Indirect evidence for cellulase production by Rhizobium in pea root nodules during bacteroid differentiation: cytochemical aspects of cellulose breakdown in rhizobial droplets

1989 ◽  
Vol 35 (9) ◽  
pp. 821-829 ◽  
Author(s):  
François-P. Chalifour ◽  
Nicole Benhamou
Keyword(s):  
Rhizobium Leguminosarum ◽  
Root Nodules ◽  
Hydrolytic Enzyme ◽  
Indirect Evidence ◽  
Cellulase Production ◽  
Cytochemical Localization ◽  
Thin Walls ◽  
Sequence Of Events ◽  
Infection Threads ◽  
Cellulose Breakdown

Cytochemical localization of cellulosic β-(1–4) glucans in pea (Pisum sativum L.) nodules at different stages of infection by an effective isolate of Rhizobium leguminosarum biovar viceae was studied using a gold-complexed exoglucanase. Cellulose subunits were present in great amounts in root cell walls, as shown by intense and regular labeling by gold particles. Labeling was unevenly distributed over the thin walls of emerging infection threads. In more developed infection threads, labeling was more intense and evenly distributed than in emerging threads, although slightly altered, unlabeled wall areas were frequently observed at the growing tips. Droplets containing rhizobia, which originated from infection threads, were surrounded by labeled wall-like material. Rhizobial droplets were either single- or multi-celled, and were sometimes separated by inner, unevenly labeled compartments. The surrounding wall-like material was irregularly labeled, and unlabeled wall areas, neighbouring intensely labeled ones, were observed frequently. There was an absence of labeling ahead of the rhizobia that escaped from the droplets, but degenerating wall-like material was present around the escaping rhizobia, mainly on their sides. At more advanced stages of development, labeling was present only over the outermost wall layers of rhizobial droplets, indicating that inner portions were degraded first. These observations suggest that a hydrolytic enzyme is involved in the sequence of events from infection thread formation through rhizobial release in the host cell cytoplasm, and that the hydrolytic enzyme is of rhizobial origin.Key words: Rhizobium–Pisum symbiosis, root nodules, rhizobial droplets, cellulose, colloidal gold.

scholarly journals Morphogenesis of root nodules in white clover. II. The effect of mutation in genes nod IJ of the microsymbiont upon the nodule structure

2014 ◽  
Vol 67 (1) ◽  
pp. 5-22
Author(s):  
Barbara Łotocka ◽  
Joanna Kopcińska ◽  
Władysław Golinowski
Keyword(s):  
White Clover ◽  
Rhizobium Leguminosarum ◽  
Root Nodules ◽  
Single Cells ◽  
Wild Strain ◽  
Root Hairs ◽  
Nodule Development ◽  
Wild Type ◽  
Delayed Reaction ◽  
Infection Threads

Morphogenesis of ineffective root nodules initiated on the roots of white clover 'Astra' by the <em>Rhizobium leguminosarum</em> biovar. <em>trifolii</em> strains ANU261 (Tn5 insertion in nod 1 gene) and ANU262 (Tn5 insertion in nod J gene) was investigated. Following changes were observed, as compared to the wild-type nodulation: the exaggerated, not delayed reaction of root hairs; the delay in nodulation with the number of nodules the same as in plants inoculated with a wild strain; the formation and organization of the nodule primordium not changed in comparison with the wild-type nodules; infection threads abnormally branched and diffusing with bacteria deprived of light zone and enriched with storage material; infected cells of bacteroidal tissue abnormally strongly osmiophilic and only slightly vacuolated; symbiosomes with very narrowed peribacteroidal space, subject to premature degradation; abnormal accumulation of starch in the nodule tissues; nodule development blocked at the stage of laterally situated meristem and single nodule bundle; inhibition of divisions in the meristem and vacuolation of its cells; the appearance of single cells with colonies of saprophytic rhizobia embedded in the fibrillar matrix in the old, degraded regions of the bacteroidal tissue.

