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.

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 Morphogenesis of root nodules in white clover. I. Effective root nodules induced by the wild type Rhizobium leguminosarium biovar. trifolii

2014 ◽  
Vol 66 (3-4) ◽  
pp. 273-292 ◽  
Author(s):  
Barbara Łotocka ◽  
Joanna Kopcińska ◽  
Władysław Golinowski
Keyword(s):  
White Clover ◽  
Trifolium Repens ◽  
Rhizobium Leguminosarum ◽  
Root Nodules ◽  
Primary Root ◽  
Wild Type ◽  
Apical Position ◽  
Root Cortex ◽  
Vascular Tissues ◽  
Transverse Division

The research aimed at investigating the morphogenesis of cylindrical root nodules in <em>Trifolium repens</em> L. induced by the wild type <em>Rhizobium leguminosarum</em> biovar. <em>trifolii</em> strain 24. It has been demonstrated that the ontogenesis of a nodule begins with a transverse division of cells of the pericycle followed by the dedifferentiation and divisions of cells of the endodermis and inner layers of the primary root cortex. Shifting of the nodule meristem from its initially lateral to the apical position characteristic for cylindrical nodules was observed. Bacteroidal, cortical and vascular tissues of the nodule are described up to 42 days after inoculation. At that time typical degraded zone had not yet appeared in the nodules.

scholarly journals Role of Cellulose Fibrils and Exopolysaccharides of Rhizobium leguminosarum in Attachment to and Infection of Vicia sativa Root Hairs

2005 ◽  
Vol 18 (6) ◽  
pp. 533-538 ◽  
Author(s):  
M. C. Laus ◽  
A. A. N. van Brussel ◽  
J. W. Kijne
Keyword(s):  
Root Hair ◽  
Rhizobium Leguminosarum ◽  
Developmental Stages ◽  
Cortical Cell ◽  
Root Hairs ◽  
Infection Thread ◽  
Vicia Sativa ◽  
Wild Type ◽  
Infection Threads ◽  
Cellulose Fibrils

Infection and subsequent nodulation of legume host plants by the root nodule symbiote Rhizobium leguminosarum usually require attachment of the bacteria to root-hair tips. Bacterial cellulose fibrils have been shown to be involved in this attachment process but appeared not to be essential for successful nodulation. Detailed analysis of Vicia sativa root-hair infection by wild-type Rhizobium leguminosarum RBL5523 and its cellulose fibril-deficient celE mutant showed that wild-type bacteria infected elongated growing root hairs, whereas cellulose-deficient bacteria infected young emerging root hairs. Exopolysaccharide-deficient strains that retained the ability to produce cellulose fibrils could also infect elongated root hairs but infection thread colonization was defective. Cellulose-mediated agglutination of these bacteria in the root-hair curl appeared to prevent entry into the induced infection thread. Infection experiments with V. sativa roots and an extracellular polysaccharide (EPS)- and cellulose-deficient double mutant showed that cellulose-mediated agglutination of the EPS-deficient bacteria in the infection thread was now abolished and that infection thread colonization was partially restored. Interestingly, in this case, infection threads were initiated in root hairs that originated from the cortical cell layers of the root and not in epidermal root hairs. Apparently, surface polysaccharides of R. leguminosarum, such as cellulose fibrils, are determining factors for infection of different developmental stages of root hairs.

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.

Diffusible factors from Frankia modify nodulation kinetics in Discaria trinervis, an intercellular root-infected actinorhizal symbiosis

2011 ◽  
Vol 38 (9) ◽  
pp. 662 ◽  
Author(s):  
Luciano Andrés Gabbarini ◽  
Luis Gabriel Wall
Keyword(s):  
Root Hair ◽  
Root Nodules ◽  
Root Hairs ◽  
Basic Structure ◽  
Peptide Bond ◽  
Nodule Development ◽  
Actinorhizal Symbiosis ◽  
Biochemical Characterisation ◽  
Clade 1 ◽  
Discaria Trinervis

Frankia BCU110501 induces nitrogen-fixing root nodules in Discaria trinervis (Gillies ex Hook. & Arn.) Reiche (Rhamnaceae) via intercellular colonisation, without root hair deformation. It produces diffusible factors (DFs) that might be involved in early interactions with the D. trinervis roots, playing a role in the nodulation process. The induction of root nodule development in actinorhizal symbiosis would depend on the concentration of factors produced by the bacteria and the plant. A detailed analysis of nodulation kinetics revealed that these DFs produce changes at the level of initial rate of nodulation and also in nodulation profile. Diluted Frankia BCU110501 inoculum could be activated in less than 96 h by DFs produced by Frankia BCU110501 cells that had been previously washed. Biochemical characterisation showed that Frankia BCU110501 DFs have a molecular weight of <12 kDa, are negatively charged at pH 7.0 and seem to contain a peptide bond necessary for their activity. Frankia BCU110501, belonging to Frankia Clade 3, does not induce nodules in Alnus acuminata H.B.K. ssp. acuminata but is able to deform root hairs, as do Frankia strains from Clade 1. The root hair deforming activity of Frankia BCU110501 DFs show the same biochemical characteristics of the DFs involved in nodulation of D. trinervis. These results suggest that Frankia symbiotic factors have a basic structure regardless of the infection pathway of the host plant.

scholarly journals Glucomannan-Mediated Attachment of Rhizobium leguminosarum to Pea Root Hairs Is Required for Competitive Nodule Infection

