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Rhizobium meliloti NOD factors elicit cell-specific transcription of the ENOD12 gene in transgenic alfalfa
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Extracellular lipo-oligosaccharides of Rhizobium, known as Nod factors, play a key role in the molecular signal exchange which leads to the specific nitrogen-fixing symbiotic association between the soil microbe and its host legume. The biological activity of Nod factors and their perception by the host plant during the earliest stages of the Rhizobium/legume interaction have been studied using transgenic alfalfa carrying a fusion between the promoter of the early nodulin gene MtENOD12 and the beta-glucuronidase (GUS) reporter gene. Histochemical staining has shown that GUS accumulates specifically in the differentiating root epidermis, prior to and during root hair emergence, within 2-3 h following the addition of purified Rhizobium meliloti Nod factors. This precocious transcriptional activation of the MtENOD12 gene, reminiscent of that observed after inoculation with intact Rhizobium, implies that the Nod factor signal can be perceived at a developmental stage preceding root hair formation. GUS activity can be detected following treatment with a wide range of R. meliloti Nod factor concentrations down to 10(-13) M, and furthermore, this rapid response to the bacterial elicitor appears to be non-systemic. Significantly, MtENOD12-GUS expression is not observed after inoculation with a R. meliloti nodH mutant which synthesizes exclusively non-sulphated Nod factors. Indeed purified Nod factors which lack the sulphate substituent are approximately 1000-fold less active than their sulphated counterparts. Thus, the triggering of ENOD12 transcription in the alfalfa root epidermis is a rapid molecular response which is subject to the same host-specificity determinant (Nod factor sulphation) that governs the interaction between alfalfa and its bacterial symbiont.
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... It should be noted that our work used a non-O-acetylated S. meliloti NF as a ligand, whereas at least part of the NFs from this species is O-acetylated on the terminal non-reducing sugar (specified by the NodL gene; Ardourel et al. 1994). This decoration is not essential for MtNFP-dependent signaling as a S. meliloti nodL mutant is able to nodulate Mt, although with a slightly lower efficiency than the WT (Smit et al. 2007), and nodL NFs are active on Medicago roots down to pM concentrations in biological assays (Ardourel et al. 1994, Journet et al. 1994, Wais et al. 2002. Even if such NFs have a 10-fold lower affinity for MtNFP compared to O-acetylated NFs, as suggested by Gysel et al. (2021), we should still be able to detect affinities in the 100-nM range, as shown in previous studies on other proteins (Fliegmann et al. 2013, Girardin et al. 2019. ...
Lysin motif receptor like kinases (LysM-RLKs) are involved in the perception of chitooligosaccharides (COs) and related lipochitooligosaccharides (LCOs) in plants. Expansion and divergence of the gene family during evolution have led to various roles in symbiosis and defence. By studying proteins of the LYR-IA subclass of LysM-RLKs of the Poaceae, we show here that they are high affinity LCO binding proteins with a lower affinity for COs, consistent with a role in LCO perception to establish arbuscular mycorrhiza (AM). In Papilionoid legumes whole genome duplication has resulted in two LYR-IA paralogs, MtLYR1 and MtNFP in Medicago truncatula, with MtNFP playing an essential role in the root nodule symbiosis with nitrogen-fixing rhizobia. We show that MtLYR1 has retained the ancestral LCO binding characteristic and is dispensable for AM. Domain swapping between the three Lysin motifs (LysMs) of MtNFP and MtLYR1 and mutagenesis in MtLYR1 suggest that the MtLYR1 LCO binding site is on the second LysM, and that divergence in MtNFP led to better nodulation, but surprisingly with decreased LCO binding. These results suggest that divergence of the LCO binding site has been important for the evolution of a role of MtNFP in nodulation with rhizobia.
... NodH catalyzes the transfer of sulfate from 3 -phosphoadenosine 5 -phosphosulfate to the terminal 6-O [28]. In alfalfa, the expression of nodulin gene MtENOD12 was significantly inhibited after inoculation with a nodH mutant compared to the wild-type Rhizobium meliloti, suggesting the sulfated Nod factors are required for nodulation by R. meliloti [29]. However, alfalfa nodulation by Sinorhizobium fredii does not required sulfated Nod-factors [30]. ...