Formation and structure of root nodules induced on Macroptilium atropurpureum inoculated with various species of Rhizobium

1991 ◽  
Vol 69 (7) ◽  
pp. 1520-1532 ◽  
Author(s):  
Michael J. Trinick ◽  
Celia Miller ◽  
Paul A. Hadobas
Keyword(s):  
Rhizobium Leguminosarum ◽  
Rhizobium Meliloti ◽  
Root Nodules ◽  
Matrix Material ◽  
Immunogold Labelling ◽  
Infection Threads ◽  
Glass Tubes ◽  
Infected Cells ◽  
Macroptilium Atropurpureum ◽  
Nodule Tissue

Fifteen strains of Rhizobium leguminosarum biovar trifolii formed ineffective nodules and (or) nodule-like structures (rhizobia were re-isolated from both structures) on Macroptilium atropurpureum grown in enclosed glass tubes. Bacteria were observed among the parenchyma cells surrounding the nodule-like structures. One variant of R. leguminosarum biovar trifolii (NGR66/ST) isolated from M. atropurpureum formed nodules on this host that exhibited abnormal intercellular and intracellular infection. The bacteria (NGR66/ST) were contained within threadlike structures, surrounded by matrix material. The identities of the Rhizobium strains were confirmed serologically after reisolation and in sections of nodule tissue using immunogold labelling. Rhizobium leguminosarum biovar phaseoli strain NGR76 isolated from Phaseolus vulgaris formed nodules on M. atropurpureum resembling those formed by effective Bradyrhizobium strains. The association was partially effective in nitrogen fixation, and this was reflected in the nodule structure. The percentage of cells infected was lower than that in fully effective nodules. There was a high frequency of infected cells showing degeneration; these were located throughout the nodule tissue and were often adjacent to healthy infected cells. The rhizobia appeared to infect new nodule cells via infection threads, which were abundant both intercellularly and intracellularly in young, mature, and degenerating host nodule cells. Strains of R. leguminosarum biovar viceae and Rhizobium meliloti were unable to induce nodule-like structures on M. atropurpureum. Key words: Macroptilium, Bradyrhizobium, Rhizobium, microscopy, nodule, structure.

scholarly journals Identification of a Family of Extensin-Like Glycoproteins in the Lumen of Rhizobium-Induced Infection Threads in Pea Root Nodules

2002 ◽  
Vol 15 (4) ◽  
pp. 350-359 ◽  
Author(s):  
Elizabeth A. Rathbun ◽  
Michael J. Naldrett ◽  
Nicholas J. Brewin
Keyword(s):  
Posttranscriptional Regulation ◽  
Rhizobium Leguminosarum ◽  
Root Nodules ◽  
Regulation Of Gene Expression ◽  
Pcr Primers ◽  
Amino Acid Sequences ◽  
Sequence Conservation ◽  
Host Cells ◽  
Cdna Clones ◽  
Infection Threads

Rhizobium leguminosarum bv. viciae normally gains access to pea host cells through tubular cell wall ingrowths termed infection threads. Matrix glycoprotein (MGP), a major component of the infection thread lumen, is also secreted from the tips of uninoculated roots and can be released into solution under reducing conditions. Monoclonal antibody MAC265, which recognizes MGP through a carbohydrate epitope, was used for immunoaffinity purification of the glycoprotein from pea roots. Following treatment with chymotrypsin, a peptide fragment was obtained and subjected to N-terminal sequencing. Using PCR primers based on this sequence, cDNA clones were isolated with RNA from inoculated roots and nodules. DNA sequencing of 30 of these clones revealed a family of closely related and repetitive polypeptides with (hydroxy)proline-rich motifs. The cDNA sequences showed over 70% identity with the deduced amino acid sequences of plant extensins, particularly with VfNDS-E from Vicia faba and MtN12 from Medicago truncatula, both of which are strongly upregulated in legume root nodules. Root nodule extensins from pea were of variable length but showed strong sequence conservation of the N-terminus, of the C-terminus, and of a central domain comprising 33 amino acids that were sometimes reiterated. The distribution of tyrosine residues suggested the possible importance of intramolecular and intermolecular cross-linking. There was strong sequence conservation with MtN12 in the 3′-untranslated region, suggesting a possible involvement in posttranscriptional regulation of gene expression.

