MtENOD20, a Nod Factor-Inducible Molecular Marker for Root Cortical Cell Activation

The spatio-temporal expression pattern of the Medicago truncatula ENOD20 gene during early stages of nodulation has been analyzed with transgenic alfalfa (M. varia) expressing a pMtENOD20-GUS chimeric fusion. Our results show that transcriptional activation of this gene occurs initially in dividing inner cortical cells corresponding to sites of nodule primordium formation and subsequently in root hairs containing infection threads. Use of Sinorhizobium meliloti nod gene mutants that uncouple nodule organogenesis from infection has confirmed that early MtENOD20 transcription is tightly linked to cortical cell activation (CCA). Furthermore, these experiments have revealed that an S. meliloti nodH mutant, defective in Nod factor sulfation and epidermal cell activation, is nevertheless able to elicit low-level CCA. Purified S. meliloti Nod factors trigger MtENOD20 transcription very rapidly (within 12 to 24 h) in the root cortex, and studies with transgenic alfalfa show that Nod factors are able to elicit gene expression coupled to CCA at concentrations as low as 10-11 M. Finally, we have made use of a range of synthetic lipo-chitooligosaccharides to show that fatty acid chain length is an important structural parameter in the capacity of Nod factors to elicit CCA. Taken together, our results suggest that pMtENOD20-GUS transgenic lines should prove valuable tools in future studies of Rhizobium and Nod factor-elicited CCA.

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Effects of a Nod-factor-overproducing strain of Sinorhizobium meliloti on the expression of the ENOD40 gene in Melilotus alba

Canadian Journal of Botany ◽

10.1139/b02-076 ◽

2002 ◽

Vol 80 (9) ◽

pp. 907-915 ◽

Cited By ~ 6

Author(s):

Walter F Giordano ◽

Michelle R Lum ◽

Ann M Hirsch

Keyword(s):

Lateral Root ◽

Sinorhizobium Meliloti ◽

Cortical Cell ◽

Host Cells ◽

Nodule Development ◽

Nodule Formation ◽

Additional Increase ◽

Nod Factor ◽

Melilotus Alba ◽

Different Strains

We have initiated studies on the molecular biology and genetics of white sweetclover (Melilotus alba Desr.) and its responses to inoculation with the nitrogen-fixing symbiont Sinorhizobium meliloti. Early nodulin genes such as ENOD40 serve as markers for the transition from root to nodule development even before visible stages of nodule formation are evident. Using Northern blot analysis, we found that the ENOD40 gene was expressed within 6 h after inoculation with two different strains of S. meliloti, one of which overproduces symbiotic Nod factors. Inoculation with this strain resulted in an additional increase in ENOD40 gene expression over a typical wild-type S. meliloti strain. Moreover, the increase in mRNA brought about by the Nod-factor-overproducing strain 24 h after inoculation was correlated with lateral root formation by using whole-mount in situ hybridization to localize ENOD40 transcripts in lateral root meristems and by counting lateral root initiation sites. Cortical cell divisions were not detected. We also found that nodulation occurred more rapidly on white sweetclover in response to the Nod-factor-overproducing strain, but ultimately there was no difference in nodulation efficiency in terms of nodule number or the number of roots nodulated by the two strains. Also, the two strains could effectively co-colonize the host when inoculated together, although a few host cells were occupied by both strains.Key words: ENOD40, Nod factor, Melilotus, Sinorhizobium, symbiosis.

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Biological activity of Nod factors

Acta Biochimica Polonica ◽

10.18388/abp.2020_5353 ◽

2020 ◽

Author(s):

Dominika Kidaj ◽

Mikolaj Krysa ◽

Katarzyna Susniak ◽

Joanna Matys ◽

Iwona Komaniecka ◽

...

