EXPRESSION OF RECOMBINANT RECEPTOR PROTEINS SYM10 AND SYM37 PISUM SATIVUM INVOLVED IN PERCEPTION OF LIPO-CHITOOLIGOSACCHARIDES NOD FACTORS

In the legume-Rhizobium interaction Nod factors emanating from rhizobia trigger a complex of specific responses in epidermis, pericycle and root cortex of the plant, thereby providing the basis for subsequent bacterial entry and organogenesis of root nodules. Since Nod factors are biologically active at pico-nanomolar concentrations and their activity depends on Nod factor structural features, it suggests the presence of high affinity receptors to these molecules. Genetic analysis of pea mutants allowed to identify genes that are essential for symbiosis development and among of them the PsSym10 and PsSym37. These genes are predicted to encode LysM-receptor-like kinases with LysM motifs in extracellular domain (LysM-RLKs). These proteins may be potential receptors to Nod factors. However experimental evidence of Nod factor binding to the putative receptors is needed to confirm the biochemical function of receptors. Mainly, it depends on the problems with receiving of membrane receptors. In this work the heterologous expression of SYM10 and SYM37 was conducted in bacterial cells. We have also optimized the conditions for recombinant proteins purification and obtained specific antibodies for next immunoenzyme analysis of two LysM-RLKs in legume plants.

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Identical Accumulation and Immobilization of Sulfated and Nonsulfated Nod Factors in Host and Nonhost Root Hair Cell Walls

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2003.16.10.884 ◽

2003 ◽

Vol 16 (10) ◽

pp. 884-892 ◽

Cited By ~ 15

Author(s):

Joachim Goedhart ◽

Jean-Jacques Bono ◽

Ton Bisseling ◽

Theodorus W. J. Gadella

Keyword(s):

Hair Cell ◽

Root Hair ◽

Cell Walls ◽

Root Hairs ◽

Sharp Decrease ◽

Biologically Active ◽

Nod Factors ◽

Nod Factor ◽

Root Hair Cell ◽

Root Hair Deformation

Nod factors are signaling molecules secreted by Rhizobium bacteria. These lipo-chitooligosaccharides (LCOs) are required for symbiosis with legumes and can elicit specific responses at subnanomolar concentrations on a compatible host. How plants perceive LCOs is unclear. In this study, using fluorescent Nod factor analogs, we investigated whether sulfated and nonsulfated Nod factors were bound and perceived differently by Medicago truncatula and Vicia sativa root hairs. The bioactivity of three novel sulfated fluorescent LCOs was tested in a root hair deformation assay on M. truncatula, showing bioactivity down to 0.1 to 1 nM. Fluorescence microscopy of plasmolyzed M. truncatula root hairs shows that sulfated fluorescent Nod factors accumulate in the cell wall of root hairs, whereas they are absent from the plasma membrane when applied at 10 nM. When the fluorescent Nod factor distribution in medium surrounding a root was studied, a sharp decrease in fluorescence close to the root hairs was observed, visualizing the remarkable capacity of root hairs to absorb Nod factors from the medium. Fluorescence correlation microscopy was used to study in detail the mobilities of sulfated and nonsulfated fluorescent Nod factors which are biologically active on M. truncatula and V. sativa, respectively. Remarkably, no difference between sulfated and nonsulfated Nod factors was observed: both hardly diffuse and strongly accumulate in root hair cell walls of both M. truncatula and V. sativa. The implications for the mode of Nod factor perception are discussed.

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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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The pleiotropic effects of extract containing rhizobial Nod factors on pea growth and yield

Open Life Sciences ◽

10.2478/s11535-013-0277-7 ◽

2014 ◽

Vol 9 (4) ◽

pp. 396-409 ◽

Cited By ~ 1

Author(s):

Janusz Podleśny ◽

Jerzy Wielbo ◽

Anna Podleśna ◽

Dominika Kidaj

Keyword(s):

Foliar Application ◽

Root Nodules ◽

Growth Dynamics ◽

Net Photosynthesis ◽

Growth Stimulation ◽

Dry Mass ◽

Growth And Yield ◽

Nod Factors ◽

Nod Factor ◽

Beneficial Effects

AbstractRhizobial lipochitooligosacharides (Nod factors) influence the development of legume roots, including growth stimulation, nodule induction and root hair curling. However, their effect on the green parts of plants is less known, therefore we evaluated seed and foliar application of an extract containing Nod factors on pea growth and yield. Pea plants were examined from emergence to full maturity, including growth dynamics and morphological (nodule number and weight, the quantity and surface area of leaves) or physiological (photosynthesis and transpiration intensity, chlorophyll and nitrogen content) parameters. The foliar application Nod factor extract, or seed dressing followed by foliar application, resulted in the best outcomes. The number and weight of root nodules, the chlorophyll content in leaves, and the intensity of net photosynthesis were all elevated. As a consequence of Nod factor treatment, the dynamics of dry mass accumulation of pea organs were improved and the pod number was increased. A significant increase in pea yield was observed after Nod factor application. Increase of nodule and pod numbers and improved growth of roots appear to be amongst the beneficial effects of Nod factor extract on the activation of secondary plant meristems.

