Early Nodulin Gene Research Articles

Exploring a rhizobium to fix nitrogen in non-leguminous plants by using a tumor-formation root pathogen

Over 110 million tons of nitrogen fertilizer every year is used for crop production. Scientists have dreamed of enabling rhizobial nitrogen fixation in non-leguminous crops to mitigate the increasing demand for nitrogen fertilizer. However, despite decades of research, rhizobial nitrogen fixation in non-host plants has not been demonstrated. Here, we reported that an N-fixing rhizobium and a clubroot pathogen Plasmodiophora brassicae exhibited a synergistic effect on fixing nitrogen in cruciferous plants. Rhizobia were found to invade P. brassicae-infected rapeseed (Brassica napus) roots in the field. The colonization of rhizobium on rapeseed roots was confirmed by co-inoculating Mesorhizobium huakuii with P. brassicae under controlled laboratory conditions. M. huakuii infection could alleviate clubroot symptoms and promote the growth of diseased rapeseeds. M. huakuii could fix nitrogen in P. brassicae-infected plants based on the results of 15N isotope dilution tests. The expression of homologs of legume genes required for symbiosis and early-nodulin genes was significantly upregulated in Arabidopsis during early infection by P. brassicae. More importantly, M. huakuii could even fix nitrogen in P. brassicae-resistant rapeseed cultivar and promote plant growth when co-inoculated with P. brassicae. Our findings provide a new avenue to understand the interaction of rhizobia with non-host plants, stimulate the exploration of fixing nitrogen in non-leguminous plants by nitrogen-fixing rhizobia, and develop a strategy for both disease control and nitrogen fixation on non-host crops.

SHORT-ROOT paralogs mediate feedforward regulation of D-type cyclin to promote nodule formation in soybean

Nitrogen fixation in soybean takes place in root nodules that arise from de novo cell divisions in the root cortex. Although several early nodulin genes have been identified, the mechanism behind the stimulation of cortical cell division during nodulation has not been fully resolved. Here we provide evidence that two paralogs of soybean SHORT-ROOT (GmSHR) play vital roles in soybean nodulation. Expression of GmSHR4 and GmSHR5 (GmSHR4/5) is induced in cortical cells at the beginning of nodulation, when the first cell divisions occur. The expression level of GmSHR4/5 is positively associated with cortical cell division and nodulation. Knockdown of GmSHR5 inhibits cell division in outer cortical layers during nodulation. Knockdown of both paralogs disrupts the cell division throughout the cortex, resulting in poorly organized nodule primordia with delayed vascular tissue formation. GmSHR4/5 function by enhancing cytokinin signaling and activating early nodulin genes. Interestingly, D-type cyclins act downstream of GmSHR4/5, and GmSHR4/5 form a feedforward loop regulating D-type cyclins. Overexpression of D-type cyclins in soybean roots also enhanced nodulation. Collectively, we conclude that the GmSHR4/5-mediated pathway represents a vital module that triggers cytokinin signaling and activates D-type cyclins during nodulation in soybean.

Amino Acid Polymorphisms in the VHIID Conserved Motif of Nodulation Signaling Pathways 2 Distinctly Modulate Symbiotic Signaling and Nodule Morphogenesis in Medicago truncatula.

Legumes establish an endosymbiotic association with nitrogen-fixing soil bacteria. Following the mutual recognition of the symbiotic partner, the infection process is controlled by the induction of the signaling pathway and subsequent activation of symbiosis-related host genes. One of the protein complexes regulating nitrogen-fixing root nodule symbiosis is formed by GRAS domain regulatory proteins Nodulation Signaling Pathways 1 and 2 (NSP1 and NSP2) that control the expression of several early nodulation genes. Here, we report on a novel point mutant allele (nsp2-6) affecting the function of the NSP2 gene and compared the mutant with the formerly identified nsp2-3 mutant. Both mutants carry a single amino acid substitution in the VHIID motif of the NSP2 protein. We found that the two mutant alleles show dissimilar root hair response to bacterial infection. Although the nsp2-3 mutant developed aberrant infection threads, rhizobia were able to colonize nodule cells in this mutant. The encoded NSP2 proteins of the nsp2-3 and the novel nsp2 mutants interact with NSP1 diversely and, as a consequence, the activation of early nodulin genes and nodule organogenesis are arrested in the new nsp2 allele. The novel mutant with amino acid substitution D244H in NSP2 shows similar defects in symbiotic responses as a formerly identified nsp2-2 mutant carrying a deletion in the NSP2 gene. Additionally, we found that rhizobial strains induce delayed nodule formation on the roots of the ns2-3 weak allele. Our study highlights the importance of a conserved Asp residue in the VHIID motif of NSP2 that is required for the formation of a functional NSP1-NSP2 signaling module. Furthermore, our results imply the involvement of NSP2 during differentiation of symbiotic nodule cells.