Keyword(s):

Molecular Level ◽

Root Nodules ◽

Cortical Cell ◽

Plant Tissues ◽

Nod Factors ◽

Nod Factor ◽

Expression Of Genes ◽

Root Hair Deformation ◽

Low Concentrations ◽

Genes Encoding

Chemically, the Nod factors (NFs) are lipochitooligosaccharides, produced mainly by bacteria of the Rhizobium genus. They are the main signaling molecules involved in the initiation of symbiosis between rhizobia and legume plants. Nod factors affect plant tissues at very low concentrations, even as low as 10–12 mol/L. They induce root hair deformation, cortical cell division, and root nodules’ formation in the host plant. At the molecular level, the cytoskeleton is reorganized and expression of genes encoding proteins called nodulins is induced in response to Nod factors in the cell. Action of Nod factors is highly specific because it depends on the structure of a particular Nod factor involved, as well as the plant receptor reacting with it.

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A plant chitinase controls cortical infection thread progression and nitrogen-fixing symbiosis

eLife ◽

10.7554/elife.38874 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 9

Author(s):

Anna Malolepszy ◽

Simon Kelly ◽

Kasper Kildegaard Sørensen ◽

Euan Kevin James ◽

Christina Kalisch ◽

...

Keyword(s):

Root Nodules ◽

Lotus Japonicus ◽

Positional Information ◽

Dependent Manner ◽

Nitrogen Fixing ◽

Nod Factors ◽

Nod Factor ◽

Infection Threads ◽

Bacterial Mutants ◽

Multicellular Organisms

Morphogens provide positional information and their concentration is key to the organized development of multicellular organisms. Nitrogen-fixing root nodules are unique organs induced by Nod factor-producing bacteria. Localized production of Nod factors establishes a developmental field within the root where plant cells are reprogrammed to form infection threads and primordia. We found that regulation of Nod factor levels by Lotus japonicus is required for the formation of nitrogen-fixing organs, determining the fate of this induced developmental program. Our analysis of plant and bacterial mutants shows that a host chitinase modulates Nod factor levels possibly in a structure-dependent manner. In Lotus, this is required for maintaining Nod factor signalling in parallel with the elongation of infection threads within the nodule cortex, while root hair infection and primordia formation are not influenced. Our study shows that infected nodules require balanced levels of Nod factors for completing their transition to functional, nitrogen-fixing organs.

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Evidence for the Involvement in Nodulation of the Two Small Putative Regulatory Peptide-Encoding Genes MtRALFL1 and MtDVL1

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-21-8-1118 ◽

2008 ◽

Vol 21 (8) ◽

pp. 1118-1127 ◽

Cited By ~ 44

Author(s):

Jean-Philippe Combier ◽

Helge Küster ◽

Etienne-Pascal Journet ◽

Natalija Hohnjec ◽

Pascal Gamas ◽

...

Keyword(s):

Nodule Development ◽

Strong Increase ◽

Nod Factors ◽

Plant Host ◽

Symbiotic Interaction ◽

Nod Factor ◽

Infection Threads ◽

Genes Encoding ◽

Supernodulating Mutant ◽

Increased Sensitivity

Nod factors are key bacterial signaling molecules regulating the symbiotic interaction between bacteria known as rhizobia and leguminous plants. Studying plant host genes whose expression is affected by Nod factors has given insights into early symbiotic signaling and development. Here, we used a double supernodulating mutant line that shows increased sensitivity to Nod factors to study the Nod factor-regulated transcriptome. Using microarrays containing more than 16,000 70-mer oligonucleotide probes, we identified 643 Nod-factor-regulated genes, including 225 new Nod-factor-upregulated genes encoding many potential regulators. Among the genes found to be Nod factor upregulated, we identified and characterized MtRALFL1 and MtDVL1, which code for two small putative peptide regulators of 135 and 53 amino acids, respectively. Expression analysis confirmed that these genes are upregulated during initial phases of nodulation. Overexpression of MtRALFL1 and MtDVL1 in Medicago truncatula roots resulted in a marked reduction in the number of nodules formed and in a strong increase in the number of aborted infection threads. In addition, abnormal nodule development was observed when MtRALFL1 was overexpressed. This work provides evidence for the involvement of new putative small-peptide regulators during nodulation.

