scholarly journals The study of pathogenicity genes in ascomycete fungus Verticillium dahliae

2012 ◽  
Author(s):  
Lugard Eboigbe
Keyword(s):  
Cell Wall ◽  
Verticillium Dahliae ◽  
Plant Cell Wall ◽  
Genomic Library ◽  
Wild Type ◽  
Tomato Plants ◽  
Knock Out ◽  
Race 2

When Verticillium dahliae enters the host plant, the first line of defense that it encounters is the cell wall. Plant pathogenic fungi (including V. dahliae) produce extracellular enzymes which degrade plant cell wall components in a coordinated action. Some of the genes that encode these cell wall degrading enzymes, i.e., xylanases, cellulases and non-specific-action genes, were the major focus of this Ph.D. programme. Gene inactivation bears a remarkable model for the determination of the functions of genes in any organism. In this research, this molecular genetic tool has been applied to examine the functional role of β-1,6-endoglucanase and β-1,4-endoxylanase genes in the pathogenicity of filamentous fungus Verticillium dahliae. The gene coding for β-1,6-endoglucanase is considered as one of the important genes that code for hydrolyzing enzymes released at the initial stages of infection by fungi for the depolymerization of the cell wall. To address the hypothesis that these enzymes are important in V. dahliae virulence, a gene encoding a β-1,6-endoglucanase (vdg6) was isolated from V. dahliae using genome walking technique. Nucleotide sequence analysis of the 3’ and 5’ ends of clones from a genomic library of the fungus showed the presence of part of the endo-1,6-β-glucanase gene in a 3.5kb genomic fragment. Using this clone as probe and by employing genome walking approaches the 3’ and 5’; of the gene were determined bringing the entire gene (vdg6) size to ~1800 bp. An internal fragment (1.2kb) of vdg6 was used to disrupt the wild-type gene of the tomato race 2 V. dahliae strain 123V and the knock-out mutant (VdB) strain was tested for pathogenicity on tomato plants. The result showed a 7.5% reduction in disease symptoms caused on tomato plants in comparison with the wild type. Growth on minimal medium supplemented with different carbon sources showed reduced ability of the mutant to breakdown cellulose, whereas growth on glucose, pectin and sucrose were similar to the wild type. Endo-β-1,4-xylanase catalyze the endohydrolysis of xylan, the major structural polysaccharide of the plant cell wall. In order to investigate the role of the β-1,4-endoxylanase gene(xylA) in virulence of V. dahliae, through the analysis of clones from a genomic library of V. dahliae strain 76 and shotgun ESTs from xylem sap growing fungus the xylA gene was isolated. Its nucleotide sequence was determined and the predicted amino acid sequence showed significant homology with family 11 xylanases. The gene was disrupted by targeted inactivation due to a single cross-over event in a V. dahliae race 2 tomato strain. The knock-out mutant (XA) was compared with the wild type strain for disease symptoms on tomato plants. The result showed a small (7%) reduction in disease severity in the mutant strain. Growth of the mutant strain on minimal medium containing cellulose as the sole carbon source was reduced compared to the wild type indicating for a role of xylA in the breakdown of complex components of the cell wall. Other cell wall degrading genes cloned were β-1,3-exoglucanase, β-1,4-endoglucanse and endoglucanase II. The major obstacle to the determination of the genes involved in the depolymerisation of cell wall and pathogenicity is function redundancy. In an attempt to overcome the hurdle created by this function redundancy in analyzing the functions of the above genes, first, the regulation of vdg6 by sucrose non-fermenting gene (VdSNF1) was checked. The results showed that vdg6 gene is under catabolite repression, it is expressed during pathogencity and is important for the virulence of V. dahliae. Secondly, double disruption mutants were constructed from the single VdB mutant and the signalling PKA (cAMP-mediated protein kinase A) gene, namely VP1, VP2, VP3 and VP4. Analysis of these double mutants showed an obvious link between vdg6 gene and cAMP-mediated PKA (VdPKAC1) and that the β-1,6-endoglucanase cell wall degrading gene contributes to the pathogenicity of the fungus. In conclusion, all experimental evidence from this study showed that cell wall degrading genes contribute to virulence and pathogenicity of the fungus, however, since most of them belong to families of genes with similar functions the system is very complex to unravel and fully understand the genetic basis of pathogenicity.

scholarly journals Pyramiding Unmarked Deletions in Ralstonia solanacearum Shows That Secreted Proteins in Addition to Plant Cell-Wall-Degrading Enzymes Contribute to Virulence

