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

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.

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

Acta Societatis Botanicorum Poloniae ◽

10.5586/asbp.1998.001 ◽

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 Rhizobium leguminosarum biovar. trifolii 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.

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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

Acta Societatis Botanicorum Poloniae ◽

10.5586/asbp.1998.002 ◽

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 Rhizobium leguminosarum biovar. trifolii, 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 (nod I*) and ANU262 (nod J*). 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.

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Electron microscopic observations of infection threads in driselase treated nodules of Astragalus sinicus

Canadian Journal of Microbiology ◽

10.1139/m86-175 ◽

1986 ◽

Vol 32 (12) ◽

pp. 947-952 ◽

Cited By ~ 20

Author(s):

Shiro Higashi ◽

Kazuya Kushiyama ◽

Mikiko Abe

Keyword(s):

Cell Wall ◽

Cell Walls ◽

Plant Cell Wall ◽

Root Nodules ◽

Morphological Characteristics ◽

Enzyme Treatment ◽

Vascular Bundles ◽

Electron Microscopic ◽

Astragalus Sinicus ◽

Infection Threads

The morphological characteristics of infection threads in the root nodules of Astragalus sinicus were examined by scanning and transmission electron microscopy. The infection threads, epidermal cell walls, and vascular bundles of the nodule were not altered when a nodule was treated with driselase (a plant cell wall degrading enzyme), although the cell walls of meristematic and bacteroid-including zones were completely decomposed by the enzyme treatment. Some infection threads were funnel shaped at the site of attachment of the infection thread to the host cell wall.

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

Acta Societatis Botanicorum Poloniae ◽

10.5586/asbp.1997.032 ◽

2014 ◽

Vol 66 (3-4) ◽

pp. 273-292 ◽

Cited By ~ 11

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 Trifolium repens L. induced by the wild type Rhizobium leguminosarum biovar. trifolii 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.

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Localizatlon of expansin-like protein in apoplast of pea (Pisum sativum L.) root nodules during interaction with Rhizobium leguminosarum bv. viciae

Acta Societatis Botanicorum Poloniae ◽

10.5586/asbp.2007.002 ◽

2011 ◽

Vol 76 (1) ◽

pp. 17-26 ◽

Cited By ~ 6

Author(s):

Marzena Sujkowska ◽

Wojciech Borucki ◽

Władysław Golinowski

Keyword(s):

Cell Wall ◽

Plant Cell ◽

Rhizobium Leguminosarum ◽

Plant Cell Wall ◽

Root Nodules ◽

Infection Thread ◽

Nodule Development ◽

Cell Enlargement ◽

Pea Root ◽

Pea Roots

During nodule development on pea roots, apoplast undergoes changes in activity of plant cell wall proteins such as expansins (EXPs). Because the accumulation of EXP protein has been correlated with the growth of various plant organs, we investigated using Western Blot and immunolocalization studies with antibody against PsEXP1, whether this protein was accumulated in the expanding cells of nodule. Immunoblot results indicated the presence of a 30-kDa band specific for pea root nodules. The EXP proteins content rose during growth of pea root nodules. Expansin(s) protein was localized in nodule apoplast as well as in the infection thread walls. The enhanced amount of expansin-like proteins in meristematic part of nodule, root and shoot was shown. The localization of this protein in the meristematic cell walls can be related to the loosening of plant cell wall before cell enlargement. Both, plant cell enlargement and infection thread growth require activity of expansin(s). Possible involvement of EXPs in the process of pea root nodule development is also discussed.