Microrna Regulation of Nodule Zone-Specific Gene Expression In Soybean

Microrna Regulation of Nodule Zone-Specific Gene Expression In Soybean

The NMN Module Conducts Nodule Number Orchestra.

Legumes control nodule number through nodulation and autoregulation of nodulation (AON) pathways. Nodule Inception (NIN) is essential for rhizobial infection and nodule organogenesis in legumes. The GmNINa-miR172c-NNC1 (NMN) module, which consists of two positive regulators, GmNINa and miR172c, and a suppressor, NNC1, integrates both pathways. GmNINa activates miR172c to downregulate NNC1, leading to nodulation, while NNC1 inhibits miR172c expression, forming a negative feedback loop. GmNINa and NNC1 interact with each other and antagonistically fine-tune GmRIC1/RIC2 expression, turning AON on and off. Conversely, activation of AON inhibits GmNINa and miR172c expression, thereby reducing their inhibitory effects on NNC1 to attenuate both nodulation signaling and AON. The NMN module functions not only as an “accelerator” of the nodulation signal to promote nodulation but also as a “brake” on the signal by activating AON to orchestrate nodule number.

Genomic dissection of an extended phenotype: Oak galling by a cynipid gall wasp.

Galls are plant tissues whose development is induced by another organism for the inducer's benefit. 30,000 arthropod species induce galls, and in most cases the inducing effectors and target plant systems are unknown. Cynipid gall wasps are a speciose monophyletic radiation that induce structurally complex galls on oaks and other plants. We used a model system comprising the gall wasp Biorhiza pallida and the oak Quercus robur to characterise inducer and host plant gene expression at defined stages through the development of galled and ungalled plant tissues, and tested alternative hypotheses for the origin and type of galling effectors and plant metabolic pathways involved. Oak gene expression patterns diverged markedly during development of galled and normal buds. Young galls showed elevated expression of oak genes similar to legume root nodule Nod factor-induced early nodulin (ENOD) genes and developmental parallels with oak buds. In contrast, mature galls showed substantially different patterns of gene expression to mature leaves. While most oak transcripts could be functionally annotated, many gall wasp transcripts of interest were novel. We found no evidence in the gall wasp for involvement of third-party symbionts in gall induction, for effector delivery using virus-like-particles, or for gallwasp expression of genes coding for plant hormones. Many differentially and highly expressed genes in young larvae encoded secretory peptides, which we hypothesise are effector proteins exported to plant tissues. Specifically, we propose that host arabinogalactan proteins and gall wasp chitinases interact in young galls to generate a somatic embryogenesis-like process in oak tissues surrounding the gall wasp larvae. Gall wasp larvae also expressed genes encoding multiple plant cell wall degrading enzymes (PCWDEs). These have functional orthologues in other gall inducing cynipids but not in figitid parasitoid sister groups, suggesting that they may be evolutionary innovations associated with cynipid gall induction.

Advances in the mechanism of cereal/legume intercropping promotion of symbiotic nitrogen fixation