SnRK1 protein kinase plays hub roles in plant carbon and nitrogen metabolism. However, the function of SnRK1 in legume nodulation and symbiotic nitrogen fixation is still elusive. In this study, we identified GmNodH, a putative sulfotransferase, as an interacting protein of GmSnRK1 by yeast two-hybrid screen. The qRT-PCR assays indicate that GmNodH gene is highly expressed in soybean roots and could be induced by rhizobial infection and nitrate stress. Fluorescence microscopic analyses showed that GmNodH was colocalized with GsSnRK1 on plasma membrane. The physical interaction between GmNodH and GmSnRK1 was further verified by using split-luciferase complementary assay and pull-down approaches. In vitro phosphorylation assay showed that GmSnRK1 could phosphorylate GmNodH at Ser193. To dissect the function and genetic relationship of GmSnRK1 and GmNodH in soybean, we co-expressed the wild-type and mutated GmSnRK1 and GmNodH genes in soybean hairy roots and found that co-expression of GmSnRK1/GmNodH genes significantly promoted soybean nodulation rates and the expression levels of nodulation-related GmNF5α and GmNSP1 genes. Taken together, this study provides the first biological evidence that GmSnRK1 may interact with and phosphorylate GmNodH to synergistically regulate soybean nodulation.
... viciae (267). Another indication comes from the observation that the transcription of some early nodulins, which are specifically expressed in the infection thread, is also induced with isolated Nod metabolites (117,128,177,215). ...
Rhizobium, Bradyrhizobium, and Azorhizobium species are able to elicit the formation of unique structures, called nodules, on the roots or stems of the leguminous host. In these nodules, the rhizobia convert atmospheric N2 into ammonia for the plant. To establish this symbiosis, signals are produced early in the interaction between plant and rhizobia and they elicit discrete responses by the two symbiotic partners. First, transcription of the bacterial nodulation (nod) genes is under control of the NodD regulatory protein, which is activated by specific plant signals, flavonoids, present in the root exudates. In return, the nod-encoded enzymes are involved in the synthesis and excretion of specific lipooligosaccharides, which are able to trigger on the host plant the organogenic program leading to the formation of nodules. An overview of the organization, regulation, and function of the nod genes and their participation in the determination of the host specificity is presented.
... Ces oscillations de calcium se traduisent par une succession d'élévations de la concentration en calcium suivies d'un retour au niveau initial, répétées avec une fréquence déterminée, et ont été décrites chez plusieurs espèces de légumineuses symbiotiques (Wais et al., 2002;Miwa et al., 2006). Il est proposé qu'elles soient un marqueur caractéristique de la transduction du signal symbiotique (Oldroyd, 2013) essentiel pour l'induction de l'expression de gènes associés aux phases initiales de la nodulation, tels que les nodulines précoces, ou « Early Nodulins », ENOD11 et ENOD12 (Journet et al., 1994(Journet et al., , 2001Charron et al., 2004). Etant donné que des mutants ne produisant pas d'oscillations de calcium peuvent conserver la capacité à induire l'influx initial de calcium dans les poils absorbants, un découplage de ces deux réponses calciques a été mis en évidence, permettant de positionner d'autres acteurs moléculaires dans la voie de signalisation comme ceux qui sont décrits dans la section suivante. ...