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Root nodulation of Sesbania rostrata suppresses stem nodulation by Sinorhizobium teranga but not Azorhizobium caulinodans

Canadian Journal of Microbiology ◽

10.1139/m96-028 ◽

1996 ◽

Vol 42 (2) ◽

pp. 187-190 ◽

Cited By ~ 9

Author(s):

Kodjo Tomekpe ◽

Marcelle Holsters ◽

Bernard Dreyfus

Keyword(s):

Host Plant ◽

Root Nodules ◽

Sesbania Rostrata ◽

Efficient Production ◽

Nod Factors ◽

Azorhizobium Caulinodans ◽

Experimental Conditions ◽

Nod Factor ◽

Stem Nodulation ◽

Azorhizobium Caulinodans Ors571

Azorhizobium caulinodans ORS571 and Sinorhizobium teranga ORS51 and ORS52 are symbionts of the same host plant Sesbania rostrata. In nature, A. caulinodans nodulates more competitively the stem-located infection sites of Sesbania rostrata. Sinorhizobium strains, although frequently present in root nodules, are seldom found in stem nodules. One probable explanation for this phenomenon is the more abundant presence of Azorhizobium on the leaf and stem surfaces of the host plant. Work presented here hints at other plausible factors that determine the greater "stem specificity" of Azorhizobium. We found that under experimental conditions in which roots are not inoculated, all strains nodulated stems very well. However, ORS51 and ORS52 were much more sensitive than ORS571 to suppression of stem nodulation by previous root inoculation. The introduction of the regulatory nodD gene from A. caulinodans diminished the sensitivity to this suppression, probably by enhanced nod gene expression and subsequent Nod factor production. Our hypothesis is that the greater infectivity of ORS571 is due to a more efficient production of mitogenic Nod factors at stem-located infection sites, thereby more readily overcoming systemic suppression caused by previous root inoculations.Key words: autoregulation, nitrogen fixation, rhizobial ecology, systemic suppression of nodulation.

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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1999.12.7.604 ◽

1999 ◽

Vol 12 (7) ◽

pp. 604-614 ◽

Cited By ~ 43

Author(s):

Vanessa Vernoud ◽

Etienne-Pascal Journet ◽

David G. Barker

Keyword(s):

Transcriptional Activation ◽

Sinorhizobium Meliloti ◽

Cell Activation ◽

Cortical Cell ◽

Structural Parameter ◽

Nod Factors ◽

Root Cortex ◽

Nod Factor ◽

Transgenic Alfalfa ◽

Infection Threads

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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The Effect of a Preparation Containing Rhizobial Nod Factors on Pea Morphological Traits and Physiology

Agronomy ◽

10.3390/agronomy11081457 ◽

2021 ◽

Vol 11 (8) ◽

pp. 1457

Author(s):

Karolina Smytkiewicz ◽

Janusz Podleśny ◽

Jerzy Wielbo ◽

Anna Podleśna

Keyword(s):

Symbiotic Nitrogen Fixation ◽

Root Nodules ◽

Nod Factors ◽

Water Control ◽

Gas Exchange Parameters ◽

Nod Factor ◽

Plant Emergence ◽

Growth Development ◽

Positive Effect ◽

Exchange Parameters

The aim of the study was to determine the possibility of increasing the pea yields by improving the symbiotic nitrogen fixation through the use of a preparation containing bacterial Nod factors (NFs). Two pea cultivars were included in the experiment: Wiato (with traditional foliage) and Model (afila type). Before sowing, the seeds were soaked in distilled water (control) and in a preparation of Nod factors at a concentration of 10−12 M dm−3 H2O. As a result, of the Nod factor preparation use, an acceleration of the date and uniformity of pea plant emergence was observed. The treatment had also a positive effect on the number and weight of root nodules, which resulted in a significant increase in the yield of vegetative and generative plant organs. A positive effect of seed soaking with NFs preparation was also observed in the dynamics of pea weight increase, chlorophyll content in leaves and the values of gas exchange parameters. Model cultivar of pea had generally higher values of the analysed traits than Wiato, but the response of both cultivars to Nod factors was similar. This means that application of the preparation containing NFs, may improve the growth, development, and yield of both types of pea.

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On the diagnostics of Adonis L. species (Ranunculaceae) of the Russian flora

10.33920/med-06-2002-01 ◽

2020 ◽

pp. 3-14

Author(s):

Aleksandr Luferov

Keyword(s):