2008 ◽  
Vol 190 (13) ◽  
pp. 4706-4715 ◽  
Author(s):  
Alan Williams ◽  
Adam Wilkinson ◽  
Martin Krehenbrink ◽  
Daniela M. Russo ◽  
Angeles Zorreguieta ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Rhizobium Leguminosarum ◽  
Root Hairs ◽  
The Other ◽  
Plant Interactions ◽  
Wild Type ◽  
Polysaccharide Synthesis ◽  
Surface Polysaccharides

ABSTRACT The Rhizobium leguminosarum biovar viciae genome contains several genes predicted to determine surface polysaccharides. Mutants predicted to affect the initial steps of polysaccharide synthesis were identified and characterized. In addition to the known cellulose (cel) and acidic exopolysaccharide (EPS) (pss) genes, we mutated three other loci; one of these loci (gmsA) determines glucomannan synthesis and one (gelA) determines a gel-forming polysaccharide, but the role of the other locus (an exoY-like gene) was not identified. Mutants were tested for attachment and biofilm formation in vitro and on root hairs; the mutant lacking the EPS was defective for both of these characteristics, but mutation of gelA or the exoY-like gene had no effect on either type of attachment. The cellulose (celA) mutant attached and formed normal biofilms in vitro, but it did not form a biofilm on root hairs, although attachment did occur. The cellulose-dependent biofilm on root hairs appears not to be critical for nodulation, because the celA mutant competed with the wild-type for nodule infection. The glucomannan (gmsA) mutant attached and formed normal biofilms in vitro, but it was defective for attachment and biofilm formation on root hairs. Although this mutant formed nodules on peas, it was very strongly outcompeted by the wild type in mixed inoculations, showing that glucomannan is critical for competitive nodulation. The polysaccharide synthesis genes around gmsA are highly conserved among other rhizobia and agrobacteria but are absent from closely related bacteria (such as Brucella spp.) that are not normally plant associated, suggesting that these genes may play a wide role in bacterium-plant interactions.

Starch content and activities of starch-metabolizing enzymes in effective and ineffective root nodules of soybean

1991 ◽  
Vol 69 (4) ◽  
pp. 697-701 ◽  
Author(s):  
Sharon I. Forrest ◽  
Desh Pal S. Verma ◽  
Rajinder S. Dhindsa
Keyword(s):  
Nitrogen Fixation ◽  
Starch Content ◽  
Root Nodules ◽  
Starch Synthase ◽  
Nodule Development ◽  
Wild Type ◽  
Starch Phosphorylase ◽  
High Concentration ◽  
Key Words Nitrogen Fixation ◽  
Glycine Max L

Starch content and activities of some enzymes of starch metabolism were determined in wild-type, N2-fixing (fix+) nodules and in two non-N2-fixing (fix−) nodules induced by Bradyrhizobium japonicum mutant strains, T5-95 and T8-1, on soybean (Glycine max L.) roots. The T5-95 nodules are similar to wild type in ultrastructure, but the T8-1 nodules are different in that the bacteroids are not released from the infection thread. After initial accumulation to relatively high concentration, starch was depleted during nitrogen fixation in fix+ nodules. However, in fix− nodules, the accumulated starch was not metabolized. The activity of starch-bound starch synthase (EC 2.4.1.21) declined in fix+ nodules but remained high in fix− nodules. The activity of α-amylase (EC 3.2.1.1) was only slightly higher than wild type in T5-95 but was four times higher than wild type in T8-1 nodules. The activity of starch phosphorylase (EC 2.4.1.1) increased in all nodule types from 14 to 21 days postinfection. A positive correlation was observed between the capacity of nodules to fix N2 and their capacity to degrade starch. Collectively, these results support the concept that starch accumulated during early stages of nodule development is metabolized to supply energy for nitrogen fixation and to meet the metabolic demands of bacteroids. Key words: nitrogen fixation, starch content, effective and ineffective nodules, starch synthase, starch phosphorylase, α-amylase.

Cytological studies of the infection process in nodulating and nonnodulating pea genotypes

1989 ◽  
Vol 67 (8) ◽  
pp. 2435-2443 ◽  
Author(s):  
M. F. Le Gal ◽  
S. L. A. Hobbs
Keyword(s):  
North American ◽  
Rhizobium Leguminosarum ◽  
Root Hairs ◽  
Infection Process ◽  
High Concentration ◽  
Cortical Cells ◽  
Pisum Sativum L ◽  
Infection Threads ◽  
American Strain ◽  
Plant Effect

Pisum sativum L., cv. Afghanistan, does not form nodules with 128C52, a North American strain of Rhizobium leguminosarum. Timing of the abortion of the nodulation process was determined by microscopy in both 'Afghanistan' and nonnodulating 'Trapper,' produced by backcrossing the nonnodulating genes of 'Afghanistan' into 'Trapper,' a North American variety. Three to 5 days after inoculation, we observed deformed roots and localized swellings as well as loosely curled root hairs in these nonnodulating combinations. Rhizobia entered root hairs and epidermal cells, but no infection threads were seen. Cortical cells divided and a nodule meristem was initiated. Some meristematic cells showed abnormal features such as a high concentration of free ribosomes, dilated endoplasmic reticulum often connected to a dilated nuclear envelope, and disrupted mitochondria. Cortical cells around the nodule meristem were devoid of starch grains. Such phenotypes are known to be associated with rhizobial mutants, but in this case a plant effect is responsible.

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.

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