Biological nitrogen fixation by rhizobia in symbiotic association with legumes is a valuable N source for agroecosystems, and thus it is crucial for increased sustainability of agricultural production. Previous studies found that legume and cereal intercropping can promote symbiotic nitrogen fixation mainly due to rhizosphere nitrogen depletion by the cereal and that soil mineral nitrogen is absorbed more by cereals than legumes, which forces the legume to increase its reliance on symbiotic nitrogen fixation. However, in addition to the nitrogen depletion theory in the rhizosphere, stoichiometry (such as nitrogen and phosphorus coupling) and molecular regulation mechanisms are also important in understanding legume nitrogen fixation. The legume usually maintains a stable stoichiometric relationship between nitrogen and phosphorus. Symbiotic nitrogen fixation of legumes tends to have a high demand for phosphorus, mainly because nitrogen fixation by legumes is a high energy-consuming process, and the required energy is directly provided by ATP. Therefore, when the phosphorus obtained by the intercropped legume is insufficient to meet the increase in symbiotic nitrogen fixation, legume growth is limited by phosphorus. Under phosphorus limitation, the phosphorus uptake strategy of the legume can be stimulated, i.e. by increasing the infection of arbuscular mycorrhizal fungi (AMF) and secretion of root exudates. The AMF mycelia of legumes can extend beyond the rhizosphere, linking the roots to the surrounding soil microhabitats and expanding the area of phosphorus absorbed by the roots. On the other hand, mobilizing rhizosphere insoluble phosphorus through root exudates is also an important pathway for improving phosphorus utilization. Increased soil phosphorus availability promotes plant growth and increased allocation of carbon (C) sources to roots and nodules, resulting in a larger root system or greater nodule formation, finally higher symbiotic nitrogen fixation. In addition, the formation of root nodules is influenced by rhizosphere talk between the cereal and legume, which are directly related to the level of nitrogen. A recent study found that under low N concentration conditions, cereal root exudates stimulated flavonoid biosynthesis and secretion partly through upregulating expression of the Chalcone-flavanone isomerase gene ( CFI ) in legumes. Meanwhile, an auxin-responsive GH3 family gene ( GH3.1 ) and the nodulation gene ENODL2 displayed significant increases, suggesting that nodulation continues under the regulation of auxin signaling and the nodulin-like protein. At nodule maturity, cereal root exudates continue to up-regulate key nodulation genes, such as NODL4 , ENOD93 and the N fixation gene FixI3 , finally promoting symbiotic nitrogen fixation. Moreover, a high concentration of soil mineral N significantly suppressed expression of the early nodulin gene NIN in Lotus corniculatus , which inhibited nodulation and symbiotic nitrogen fixation. In the cereal/legume intercropping system, rhizosphere nitrogen depletion can relieve the inhibition of nitrate on the expression of key nodulation and N fixation genes, thus promoting nodulation and symbiotic nitrogen fixation. In conclusion, intercropping can promote nitrogen uptake and optimize nitrogen use efficiency through the coupling effects of rhizosphere nitrogen depletion, phosphorus mobilization and the regulation of nitrogen fixation genes. This review will help improve our understanding of the mechanism of symbiotic nitrogen fixation in the cereal/legume intercropping system.

Heterologous Expression of Rhizobial CelC2 Cellulase Impairs Symbiotic Signaling and Nodulation in Medicago truncatula.

The infection of legume plants by rhizobia is tightly regulated to ensure accurate bacterial penetration, infection, and development of functionally efficient nitrogen-fixing root nodules. Rhizobial Nod factors (NF) have key roles in the elicitation of nodulation signaling. Infection of white clover roots also involves the tightly regulated specific breakdown of the noncrystalline apex of cell walls in growing root hairs, which is mediated by Rhizobium leguminosarum bv. trifolii cellulase CelC2. Here, we have analyzed the impact of this endoglucanase on symbiotic signaling in the model legume Medicago truncatula. Ensifer meliloti constitutively expressing celC gene exhibited delayed nodulation and elicited aberrant ineffective nodules, hampering plant growth in the absence of nitrogen. Cotreatment of roots with NF and CelC2 altered Ca2+ spiking in root hairs and induction of the early nodulin gene ENOD11. Our data suggest that CelC2 alters early signaling between partners in the rhizobia-legume interaction.

In BPS1 Downregulated Roots, the BYPASS1 Signal Disrupts the Induction of Cortical Cell Divisions in Bean-Rhizobium Symbiosis.