scholarly journals Morphogenesis of root nodules in white clover. III. The effect of mutation in nod IJ genes of the microsymbiont upon the DNA level in the host tissue

2014 ◽  
Vol 67 (1) ◽  
pp. 23-29
Author(s):  
Barbara Łotocka ◽  
Władysław Golinowski
Keyword(s):  
White Clover ◽  
Rhizobium Leguminosarum ◽  
Root Nodule ◽  
Nuclear Dna ◽  
Root Nodules ◽  
Root Hairs ◽  
Infection Threads ◽  
Dna Contents ◽  
Mutant Strains ◽  
Nuclear Dna Contents

On the basis of cytophotometric measurements a slightly increased DNA level in the nuclei of curled root hairs containing infection threads was observed in white clover inoculated with wild and mutant strains of <em>Rhizobium leguminosarum</em> biovar. <em>trifolii</em>, as compared to normal root hairs of te same plants. Cells of the root nodule primordia in 72 h after the inoculation, as compared to the root primary cortex, demonstrated an increased level of the nuclear DNA. No differences were observed in the nuclear DNA contents in individual layers of the cortex of the 28 day-old nodules. Generally it was low, varying from 2c to 4c. The meristematic and bacteroidal tissues in the effective nodules were characterized by a higher DNA level, as compared to the respective zones in ineffective nodules induced with the strains ANU261 (<em>nod I<sup>*</sup></em>) and ANU262 (<em>nod J<sup>*</sup></em>). The DNA level in the effective bacteroidal tissue varied from 4c to 32c, while in the tissue containing the strain ANU26l only the 2c-8c nuclei could be found and in the tissue with the strain ANU262 - the 4c-16c nuclei.

scholarly journals Development of Functional Symbiotic White Clover Root Hairs and Nodules Requires Tightly Regulated Production of Rhizobial Cellulase CelC2

2011 ◽  
Vol 24 (7) ◽  
pp. 798-807 ◽  
Author(s):  
Marta Robledo ◽  
José I. Jiménez-Zurdo ◽  
M. José Soto ◽  
Encarnación Velázquez ◽  
Frank Dazzo ◽  
...  
Keyword(s):  
Cell Wall ◽  
White Clover ◽  
Rhizobium Leguminosarum ◽  
Plant Cell Wall ◽  
Root Nodules ◽  
Root Hairs ◽  
Nitrogen Fixing ◽  
Plant Host ◽  
Root Cortex ◽  
Infection Threads

The establishment of rhizobia as nitrogen-fixing endosymbionts within legume root nodules requires the disruption of the plant cell wall to breach the host barrier at strategic infection sites in the root hair tip and at points of bacterial release from infection threads (IT) within the root cortex. We previously found that Rhizobium leguminosarum bv. trifolii uses its chromosomally encoded CelC2 cellulase to erode the noncrystalline wall at the apex of root hairs, thereby creating the primary portal of its entry into white clover roots. Here, we show that a recombinant derivative of R. leguminosarum bv. trifolii ANU843 that constitutively overproduces the CelC2 enzyme has increased competitiveness in occupying aberrant nodule-like root structures on clover that are inefficient in nitrogen fixation. This aberrant symbiotic phenotype involves an extensive uncontrolled degradation of the host cell walls restricted to the expected infection sites at tips of deformed root hairs and significantly enlarged infection droplets at termini of wider IT within the nodule infection zone. Furthermore, signs of elevated plant host defense as indicated by reactive oxygen species production in root tissues were more evident during infection by the recombinant strain than its wild-type parent. Our data further support the role of the rhizobial CelC2 cell wall–degrading enzyme in primary infection, and show evidence of its importance in secondary symbiotic infection and tight regulation of its production to establish an effective nitrogen-fixing root nodule symbiosis.

scholarly journals NodC-based evaluation of the occurrence of bacteria in nodules of Pisum sativum isolated on YEM agar