2005 ◽  
Vol 18 (12) ◽  
pp. 1296-1305 ◽  
Author(s):  
Huanli Liu ◽  
Shuping Zhang ◽  
Mark A. Schell ◽  
Timothy P. Denny
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Ralstonia Solanacearum ◽  
Plant Cell Wall ◽  
Secreted Proteins ◽  
Cellulolytic Enzymes ◽  
Wild Type ◽  
Cell Wall Degrading Enzymes ◽  
Tomato Plants ◽  
Degrading Enzymes

Ralstonia solanacearum, like many phytopathogenic bacteria, makes multiple extracellular plant cell-wall-degrading enzymes (CWDE), some of which contribute to its ability to cause wilt disease. CWDE and many other proteins are secreted to the milieu via the highly conserved type II protein secretion system (T2SS). R. solanacearum with a defective T2SS is weakly virulent, but it is not known whether this is due to absence of all the CWDE or the loss of other secreted proteins that contribute to disease. These alternatives were investigated by creating mutants of wild-type strain GMI1000 lacking either the T2SS or up to six CWDE and comparing them for virulence on tomato plants. To create unmarked deletions, genomic regions flanking the target gene were polymerase chain reaction (PCR)-amplified, were fused using splice overlap extension PCR, were cloned into a suicide plasmid harboring the sacB counter-selectable marker, and then, were site-specifically introduced into the genome. Various combinations of five deletions (δpehA, δpehB, δpehC, δpme, and δegl) and one inactivated allele (cbhA::aphA-3) resulted in 15 mutants missing one to six CWDE. In soil-drench inoculation assays, virulence of mutants lacking only pectic enzymes (PehA, PehB, PehC, and Pme) was not statistically different from GMI1000, but all the mutants lacking one or both cellulolytic enzymes (Egl or CbhA) wilted plants significantly more slowly than did the wild type. The GMI-6 mutant that lacks all six CWDE was more virulent than the mutant lacking only its two cellulolytic enzymes, and both were significantly more virulent than the T2SS mutant (GMI-D). Very similar results were observed in wounded-petiole inoculation assays, so GMI-6 and GMI-D appear to be less capable of colonizing tomato tissues after invasion. Because the T2SS mutant was much less virulent than the sixfold CWDE mutant, we conclude that other secreted proteins contribute substantially to the ability of R. solanacearum GMI1000 to systemically colonize tomato plants.

scholarly journals Ralstonia solanacearum Pectin Methylesterase Is Required for Growth on Methylated Pectin but Not for Bacterial Wilt Virulence

1998 ◽  
Vol 64 (12) ◽  
pp. 4918-4923 ◽  
Author(s):  
Julie Tans-Kersten ◽  
Yanfen Guan ◽  
Caitilyn Allen
Keyword(s):  
Cell Wall ◽  
Bacterial Wilt ◽  
Plant Cell Wall ◽  
Genomic Library ◽  
Response Regulator ◽  
Disease Process ◽  
Pectin Methylesterase ◽  
Wild Type ◽  
Cell Wall Degrading Enzymes

ABSTRACT Ralstonia (Pseudomonas)solanacearum causes bacterial wilt, a serious disease of many crop plants. The pathogen produces several extracellular plant cell wall-degrading enzymes, including polygalacturonases (PGs) and pectin methylesterase (Pme). Pme removes methyl groups from pectin, thereby facilitating subsequent breakdown of this cell wall component by PGs, which are known bacterial wilt virulence factors. R. solanacearum PGs could not degrade 93% methylated pectin unless the substrate was first demethylated by Pme, but as the degree of methylation of the pectin substrate decreased, PG activity increased. Primers derived from a published pme sequence generated an 800-bp DNA probe fragment, which identified Pme-encoding plasmids from a R. solanacearum genomic library. A pmechromosomal mutant had no detectable Pme activity in vitro and no longer grew on 93% methylated pectin as a carbon source. Curiously, the pme mutant, which had no detectable PG activity on highly methylated pectin, was just as virulent as the wild-type strain on tomato, eggplant (aubergine), and tobacco. Since PG activity is required for full virulence, this result suggests that the pectin in these particular hosts may not be highly methylated, or that the breakdown of highly methylated pectin is not a significant factor in the disease process in general. A positive response regulator of PG production called PehR was not required for wild-type Pme production. However, a mutant strain lacking PhcA, which is a global regulator of several virulence genes, produced no detectable Pme activity. Thus,pme expression is directly or indirectly regulated by PhcA but not by PehR.

scholarly journals Arabidopsis Response Regulator 6 (ARR6) Modulates Plant Cell-Wall Composition and Disease Resistance

2020 ◽  
Vol 33 (5) ◽  
pp. 767-780 ◽  
Author(s):  
Laura Bacete ◽  
Hugo Mélida ◽  
Gemma López ◽  
Patrick Dabos ◽  
Dominique Tremousaygue ◽  
...  
Keyword(s):  
Cell Wall ◽  
Disease Resistance ◽  
Immune Responses ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Response Regulator ◽  
Cell Wall Composition ◽  
Wild Type ◽  
Molecular Patterns