Les légumineuses adaptent l’architecture de leur système racinaire aux conditions environnementales en modifiant la croissance et le nombre de leurs racines latérales mais aussi en développant des nodosités fixatrices d’azote en condition de carence azotée et en présence de bactéries symbiotiques (rhizobia). Des voies de régulation systémique impliquant des peptides de signalisation perçus par des récepteurs kinases permettent la coordination du développement de ces organes racinaires en fonction de la disponibilité en azote dans le sol et des besoins de la plante. Les objectifs de cette thèse était d’une part d’identifier quel signal peptidique agit via l’un de ces récepteurs, appelé CRA2, pour induire la nodulation et réprimer la formation des racines latérales ; et d’autre part de déterminer comment cette voie s’intègre avec d’autres voies de signalisation régulant l’architecture racinaire, ie (i) la voie systémique impliquant des peptides de signalisation CLE et le récepteur SUNN régulant négativement la nodulation et (ii) la voie des phytohormones cytokinines régulant l’architecture racinaire de manière similaire à CRA2 .Nous avons montré que le récepteur CRA2 est requis pour l’action du peptide de signalisation MtCEP1, et que les deux voies systémiques régulant de manière antagoniste la nodulation agissaient de manière indépendante Enfin, nous avons montré que le gène MtCEP7, induit par rhizobium et les cytokinines, favorise les infections rhizobiennes, et sa production est coordonnée avec celle d’un peptide CLE régulant négativement la nodulation, via les cytokinines et le facteur de transcription NIN.
Autoregulation of nodulation (AON) plays a central role in nodulation by inhibiting the formation of excess number of legume root nodules. In this study, the effect of hydroxymethylglutaryl-coenzyme A reductase 1 (GmHMGR1) gene expression on nodulation and the AON system in Glycine max (L.) Merr was investigated. Wild-type soybean (cultivar Bragg) and its near-isogenic supernodulating mutant (nitrate tolerant symbiotic) nts1007 were selected to identify the expression pattern of this gene in rootlets after inoculation by its microsymbiont Bradyrhizobium. For further analysis, the full length of GmHMGR1 and its promoter were cloned after amplification by inverse-PCR and BAC library screening. Also, we constructed an intron hairpin RNA interference (ihpRNAi) and a GmHMGR1 promoter: β-glucuronidase fusion constructs, consequently for suppression of GmHMGR1 and histochemical analysis in transgenic soybean hairy roots induced by Agrobacterium rhizogenes strain K599. The GmHMGR1 gene was functional during the early stages of nodulation with the AON system having a negative effect on GmHMGR1 expression and nodule formation in wild-type rootlets. GmHMGR1 was particularly expressed in the developing phloem within the root, nodules and nodule lenticels. Expression of GmHMGR1 in transgenic hairy roots was suppressed by RNAi silencing approximately 85% as compared to empty vector controls. This suggests that the GmHMGR1 gene has an important role in triggering nodule formation as its suppression caused a reduction of nodule formation in nts mutant lines with a deficient AON system.
Salt stress is a major agricultural concern inhibiting not only plant growth but also the symbiotic association between legume roots and the soil bacteria rhizobia. This symbiotic association is initiated by a molecular dialogue between the two partners, leading to the activation of a signaling cascade in the legume host and, ultimately, the formation of nitrogen-fixing root nodules. Here, we show that a moderate salt stress increases the responsiveness of early symbiotic genes in Medicago truncatula to its symbiotic partner, Sinorhizobium meliloti while, conversely, inoculation with S. meliloti counteracts salt-regulated gene expression, restoring one-third to control levels. Our analysis of early nodulin 11 (ENOD11) shows that salt-induced expression is dynamic, Nod-factor dependent, and requires the ionic but not the osmotic component of salt. We demonstrate that salt stimulation of rhizobium-induced gene expression requires NSP2, which functions as a node to integrate the abiotic and biotic signals. In addition, our work reveals that inoculation with S. meliloti succinoglycan mutants also hyperinduces ENOD11 expression in the presence or absence of salt, suggesting a possible link between rhizobial exopolysaccharide and the plant response to salt stress. Finally, we identify an accessory set of genes that are induced by rhizobium only under conditions of salt stress and have not been previously identified as being nodulation-related genes. Our data suggest that interplay of core nodulation genes with different accessory sets, specific for different abiotic conditions, functions to establish the symbiosis. Together, our findings reveal a complex and dynamic interaction between plant, microbe, and environment.
[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .
The soil environmental conditions and symbiotic interactions are the major determinants of legume root architecture. Investigating cues that affect root developmental adaptations to the environment as well as understanding the mechanisms underlying the control of the root architecture are crucial to improve agronomical traits, notably in the legume family. Indeed, legumes are one of the most widespread crops, in terms of number and diversity of cultivated species.