2019 ◽  
Vol 70 (1) ◽  
pp. 59-67
Author(s):  
Anna Lenart-Boroń ◽  
Tadeusz Zając ◽  
Piotr Mateusz Boroń ◽  
Agnieszka Klimek-Kopyra
Keyword(s):  
Pisum Sativum ◽  
Rhizobium Leguminosarum ◽  
Reference Strain ◽  
Root Nodules ◽  
Bacterial Species ◽  
Soil Science ◽  
Associated Bacteria ◽  
Plant Cultivation ◽  
Yeast Extract Mannitol ◽  
Diverse Community

SummaryThe bacterial nodulation (nod) genes are essential in the formation process of root nodules. This study was aimed to verify the occurrence of nodule-associated bacteria in two pea varieties (“Tarchalska” and “Klif ”) inoculated withRhizobiuminoculants – Nitragine™ and a noncommercial one produced by the Polish Institute of Soil Science and Plant Cultivation (IUNG). The number of colonies isolated on yeast extract mannitol (YEM) agar from the nodules of “Klif ” inoculated with IUNG inoculants was significantly higher than the number of colonies isolated from other variants. Species identification was based on sequencing of 16S rDNA, which revealed that despite careful sterilization of nodules, sequences of other bacterial species were detected. Among them, one sequence belonged toRhizobium leguminosarum(isolated from IUNG inoculant). To assess the presence of nodulation-capableRhizobium, amplification of thenodCgene was performed, which revealed that of 29 samples, 19 were positive. The remaining isolates, including reference strain and bacteria isolated from Nitragine™, lacked this gene. The results show that pea nodules harbor a very diverse community of bacteria. The lack ofnodCgene in some strains isolated from plants inoculated with Nitragine™ and with IUNG inoculant proves that even ifR. leguminosarumare abundant, they may not be efficient in nodulation.

Diversity of nodular bacteria ofScorpiurus muricatusin western Algeria and their impact on plant growth

2017 ◽  
Vol 63 (5) ◽  
pp. 450-463 ◽  
Author(s):  
Zoulikha Bouchiba ◽  
Zineb Faiza Boukhatem ◽  
Zohra Ighilhariz ◽  
Nouria Derkaoui ◽  
Benaissa Kerdouh ◽  
...  
Keyword(s):  
Plant Growth ◽  
Rhizobium Leguminosarum ◽  
Root Nodules ◽  
Pcr Amplification ◽  
Nucleotide Sequencing ◽  
Rrna Gene ◽  
Bacterial Strains ◽  
Single Strain ◽  
Western Algeria ◽  
First Time

A total of 51 bacterial strains were isolated from root nodules of Scorpiurus muricatus sampled from 6 regions of western Algeria. Strain diversity was assessed by rep-PCR amplification fingerprinting, which grouped the isolates into 28 different clusters. Partial nucleotide sequencing of the 16S rRNA gene and BLAST analysis revealed that root nodules of S. muricatus were colonized by different species close to Rhizobium vignae, Rhizobium radiobacter, Rhizobium leguminosarum, Phyllobacterium ifriqiyense, Phyllobacterium endophyticum, Starkeya sp., and Pseudomonas sp. However, none of these strains was able to form nodules on its host plant; even nodC was present in a single strain (SMT8a). The inoculation test showed a great improvement in the growth of inoculated plants compared with noninoculated control plants. A significant amount of indole acetic acid was produced by some strains, but only 2 strains could solubilize phosphate. In this report we described for the first time the diversity of bacteria isolated from root nodules of S. muricatus growing in different regions in western Algeria and demonstrated their potential use in promoting plant growth.

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

2018 ◽  
Vol 31 (5) ◽  
pp. 568-575 ◽  
Author(s):  
Marta Robledo ◽  
Esther Menéndez ◽  
Jose Ignacio Jiménez-Zurdo ◽  
Raúl Rivas ◽  
Encarna Velázquez ◽  
...  
Keyword(s):  
Medicago Truncatula ◽  
Cell Walls ◽  
Rhizobium Leguminosarum ◽  
Root Nodules ◽  
Root Hairs ◽  
Nod Factors ◽  
Model Legume ◽  
Ensifer Meliloti ◽  
Early Nodulin ◽  
The Impact

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.

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