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Strain-Ecotype Specificity in Sinorhizobium meliloti-Medicago truncatula Symbiosis Is Correlated to Succinoglycan Oligosaccharide Structure

Journal of Bacteriology ◽

10.1128/jb.00739-07 ◽

2007 ◽

Vol 189 (21) ◽

pp. 7733-7740 ◽

Cited By ~ 48

Author(s):

Senay Simsek ◽

Tuula Ojanen-Reuhs ◽

Samuel B. Stephens ◽

Bradley L. Reuhs

Keyword(s):

Medicago Truncatula ◽

Sinorhizobium Meliloti ◽

Donor Strain ◽

Factor Activity ◽

Nod Factors ◽

Strain Specificity ◽

Oligosaccharide Structure ◽

Nod Factor ◽

Structural Nature ◽

Molecular Signals

ABSTRACT Molecular signals, including Nod factors and succinoglycan, are necessary for the establishment of nitrogen-fixing nodules (Fix+) in Medicago truncatula-Sinorhizobium meliloti symbiosis. This report shows that M. truncatula-S. meliloti interactions involve ecotype-strain specificity, as S. meliloti Rm41 and NRG247 are Fix+ (compatible) on M. truncatula A20 and Fix− (incompatible) on M. truncatula A17, the Fix phenotypes are reversed with S. meliloti NRG185 and NRG34, and there is a correlation between the host specificity and succinoglycan oligosaccharide structure. S. meliloti NRG185 produces oligosaccharides that are almost fully succinylated, with two succinate groups per subunit, whereas the oligosaccharides produced by S. meliloti Rm41 include many nonsuccinylated subunits, as well as subunits with a single succinate group and others with malate. The results of this study demonstrated the following: (i) incompatibility is not a consequence of an avirulence factor or lack of Nod factor activity; (ii) the Fix+ phenotypes are succinoglycan dependent; (iii) there is structural variability in the succinoglycan oligosaccharide populations between S. meliloti strains; (iv) the structural nature of the succinoglycan oligosaccharides is correlated to compatibility; most importantly, (v) an S. meliloti Rm41 derivative, carrying exo genes from an M. truncatula A17-compatible strain, produced a modified population of succinoglycan oligosaccharides (similar to the donor strain) and was Fix+ on A17.

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Activity of Sinorhizobium meliloti NodAB and NodH Enzymes on Thiochitooligosaccharides

Journal of Bacteriology ◽

10.1128/jb.184.14.4039-4043.2002 ◽

2002 ◽

Vol 184 (14) ◽

pp. 4039-4043 ◽

Cited By ~ 5

Author(s):

Audrey M. Southwick ◽

Lai-Xi Wang ◽

Sharon R. Long ◽

Yuan C. Lee

Keyword(s):

Sinorhizobium Meliloti ◽

Signal Molecules ◽

Gene Products ◽

Nod Factors ◽

Nod Factor ◽

Glycosidic Bonds

ABSTRACT Rhizobium bacteria synthesize signal molecules called Nod factors that elicit responses in the legume root during nodulation. Nod factors, modified N-acylated β-(1,4)-N-acetylglucosamine, are synthesized by the nodulation (nod) gene products. We tested the ability of three Sinorhizobium meliloti nod gene products to modify Nod factor analogs with thio linkages instead of O-glycosidic bonds in the oligosaccharide backbone.