Raw Materials ◽

Natural Populations ◽

Structural Features ◽

Biologically Active ◽

Scientific Medicine ◽

Diagnostic Features ◽

Plant Raw Materials ◽

Similar Chemical ◽

Chemical Studies ◽

Alternative Sources

The article provides brief information about cardiotonic, sedative, cytostatic, diuretic, and antibacterial effects of biologically active compounds of Adonis L. (Ranunculaceae) species. Chemical studies allowed to identify the cardiac glycosides, or cardenolides: or cardenolides: adontoxin, adonitol, adonitoxigenin, acetyldigitoxin and others. In scientific medicine, it is currently allowed to use Adonis vernalis L. Other types of Adonis have a similar chemical composition and are offered as substitutes for this official species, for example, Adonis apennina L. Many Adonis species have limited natural resources, and in some regions are rare, requiring conservation of their natural populations. The search for alternative sources of medicinal plant raw materials, based on this, is relevant. The experimental part of our research was carried out using the morphological and geographical method with the involvement of information on ecology and phenology. For the first time summarizes the diagnostic features of Adonis flora of Russian flora. Previously unknown structural features (shape and size of anthers) were identified that characterize the subgenera Adonanthe and Adonis. Taxonomic study of the genus Adonis of the Russian flora allowed us to determine its species composition, clarify its systematic affiliation, and nomenclature synonyms. 9 species were identified. Of these, 6 are perennials belonging to the subgenus Adonanthe, section Consiligo, which includes 2 subsections: Amurenses (2 species) and Vernales, which is differentiated into 2 rows: Apenninae (2 species) and Vernales (2 species). Subgenus Adonis is represented by 2 sections: Adonis (1 species) and Lophocarpa with sections Aestivales (1 species) and Dentatae (1 species). For all the considered species and varieties, the main distribution areas are given. A key has been compiled to determine the wild Adonis species distributed in Russia.

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Characterization of Soluble, Disulfide Bond-Stabilized, Prokaryotically Expressed Human Thyrotropin Receptor Ectodomain*

Endocrinology ◽

10.1210/endo.138.2.4920 ◽

1997 ◽

Vol 138 (2) ◽

pp. 588-593 ◽

Cited By ~ 8

Author(s):

Y. Bobovnikova ◽

P. N. Graves ◽

H. Vlase ◽

T. F. Davies

Keyword(s):

Amino Acids ◽

Disulfide Bond ◽

Disulfide Bonds ◽

Thioredoxin Reductase ◽

Biologically Active ◽

Receptor Protein ◽

Enzyme Linked Immunosorbent Assay ◽

Fusion Partner ◽

E Coli ◽

Recombinant Receptor

Abstract To study the interaction of TSH receptor (TSHR) autoantibodies with receptor protein, it is necessary first to express the receptor in the proper conformation including the formation of correct disulfide bridges. However, the reducing environment of the Escherichia coli (E. coli) cytoplasm prevents the generation of protein disulfide bonds and limits the solubility and immunoreactivity of recombinant human TSHR (hTSHR) products. To circumvent these limitations, hTSHR complementary DNA encoding the extracellular domain (hTSHR-ecd; amino acids 21–415) was inserted into the vector pGEX-2TK by directional cloning and used to transform the thioredoxin reductase mutant strain of E. coli (Ad494), which allowed formation of disulfide bonds in the cytoplasm. After induction, the expressed soluble hTSHR-ecd fusion protein was detected by Western blot analysis using a monoclonal antibody directed against hTSHR amino acids 21–35. This showed that over 50% of the expressed hTSHR-ecd was soluble in contrast to expression in a wild-type E. coli (strain αF′), where the majority of the recombinant receptor was insoluble. The soluble recombinant receptor was affinity purified and characterized. Under nonreducing SDS-PAGE conditions, the soluble hTSHR-ecd migrated as refolded, disulfide bond-stabilized, multimeric species, whose formation was independent of fusion partner protein. This product was found to be biologically active as evidenced by the inhibition of the binding of 125I-TSH to the full-length hTSHR expressed in transfected CHO cells and was used to develop a competitive capture enzyme-linked immunosorbent assay for mapping of hTSHR antibody epitopes. Hence, hTSHR-ecd produced in bacteria with a thioredoxin reductase mutation was found to be highly soluble and biologically relevant.

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Search for active candidates in a range of flavonoids regarding SARS-CoV-2 by the method of molecular docking

10.33920/med-13-2101-02 ◽

2021 ◽

pp. 22-35

Author(s):

Stanislav V. Pechinskii ◽

Eduard T. Oganesyan ◽

Anna G. Kuregyan

Keyword(s):

Molecular Docking ◽

Antiviral Activity ◽

Cost Effective ◽

Structural Features ◽

Biologically Active ◽

Active Structures ◽

Main Protease ◽

Targeted Screening ◽

The Relationship ◽

Structure And Activity

Molecular docking is a convenient and cost-effective tool for targeted screening of biologically active structures. This method makes it possible to reveal the relationship between structure and activity, as well as to search for new active compounds. Due to the fact that the antiviral activity of flavonoids and their derivatives has been shown experimentally and clinically, the study of their antiviral activity against SARS-CoV-2 is a promising study. In an in silico experiment, the possibility of binding 20 flavonoid ligands and the main protease SARS-CoV-2 was studied. The structural features of flavone and flavanone derivatives have been determined, which determine their ability to block the main protease of the SARS-CoV-2 virus. Structures of eight new candidates that bind the main protease SARS-CoV-2, which have the prospect of synthesis and further pharmacological research, have been proposed.

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