BYPASS1 (BPS1), which is a well-conserved gene in plants, is required for normal root and shoot development. In the absence of BPS1 gene function, Arabidopsis overproduces a mobile signalling compound (the BPS1 signal) in roots, and this transmissible signal arrests shoot growth and causes abnormal root development. In addition to the shoot and root meristem activities, the legumes also possess transient meristematic activity in root cortical cells during Rhizobium symbiosis. We explored the role of Phaseolus vulgaris BPS1 during nodule primordium development using an RNA-interference (RNAi) silencing approach. Our results show that upon Rhizobium infection, the PvBPS1-RNAi transgenic roots failed to induce cortical cell divisions without affecting the rhizobia-induced root hair curling and infection thread formation. The transcript accumulation of early nodulin genes, cell cyclins, and cyclin-dependent kinase genes was affected in RNAi lines. Interestingly, the PvBPS1-RNAi root nodule phenotype was partially rescued by exogenous application of fluridone, a carotenoid biosynthesis inhibitor, which was used because the carotenoids are precursors of BPS1 signalling molecules. Furthermore, we show that the PvBPS1 promoter was active in the nodule primordia. Together, our data show that PvBPS1 plays a vital role in the induction of meristematic activity in root cortical cells and in the establishment of nodule primordia during Phaseolus-Rhizobium symbiosis.

Reactive Oxygen Species and Nitric Oxide Control Early Steps of the Legume - Rhizobium Symbiotic Interaction.

The symbiotic interaction between legumes and nitrogen-fixing rhizobium bacteria leads to the formation of a new organ, the nodule. Early steps of the interaction are characterized by the production of bacterial Nod factors, the reorientation of root-hair tip growth, the formation of an infection thread (IT) in the root hair, and the induction of cell division in inner cortical cells of the root, leading to a nodule primordium formation. Reactive oxygen species (ROS) and nitric oxide (NO) have been detected in early steps of the interaction. ROS/NO are determinant signals to arbitrate the specificity of this mutualistic association and modifications in their content impair the development of the symbiotic association. The decrease of ROS level prevents root hair curling and ITs formation, and that of NO conducts to delayed nodule formation. In root hairs, NADPH oxidases were shown to produce ROS which could be involved in the hair tip growth process. The use of enzyme inhibitors suggests that nitrate reductase and NO synthase-like enzymes are the main route for NO production during the early steps of the interaction. Transcriptomic analyses point to the involvement of ROS and NO in the success of the infection process, the induction of early nodulin gene expression, and the repression of plant defense, thereby favoring the establishment of the symbiosis. The occurrence of an interplay between ROS and NO was further supported by the finding of both S-sulfenylated and S-nitrosylated proteins during early symbiotic interaction, linking ROS/NO production to a redox-based regulation of the symbiotic process.

Overexpression of rice phosphate transporter gene OsPT2 enhances nitrogen fixation and ammonium assimilation in transgenic soybean under phosphorus deficiency

Phosphorus (P) deficiency is one of the major factors that limit legume nodulation and nitrogen (N) fixation, and thus legume productivity. In our previous study, we showed that three T2 transgenic soybean lines overexpressing rice phosphate transporter gene OsPT2 showed enhanced tolerance to low P stress. This study aimed to determine whether OsPT2 overexpression would increase N2 fixation and ammonium assimilation in three T3 homozygous transgenic lines (HTLs) under P deficiency in pot culture. Under low inorganic phosphate (Pi) conditions, the P accumulation, total N and total ureide concentrations were significantly higher in the T3 HTLs than in the wild type (WT) plants. Further, the T3 HTLs showed significantly better plant growth performance and nodule development than the WT plants under low-Pi conditions. Quantitative real-time PCR (qRT-PCR) analysis showed that the expression levels of GmENOD40-1, GmENOD40-2 (two early nodulin genes), and GmLba (one leghemoglobin gene) were significantly increased in T3 HTLs under P deficiency at 24 and 32 d after inoculation (DAI). The increased transcript levels of GmGS1β1 and GmGS1β2 (two cytosolic glutamine synthetase genes) in the T3 HTLs were consistent with the increase in glutamine synthetase (GS, EC 6.3.1.2) activity at 32 DAI. Our results indicated that the overexpression of OsPT2 in T3 HTLs enhances N2 fixation and ammonium assimilation activity under low P stress.

Comparing Gene Expression Profiles Between Bt and non-Bt Rice in Response to Brown Planthopper Infestation.