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Rapid Colorimetric Quantification of Lipo-chitooligosaccharides from Mesorhizobium loti and Sinorhizobium meliloti

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2002.15.9.859 ◽

2002 ◽

Vol 15 (9) ◽

pp. 859-865

Author(s):

Joachim Goedhart ◽

Jean-Jacques Bono ◽

Theodorus W. J. Gadella

Keyword(s):

Methylene Blue ◽

Organic Phase ◽

Sinorhizobium Meliloti ◽

Sodium Dodecyl ◽

Ion Pair ◽

Nod Factors ◽

Two Phase ◽

Nod Factor ◽

Mesorhizobium Loti ◽

Hydrolysis Of

Nod factors are lipids with a chitinlike headgroup produced by gram-negative Rhizobium bacteria. These lipo-chitooligosaccharides (LCOs) are essential signaling molecules for accomplishing symbiosis between the bacteria and roots of legume plants. Despite their important role in the Rhizobium-legume interaction, no fast and sensitive Nod factor quantification methods exist. Here, we report two different quantification methods. The first is based on the enzymatic hydrolysis of Nod factors to release N-acetylglucosamine (GlcNAc), which can subsequently be quantified. It is shown that the degrading enzyme, glusulase, releases exactly two GlcNAc units per pentameric nodulation factor from Mesorhizobium loti factor, allowing quantification of LCOs from Mesorhizobium loti. The second method is based on a specific type of Nod factors that are sulfated on the reducing GlcNAc, allowing quantification analogous to the quantification of sulfolipids. Here, a two-phase extraction method is used in the presence of methylene blue, which specifically forms an ion pair with sulfated lipids. The blue ion pair partitions into the organic phase, after which the methylene blue signal can be quantified. To enable Nod factor quantification with this method, the organic phase was modified and the partitioning was evaluated using fluorescent and radiolabeled sulfated Nod factors. It is shown that sulfated LCOs can be quantified with this method, using sodium dodecyl sulfate for calibration. Both methods allow Nod factor quantification in parallel enabling a fast and easy detection of nanomole quantities of Nod factors. Accurate Nod factor quantification will be crucial for characterization and cross-comparison of the affinity for Nod factors of newly identified Nod factor binding proteins or putative Nod factor receptors.

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The Early Nodulin Gene MtN6 Is a Novel Marker for Events Preceding Infection of Medicago truncatula Roots by Sinorhizobium meliloti

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1999.12.6.544 ◽

1999 ◽

Vol 12 (6) ◽

pp. 544-555 ◽

Cited By ~ 27

Author(s):

R. Mathis ◽

C. Grosjean ◽

F. de Billy ◽

T. Huguet ◽

P. Gamas

Keyword(s):

Medicago Truncatula ◽

Sinorhizobium Meliloti ◽

Root Nodules ◽

Cdna Clones ◽

Specific Marker ◽

Nod Factors ◽

Cortical Cells ◽

Infection Threads ◽

Early Nodulin ◽

Cell Layers

MtN6 belongs to a series of cDNA clones representing Medicago truncatula genes transcriptionally activated during nodulation by Sinorhizobium meliloti (P. Gamas, F. de Carvalho Niebel, N. Lescure, and J. V. Cullimore, Mol. Plant-Microbe Interact. 9:233–242, 1996). We show here by in situ hybridization that MtN6 transcripts specifically accumulate first at very localized regions in the outer root cell layers, corresponding to outer cortical cells containing preinfection threads. At later stages, MtN6 expression is observed ahead of growing infection threads, including in the infection zone of mature root nodules. Interestingly, regulation of MtN6 is clearly distinct from that of other early nodulins expressed in the same region of the nodule, in terms of response to bacterial symbiotic mutants and to purified Nod factors. We thus suggest that MtN6 represents the first specific marker of a pathway involved in preparation to infection, which is at least partly controlled by Nod factors. Finally, we discuss the intriguing sequence homology shown by MtN6 to a protein from Emericella (Aspergillus) nidulans, FluG, that plays a key role in controlling the organogenesis of conidiophores (B. N. Lee and T. H. Adams, Genes Dev. 8:641–651, 1994).

