a putative GRAS-type transcription factor (14), although it is not similar to NSP1 (17% identity, 32% similarity) (Fig. 2). In contrast, NSP1 is highly homologous to two putative proteins of Populus trichocarpa (poplar) (here named HOMOLOG OF NSP1; PtHNO1 and PtHNO2), SCARECROW-LIKE 29 (SCL29) of Arabidopsis, and a putative protein of rice (OsHNO) (Fig. 2; fig. S4). The occurrence of putative orthologs in nonlegume plant species and the presence of only a single NSP1-type gene in medicago suggest that NSP1-type proteins have a nonsymbiotic function and that, during evolution, NSP1 has obtained an additional function in Nod factor signaling in legume species (fig. S5F).

NSP1 is preferentially expressed in roots, and its expression does not markedly change on Rhizobium inoculation (Fig. 3; figs. S5 and S6). Because NSP1 and NSP2 (14) are expressed before Nod factor signaling, and both are essential for all known Nod factor–induced changes in gene expression, they are NRFs and probably act in a cooperative manner.

Fig. 3.

Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) analysis of NSP1 expression. NSP1 is constitutively expressed in roots and not up-regulated on inoculation with S. meliloti (2 days). Expression of all five medicago ACTIN genes was used for normalization (see also figs. S5 and S6).

Besides NSP1 and NSP2, another putative transcription factor essential for root nodule formation has been identified in lotus and pea, namely, NIN (20, 21). Whether NIN is required for Nod factor–induced gene expression is not known; however, nin mutants are blocked at a later stage of the interaction, as excessive root hair curling does occur (16, 17, 20, 21). Therefore, it is unlikely that NIN is a primary transcription factor, which is also in line with its markedly increased expression when inoculated with rhizobia (3, 20).

To determine whether NSP1 could be activated directly by DMI3, we determined its subcellular localization. We used a GFP-NSP1 fusion driven by the 4-kb NSP1 upstream region (NSP1::GFP-NSP1) (13). This construct complements both nsp1 mutants (fig. S7). In roots transformed with this fusion construct, GFP-NSP1 is located in the nucleus of all epidermal and cortical root cells (Fig. 1, B and C). The nuclear localization of GFP-NSP1 is not altered after inoculation with S. meliloti Sm2011. Because DMI3 and NSP1 are both located in the nucleus, NSP1 could be directly activated by DMI3, but such interaction remains to be demonstrated. The subcellular localization of NSP1 differs from that of NSP2. In transgenic roots expressing 35S::NSP2-GFP, the fusion protein is predominantly located in the nuclear envelope and the endoplasmic reticulum before Nod factor signaling (14). When Nod factor was added, NSP2-GFP accumulated in the nucleus (14). Because DMI3 is already localized in the nucleus in uninoculated plants, it is unlikely that the nuclear accumulation of NSP2 is controlled directly by DMI3. The colocalization of NSP1 and DMI3, together with the genetic analysis that shows that DMI3 acts directly upstream of NSP1, strongly supports that NSP1 is a target of DMI3.

Supporting Online Material

Materials and Methods

Figs. S1 to S7

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