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Extracellular Glycanases of Rhizobium leguminosarum Are Activated on the Cell Surface by an Exopolysaccharide-Related Component

Journal of Bacteriology ◽

10.1128/jb.182.5.1304-1312.2000 ◽

2000 ◽

Vol 182 (5) ◽

pp. 1304-1312 ◽

Cited By ~ 27

Author(s):

Angeles Zorreguieta ◽

Christine Finnie ◽

J. Allan Downie

Keyword(s):

Cell Wall ◽

Agrobacterium Tumefaciens ◽

Cell Surface ◽

Rhizobium Leguminosarum ◽

Plant Cell Wall ◽

Agar Medium ◽

Wild Type ◽

Close Proximity ◽

Mutant Strains ◽

Colony Surface

ABSTRACT Rhizobium leguminosarum secretes two extracellular glycanases, PlyA and PlyB, that can degrade exopolysaccharide (EPS) and carboxymethyl cellulose (CMC), which is used as a model substrate of plant cell wall cellulose polymers. When grown on agar medium, CMC degradation occurred only directly below colonies of R. leguminosarum, suggesting that the enzymes remain attached to the bacteria. Unexpectedly, when a PlyA-PlyB-secreting colony was grown in close proximity to mutants unable to produce or secrete PlyA and PlyB, CMC degradation occurred below that part of the mutant colonies closest to the wild type. There was no CMC degradation in the region between the colonies. By growing PlyB-secreting colonies on a lawn of CMC-nondegrading mutants, we could observe a halo of CMC degradation around the colony. Using various mutant strains, we demonstrate that PlyB diffuses beyond the edge of the colony but does not degrade CMC unless it is in contact with the appropriate colony surface. PlyA appears to remain attached to the cells since no such diffusion of PlyA activity was observed. EPS defective mutants could secrete both PlyA and PlyB, but these enzymes were inactive unless they came into contact with an EPS+ strain, indicating that EPS is required for activation of PlyA and PlyB. However, we were unable to activate CMC degradation with a crude EPS fraction, indicating that activation of CMC degradation may require an intermediate in EPS biosynthesis. Transfer of PlyB to Agrobacterium tumefaciens enabled it to degrade CMC, but this was only observed if it was grown on a lawn ofR. leguminosarum. This indicates that the surface ofA. tumefaciens is inappropriate to activate CMC degradation by PlyB. Analysis of CMC degradation by other rhizobia suggests that activation of secreted glycanases by surface components may occur in other species.

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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-11-17-0265-r ◽

2018 ◽

Vol 31 (5) ◽

pp. 568-575 ◽

Cited By ~ 5

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

Canadian Journal of Botany ◽

10.1139/b89-313 ◽

1989 ◽

Vol 67 (8) ◽

pp. 2435-2443 ◽

Cited By ~ 3

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.

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The Role of Glycoside Hydrolases in Phytopathogenic Fungi and Oomycetes Virulence

International Journal of Molecular Sciences ◽

10.3390/ijms22179359 ◽

2021 ◽

Vol 22 (17) ◽

pp. 9359

Author(s):

Vahideh Rafiei ◽

Heriberto Vélëz ◽

Georgios Tzelepis

Keyword(s):

Cell Wall ◽

Plant Cell Wall ◽

Hydrolytic Enzymes ◽

Fungal Species ◽

Phytopathogenic Fungi ◽

Glycoside Hydrolases ◽

Cell Wall Degrading Enzymes ◽

Plant Host ◽

Successful Infection ◽

Molecular Patterns

Phytopathogenic fungi need to secrete different hydrolytic enzymes to break down complex polysaccharides in the plant cell wall in order to enter the host and develop the disease. Fungi produce various types of cell wall degrading enzymes (CWDEs) during infection. Most of the characterized CWDEs belong to glycoside hydrolases (GHs). These enzymes hydrolyze glycosidic bonds and have been identified in many fungal species sequenced to date. Many studies have shown that CWDEs belong to several GH families and play significant roles in the invasion and pathogenicity of fungi and oomycetes during infection on the plant host, but their mode of function in virulence is not yet fully understood. Moreover, some of the CWDEs that belong to different GH families act as pathogen-associated molecular patterns (PAMPs), which trigger plant immune responses. In this review, we summarize the most important GHs that have been described in eukaryotic phytopathogens and are involved in the establishment of a successful infection.

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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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