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Sinorhizobium fredii HH103 Invades Lotus burttii by Crack Entry in a Nod Factor–and Surface Polysaccharide–Dependent Manner

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-09-16-0195-r ◽

2016 ◽

Vol 29 (12) ◽

pp. 925-937 ◽

Cited By ~ 16

Author(s):

Sebastián Acosta-Jurado ◽

Dulce-Nombre Rodríguez-Navarro ◽

Yasuyuki Kawaharada ◽

Juan Fernández Perea ◽

Antonio Gil-Serrano ◽

...

Keyword(s):

Root Hairs ◽

Broad Host Range ◽

Capsular Polysaccharides ◽

Dependent Manner ◽

Nod Factors ◽

Sinorhizobium Fredii ◽

Nod Factor ◽

Mesorhizobium Loti ◽

Infection Threads ◽

Surface Polysaccharide

Sinorhizobium fredii HH103-Rifr, a broad host range rhizobial strain, induces nitrogen-fixing nodules in Lotus burttii but ineffective nodules in L. japonicus. Confocal microscopy studies showed that Mesorhizobium loti MAFF303099 and S. fredii HH103-Rifr invade L. burttii roots through infection threads or epidermal cracks, respectively. Infection threads in root hairs were not observed in L. burttii plants inoculated with S. fredii HH103-Rifr. A S. fredii HH103-Rifr nodA mutant failed to nodulate L. burttii, demonstrating that Nod factors are strictly necessary for this crack-entry mode, and a noeL mutant was also severely impaired in L. burttii nodulation, indicating that the presence of fucosyl residues in the Nod factor is symbiotically relevant. However, significant symbiotic impacts due to the absence of methylation or to acetylation of the fucosyl residue were not detected. In contrast S. fredii HH103-Rifr mutants showing lipopolysaccharide alterations had reduced symbiotic capacity, while mutants affected in production of either exopolysaccharides, capsular polysaccharides, or both were not impaired in nodulation. Mutants unable to produce cyclic glucans and purine or pyrimidine auxotrophic mutants formed ineffective nodules with L. burttii. Flagellin-dependent bacterial mobility was not required for crack infection, since HH103-Rifr fla mutants nodulated L. burttii. None of the S. fredii HH103-Rifr surface-polysaccharide mutants gained effective nodulation with L. japonicus.

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Cell Biological Changes of Outer Cortical Root Cells in Early Determinate Nodulation

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.7.839 ◽

2001 ◽

Vol 14 (7) ◽

pp. 839-847 ◽

Cited By ~ 40

Author(s):

Paulina C. van Spronsen ◽

Mette Grønlund ◽

Cristina Pacios Bras ◽

Herman P. Spaink ◽

Jan W. Kijne

Keyword(s):

Lotus Japonicus ◽

Nod Factors ◽

Outer Cortex ◽

Root Cortex ◽

Root Cells ◽

Leguminous Plants ◽

Infection Threads ◽

Cytoplasmic Bridges ◽

Inner Cortex

In the symbiosis of leguminous plants and Rhizobium bacteria, nodule primordia develop in the root cortex. This can be either in the inner cortex (indeterminate-type of nodulation) or outer cortex (determinate-type of nodulation), depending upon the host plant. We studied and compared early nodulation stages in common bean (Phaseolus vulgaris) and Lotus japonicus, both known as determinate-type nodulation plants. Special attention was paid to the occurrence of cytoplasmic bridges, the influence of rhizobial Nod factors (lipochitin oligosaccharides [LCOs]) on this phenomenon, and sensitivity of the nodulation process to ethylene. Our results show that i) both plant species form initially broad, matrix-rich infection threads; ii) cytoplasmic bridges occur in L. japonicus but not in bean; iii) formation of these bridges is induced by rhizobial LCOs; iv) formation of primordia starts in L. japonicus in the middle root cortex and in bean in the outer root cortex; and v) in the presence of the ethylenebiosynthesis inhibitor aminoethoxyvinylglycine (AVG), nodulation of L. japonicus is stimulated when the roots are grown in the light, which is consistent with the role of cytoplasmic bridges during nodulation of L. japonicus.

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