Legume roots can develop two types of secondary root organs: lateral roots and nitrogen‐fixing nodules. Lateral root formation is common to all higher plants; however, nodules are present only on legume roots as a result of the symbiotic interaction with nitrogen‐fixing soil bacteria, collectively known as rhizobia. The only nonlegume plants described to be able to interact with rhizobia to form nitrogen‐fixing nodules are Parasponia spp.
What makes the legume root system so peculiar? The aim of this chapter is to give an overview of the current knowledge of the development of secondary root organs in legumes. A comparative analysis of their structure and ontogeny will be presented, and the specific and common regulatory mechanisms involved will be described.
Ten isoleucine+valine and three leucine auxotrophs of Sinorhizobium meliloti Rmd201 were obtained by random mutagenesis with transposon Tn5 followed by screening of Tn5 derivatives on minimal medium supplemented with modified Holliday pools. Based on intermediate feeding, intermediate accumulation and cross-feeding studies, isoleucine+valine and leucine auxotrophs were designated as ilvB/ilvG, ilvC and ilvD, and leuC/leuD and leuB mutants, respectively. Symbiotic properties of all ilvD mutants with alfalfa plants were similar to those of the parental strain. The ilvB/ilvG and ilvC mutants were Nod-. Inoculation of alfalfa plants with ilvB/ilvG mutant did not result in root hair curling and infection thread formation. The ilvC mutants were capable of curling root hairs but did not induce infection thread formation. All leucine auxotrophs were Nod+ Fix-. Supplementation of leucine to the plant nutrient medium did not restore symbiotic effectiveness to the auxotrophs. Histological studies revealed that the nodules induced by the leucine auxotrophs did not develop fully like those induced by the parental strain. The nodules induced by leuB mutants were structurally more advanced than the leuC/leuD mutant induced nodules. These results indicate that ilvB/ilvG, ilvC and one or two leu genes of S. meliloti may have a role in symbiosis. The position of ilv genes on the chromosomal map of S. meliloti was found to be near ade-15 marker.
Plant PRAF proteins contain four major protein domains. Based on sequence homology, two of them, the pleckstrin homology PH and zinc‐finger FYVE domains, have putative affinity with lipids. To date, PRAF function remains elusive in plants. In this paper, we show that PH and FYVE domains have differential in vitro binding affinity with various lipids. Overexpression of the protein domains fused to the green fluorescent protein shows that the PH::GFP protein is localized in the endosome whereas the FYVE domain::GFP fusion protein is detected in the cytosol and in the nucleus. To analyze the putative importance of the lipid domains, PH, and FYVE domains fused to GFP were overexpressed in transgenic roots. MtZR1‐PH::GFP protein overexpression led to a significant reduction of root growth meanwhile nodule development and biological‐nitrogen fixation were negatively affected by the overexpression of phosphoinositide binding domain MtZR1‐FYVE. Compared to animal models, PH and FYVE domains from MtZR1 display an atypical subcellular distribution and non‐classical patterns of interaction with phospholipids.
Shoot segments of Medicago varia genotype A2 were co-cultivated with Agrobacterium tumefaciens strain bo42 carrying pGA471, a plasmid coding for the kanamycin resistant determinant as transferable positive selection marker in plant cells (An et al., 1985). Resistant plants were regenerated at high frequency from green calli developed on inoculated stem cuttings under kanamycin selection. DNA-DNA hybridization analysis showed the presence of the structural gene of the kanamycin resistant determinant in total DNA isolated from several independent transformants. All data presented clearly demonstrate the transfer, stable maintenance and functional expression of the kanamycin resistance marker in Medicago varia cells which retain their morphogenic property.