Bt proteins are the most widely used insecticidal proteins in transgenic crops for improving insect resistance. We previously observed longer nymphal developmental duration and lower fecundity in brown planthopper (BPH) fed on Bt rice line KMD2, although Bt insecticidal protein Cry1Ab could rarely concentrate in this non-target rice pest. In the present study, we performed microarray analysis in an effort to detect Bt-independent variation, which might render Bt rice more defensive and/or less nutritious to BPH. We detected 3834 and 3273 differentially expressed probe-sets in response to BPH infestation in non-Bt parent Xiushui 11 and Bt rice KMD2, respectively, only 439 of which showed significant differences in expression between rice lines. Our analysis revealed a shift from growth to defense responses in response to BPH infestation, which was also detected in many other studies of plants suffering biotic and abiotic stresses. Chlorophyll biosynthesis and basic metabolism pathways were inhibited in response to infestation. IAA and GA levels decreased as a result of the repression of biosynthesis-related genes or the induction of inactivation-related genes. In accordance with these observations, a number of IAA-, GA-, BR-signaling genes were downregulated in response to BPH. Thus, the growth of rice plants under BPH attack was reduced and defense related hormone signaling like JA, SA and ET were activated. In addition, growth-related hormone signaling pathways, such as GA, BR, and auxin signaling pathways, as well as ABA, were also found to be involved in BPH-induced defense. On the other side, 51 probe-sets (represented 50 genes) that most likely contribute to the impact of Bt rice on BPH were identified, including three early nodulin genes, four lipid metabolic genes, 14 stress response genes, three TF genes and genes with other functions. Two transcription factor genes, bHLH and MYB, together with lipid transfer protein genes LTPL65 and early nodulin gene ENOD93, are the most likely candidates for improving herbivore resistance in plants.

The small GTPase ROP10 of Medicago truncatula is required for both tip growth of root hairs and nod factor-induced root hair deformation.

Rhizobia preferentially enter legume root hairs via infection threads, after which root hairs undergo tip swelling, branching, and curling. However, the mechanisms underlying such root hair deformation are poorly understood. Here, we showed that a type II small GTPase, ROP10, of Medicago truncatula is localized at the plasma membrane (PM) of root hair tips to regulate root hair tip growth. Overexpression of ROP10 and a constitutively active mutant (ROP10CA) generated depolarized growth of root hairs, whereas a dominant negative mutant (ROP10DN) inhibited root hair elongation. Inoculated with Sinorhizobium meliloti, the depolarized swollen and ballooning root hairs exhibited extensive root hair deformation and aberrant infection symptoms. Upon treatment with rhizobia-secreted nodulation factors (NFs), ROP10 was transiently upregulated in root hairs, and ROP10 fused to green fluorescent protein was ectopically localized at the PM of NF-induced outgrowths and curls around rhizobia. ROP10 interacted with the kinase domain of the NF receptor NFP in a GTP-dependent manner. Moreover, NF-induced expression of the early nodulin gene ENOD11 was enhanced by the overexpression of ROP10 and ROP10CA. These data suggest that NFs spatiotemporally regulate ROP10 localization and activity at the PM of root hair tips and that interactions between ROP10 and NF receptors are required for root hair deformation and continuous curling during rhizobial infection.

Soybean miR172c targets the repressive AP2 transcription factor NNC1 to activate ENOD40 expression and regulate nodule initiation.

MicroRNAs are noncoding RNAs that act as master regulators to modulate various biological processes by posttranscriptionally repressing their target genes. Repression of their target mRNA(s) can modulate signaling cascades and subsequent cellular events. Recently, a role for miR172 in soybean (Glycine max) nodulation has been described; however, the molecular mechanism through which miR172 acts to regulate nodulation has yet to be explored. Here, we demonstrate that soybean miR172c modulates both rhizobium infection and nodule organogenesis. miR172c was induced in soybean roots inoculated with either compatible Bradyrhizobium japonicum or lipooligosaccharide Nod factor and was highly upregulated during nodule development. Reduced activity and overexpression of miR172c caused dramatic changes in nodule initiation and nodule number. We show that soybean miR172c regulates nodule formation by repressing its target gene, Nodule Number Control1, which encodes a protein that directly targets the promoter of the early nodulin gene, ENOD40. Interestingly, transcriptional levels of miR172c were regulated by both Nod Factor Receptor1α/5α-mediated activation and by autoregulation of nodulation-mediated inhibition. Thus, we established a direct link between miR172c and the Nod factor signaling pathway in addition to adding a new layer to the precise nodulation regulation mechanism of soybean.

Identification of regulatory genes to improve nitrogen use efficiency

Guevara, D. R., Bi, Y.-M. and Rothstein, S. J. 2014. Identification of regulatory genes to improve nitrogen use efficiency. Can. J. Plant Sci. 94: 1009–1012. Crop production on soils containing sub-optimal levels of nitrogen (N) severely compromises yield potential. The development of crop varieties displaying high N use efficiency (NUE) is necessary in order to optimize N fertilizer use, and reduce the environmental damage caused by the current excessive application of N in agricultural areas. Genome-wide microarray analysis of rice plants grown under N-limiting environments was performed to identify NUE candidate genes. An early nodulin gene, OsENOD93-1, was strongly up-regulated during plant growth under low N. A constitutive Ubiquitin promoter was used to drive the expression of the OsENOD93-1 gene in transgenic plants to determine the importance of OsENOD93-1 for rice NUE. Transgenic rice plants over-expressing the OsENOD93-1 gene achieved ∼23% and 16% more yield and biomass, respectively, compared with wild-type plants when grown under N-limitation conditions. OsENOD93-1-OX transgenic plants accumulated a higher amount of total amino acids in the roots and xylem sap under N stress, suggesting that OsENOD93-1 plays a role in the transportation of amino acids. Taken together, we demonstrate that an effective way to identify NUE gene candidates involves both transcriptional profiling coupled with a transgenic validation approach to improve complex traits such as NUE in important crops.

Identification of regulatory genes to improve nitrogen use efficiency

Guevara, D. R., Bi, Y.-M. and Rothstein, S. J. 2014. Identification of regulatory genes to improve nitrogen use efficiency. Can. J. Plant Sci. 94: 1009-1012. Crop production on soils containing sub-optimal levels of nitrogen (N) severely compromises yield potential. The development of crop varieties displaying high N use efficiency (NUE) is necessary in order to optimize N fertilizer use, and reduce the environmental damage caused by the current excessive application of N in agricultural areas. Genome-wide microarray analysis of rice plants grown under N-limiting environments was performed to identify NUE candidate genes. An early nodulin gene, OsENOD93-1, was strongly up-regulated during plant growth under low N. A constitutive Ubiquitin promoter was used to drive the expression of the OsENOD93-1 gene in transgenic plants to determine the importance of OsENOD93-1 for rice NUE. Transgenic rice plants over-expressing the OsENOD93-1 gene achieved ~23% and 16% more yield and biomass, respectively, compared w...

Characterization of genes expressed in Casuarina equisetifolia in response to elicitation by cell wall components of Trichosporium vesiculosum

AbstractCasuarina equisetifolia has the widest distribution of all Casuarina species and is a nitrogen-fixing tree planted in tropical/subtropical littoral zones of Asia, the Pacific and Africa for coastal reclamation, charcoal making, pulp and timber. Trichosporium vesiculosum, the causal agent of blister bark disease, is a serious pathogen of C. equisetifolia. The present study was undertaken to understand the molecular mechanisms involved during pathogen cell wall elicitation in this hardy tree species. Transcript profiling during elicitation induced by cell wall components of T. vesiculosum revealed expression of resistance genes; cytochrome oxidase; trans membrane proteins; genes involved in programmed cell death like 26S proteasome and ubiquitin activating enzyme; early nodulin gene, wound inducible metallocarboxy peptidase inhibitor, glucanase, metal binding protein and signal recognition particle. The fold expression of selected transcripts including glucanase, 26 S proteasome, signal recognition particle, cytochrome oxidase and the metal binding protein using RT-qPCR revealed 12-59 fold increase in expression after 48 hours of elicitor treatment. The expression of these transcripts during abiotic stresses like heat, mechanical wounding, salt (NaCl) and drought (PEG) was also analyzed. Glucanase was up-regulated significantly during wounding and heat stress while proteasome was up-regulated 1-4 fold during NaCl, PEG and wounding stress. The novel transcript CeHMA was up-regulated under all the stress conditions. This is the first report on molecular defense in C. equisetifolia and has provided a pool of candidate genes for detailed molecular dissection to further broaden the knowledge on the response of woody perennials during pathogen cell wall elicitation.