Medicago truncatula has all the characteristics required for a concerted analysis of nitrogen-fixing symbiosis withRhizobium using the tools of molecular biology, cellular biology and genetics.M. truncatula is a diploid and autogamous plant has a relatively small genome, and preliminary molecular analysis suggests that allelic
heterozygosity is minimal compared with the cross-fertilising tetraploid alfalfa (Medicago sativa). TheM. truncatula cultivar Jemalong is nodulated by theRhizobium meliloti strain 2011, which has already served to define many of the bacterial genes involved in symbiosis with alfalfa. A genotype
of Jemalong has been identified which can be regenerated after transformation byAgrobacterium, thus allowing the analysis ofin-vitro-modified genes in an homologous transgenic system. Finally, by virtue of the diploid, self-fertilising and genetically homogeneous
character ofM. truncatula, it should be relatively straightforward to screen for recessive mutations in symbiotic genes, to carry out genetic analysis,
and to construct an RFLP map for this plant.
To study the molecular responses of the host legume during early stages of the symbiotic interaction with Rhizobium, we have cloned and characterized the infection-related early nodulin gene MtENOD12 from Medicago truncatula. In situ hybridization experiments have shown that, within the indeterminate Medicago nodule, transcription of the MtENOD12 gene begins in cell layers of meristematic origin that lie ahead of the infection zone, suggesting that these cells are undergoing preparation for bacterial infection. Histochemical analysis of transgenic alfalfa plants that express an MtENOD12 promoter-beta-glucuronidase gene fusion has confirmed this result and further revealed that MtENOD12 gene transcription occurs as early as 3 to 6 hr following inoculation with R. meliloti in a zone of differentiating root epidermal cells which lies close to the growing root tip. It is likely that this transient, nodulation (nod) gene-dependent activation of the ENOD12 gene also corresponds to the preparation of the plant for bacterial infection. We anticipate that this extremely precocious response to Rhizobium will provide a valuable molecular marker for studying early signal exchange between the two symbiotic organisms.
In a search for plant genes expressed during early symbiotic interactions between Medicago sativa and Rhizobium meliloti, we have isolated and characterized two alfalfa genes which have strong sequence similarity to members of the Enod12 gene family of Pisum sativum. The M. sativa genes, MsEnod12A and B, encode putative protein products of 8066 Da and 12849 Da, respectively, each with a signal sequence at the N-terminus followed by a repetitive proline-rich region. Based on their expression during the initial period of nodule development, MsEnod12A and B are alfalfa early nodulin genes.
RHIZOBIUM meliloti is a symbiotic bacterium that elicits the morphogenesis of nitrogen-fixing nodules, specific organs on the roots of alfalfa (Medicago sativa) 1. In R. meliloti a series of nodulation (nod) genes have been identified which are involved in root-hair curling and infection and in nodule formation 1. The nodABC genes, common to all Rhizobium sp., and the host-range nodH and nodPQ genes are involved in the production of an excreted root-hair deforming factor, NodRm-1, which is a sulphated and acylated glucosamine oligosaccharide 2-7. Here we report that purified NodRm-1 and a related compound, Ac-NodRm-1, at concentrations in the micromolar-nanomolar range, elicit cortical cell divisions and the formation of genuine nodules on aseptically grown seedlings of alfalfa. Chemical modifications of NodRm-1, such as the removal of the sulphate group, reduction of the terminal sugar or hydrogenation of the acyl chain result in a strong decrease in the morphogenic activity. A highly specific prokaryotic lipo-oligosaccharide signal is thus able to trigger a genuine organogenesis in a higher plant.
Rhizobium bacteria produce certain lipo-oligosaccharides (modified chitin oligomers) after induction of nodulation (nod) gene transcription by the host plant. The function of the rhizobial nod genes in the biosynthesis of these lipo-oligosaccharides, focusing on their host specific aspects, is discussed. The lipo-oligosaccharides can elicit various responses in the host plants, like the formation of pre-infection threads and nodule meristems. Speculating on their function in plant morphogenesis the question is raised: do the rhizobial lipo-oligosaccharides resemble unknown plant signal molecules?
Initial stages in the Rhizobium-legume symbiosis can be thought of as a reciprocal molecular conversation: transmission of a gene inducer from legume host to bacterium, with ensuing bacterial synthesis of a morphogen that is transmitted to the plant, switching the developmental fate of the legume root. These signal molecules have a key role in determining bacterium-host specificity and the purified Nod factor compounds provide useful new tools to probe plant cell function.