Genetic dissection of Rhizobium-induced infection and nodule organogenesis in pea based on ENOD12A and ENOD5 expression analysis.

In legumes, perception of rhizobial lipochitooligosacharide-based molecules (Nod factors) and subsequent signal transduction triggers transcription of plant symbiosis-specific genes (early nodulins). We present genetic dissection of Nod factor-controlled processes in Pisum sativum using two early nodulin genes PsENOD12a and PsENOD5, that are differentially up-regulated during symbiosis. A novel set of non-nodulating pea mutants in fourteen loci was examined, among which seven loci are not described in Lotus japonicus and Medicago truncatula. Mutants defective in Pssym10, Pssym8, Pssym19, Pssym9 and Pssym7 exhibited no PsENOD12a and PsENOD5 activation in response to Nod factor-producing rhizobia. Thus, a conserved signalling module from the LysM receptor kinase encoded by Pssym10 down to the GRAS transcription factor encoded by Pssym7 is essential for Nod factor-induced gene expression. Of the two investigated genes, PsENOD5 was more strictly regulated; not only requiring the SYM10-SYM7 module, but also SYM35 (NIN transcription factor), SYM14, SYM16 and SYM34. Since Pssym35, Pssym14, Pssym34 and Pssym16 mutants show arrested infection and nodule formation at various stages, PsENOD5 expression seems to be essential for later symbiotic events, when rhizobia enter into plant tissues. Activation of PsENOD12a only requires components involved in early steps of signalling and can be considered as a marker of early symbiotic events preceding infection.

A novel RNA-binding peptide regulates the establishment of theMedicago truncatula-Sinorhizobium melilotinitrogen-fixing symbiosis

Plants use a variety of small peptides for cell to cell communication during growth and development. Leguminous plants are characterized by their ability to develop nitrogen-fixing nodules via an interaction with symbiotic bacteria. During nodule organogenesis, several so-called nodulin genes are induced, including large families that encode small peptides. Using a three-hybrid approach in yeast cells, we identified two new small nodulins, MtSNARP1 and MtSNARP2 (for small nodulin acidic RNA-binding protein), which interact with the RNA of MtENOD40, an early induced nodulin gene showing conserved RNA secondary structures. The SNARPs are acidic peptides showing single-stranded RNA-binding activity in vitro and are encoded by a small gene family in Medicago truncatula. These peptides exhibit two new conserved motifs and a putative signal peptide that redirects a GFP fusion to the endoplasmic reticulum both in protoplasts and during symbiosis, suggesting they are secreted. MtSNARP2 is expressed in the differentiating region of the nodule together with several early nodulin genes. MtSNARP2 RNA interference (RNAi) transgenic roots showed aberrant early senescent nodules where differentiated bacteroids degenerate rapidly. Hence, a functional symbiotic interaction may be regulated by secreted RNA-binding peptides.

Increased nitrogen‐use efficiency in transgenic rice plants over‐expressing a nitrogen‐responsive early nodulin gene identified from rice expression profiling

Development of genetic varieties with improved nitrogen-use efficiency (NUE) is essential for sustainable agriculture. In this study, we developed a growth system for rice wherein N was the growth-limiting factor, and identified N-responsive genes by a whole genome transcriptional profiling approach. Some genes were selected to test their functionality in NUE by a transgenic approach. One such example with positive effects on NUE is an early nodulin gene OsENOD93-1. This OsENOD93-1 gene responded significantly to both N induction and N reduction. Transgenic rice plants over-expressing the OsENOD93-1 gene had increased shoot dry biomass and seed yield. This OsENOD93-1 gene was expressed at high levels in roots of wild-type (WT) plants, and its protein product was localized in mitochondria. Transgenic plants accumulated higher concentrations of total amino acids and total N in roots. A higher concentration of amino acids in xylem sap was detected in transgenic plants, especially under N stress. In situ hybridization revealed that OsENOD93-1 is expressed in vascular bundles, as well as in epidermis and endodermis. This work demonstrates that transcriptional profiling, coupled with a transgenic validation approach, is an effective strategy for gene discovery. The knowledge gained from this study could be applied to other important crops.