The cytokinin signaling pathway, which is mediated by Arabidopsis response regulator (ARR) proteins, has been involved in the modulation of some disease-resistance responses. Here, we describe novel functions of ARR6 in the control of plant disease-resistance and cell-wall composition. Plants impaired in ARR6 function (arr6) were more resistant and susceptible, respectively, to the necrotrophic fungus Plectosphaerella cucumerina and to the vascular bacterium Ralstonia solanacearum, whereas Arabidopsis plants that overexpress ARR6 showed the opposite phenotypes, which further support a role of ARR6 in the modulation of disease-resistance responses against these pathogens. Transcriptomics and metabolomics analyses revealed that, in arr6 plants, canonical disease-resistance pathways, like those activated by defensive phytohormones, were not altered, whereas immune responses triggered by microbe-associated molecular patterns were slightly enhanced. Cell-wall composition of arr6 plants was found to be severely altered compared with that of wild-type plants. Remarkably, pectin-enriched cell-wall fractions extracted from arr6 walls triggered more intense immune responses than those activated by similar wall fractions from wild-type plants, suggesting that arr6 pectin fraction is enriched in wall-related damage-associated molecular patterns, which trigger immune responses. This work supports a novel function of ARR6 in the control of cell-wall composition and disease resistance and reinforces the role of the plant cell wall in the modulation of specific immune responses.

scholarly journals Synergistic Effect of Different Plant Cell Wall–Degrading Enzymes Is Important for Virulence of Fusarium graminearum

2017 ◽  
Vol 30 (11) ◽  
pp. 886-895 ◽  
Author(s):  
Maria Chiara Paccanaro ◽  
Luca Sella ◽  
Carla Castiglioni ◽  
Francesca Giacomello ◽  
Ana Lilia Martínez-Rocha ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fusarium Graminearum ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Xylanase Activity ◽  
Cellulase Activity ◽  
Wild Type ◽  
Cell Wall Degrading Enzymes ◽  
Xylanase Production ◽  
Significant Difference

Endo-polygalacturonases (PGs) and xylanases have been shown to play an important role during pathogenesis of some fungal pathogens of dicot plants, while their role in monocot pathogens is less defined. Pg1 and xyr1 genes of the wheat pathogen Fusarium graminearum encode the main PG and the major regulator of xylanase production, respectively. Single- and double-disrupted mutants for these genes were obtained to assess their contribution to fungal infection. Compared with wild-type strain, the ∆pg mutant showed a nearly abolished PG activity, slight reduced virulence on soybean seedlings, but no significant difference in disease symptoms on wheat spikes; the ∆xyr mutant was strongly reduced in xylanase activity and moderately reduced in cellulase activity but was as virulent as wild type on both soybean and wheat plants. Consequently, the ΔpgΔxyr double mutant was impaired in xylanase, PG, and cellulase activities but, differently from single mutants, was significantly reduced in virulence on both plants. These findings demonstrate that the concurrent presence of PG, xylanase, and cellulase activities is necessary for full virulence. The observation that the uronides released from wheat cell wall after a F. graminearum PG treatment were largely increased by the fungal xylanases suggests that these enzymes act synergistically in deconstructing the plant cell wall.

scholarly journals Structural characteristics of plant cell wall elucidated by solution-state 2D NMR spectroscopy with an optimized procedure

2020 ◽  
Vol 9 (1) ◽  
pp. 650-663
Author(s):  
Wanwan Wang ◽  
Jibao Cai ◽  
Zhenyu Xu ◽  
Yi Zhang ◽  
Fanchao Niu ◽  
...  
Keyword(s):  
Cell Wall ◽  
Quantum Coherence ◽  
Nmr Spectra ◽  
Plant Cell Wall ◽  
Milling Time ◽  
Structural Characteristics ◽  
2D Nmr ◽  
2D Nmr Spectroscopy ◽  
Complete Representation

AbstractA method was developed for rapid qualitative determination of lignocellulose in the tobacco cell wall by utilizing 2D heteronuclear single quantum coherence NMR spectra (2D HSQC NMR). Traditional methods for analyzing the structure of lignocellulose involve many steps of separation and extraction, which is labor-intensive. In this work, the whole cell wall was milled and dissolved in deuterium solvent. The solvent dimethylsulfoxide (DMSO-d6) containing hexamethylphosphoramide (HMPA-d18) enhanced swelling of the sample and gave high-resolution spectra. The tobacco samples are ball milled at different ball milling times, and the state of the particles is observed through an electron microscope, and then the probability of the particles being less than 5 µm is counted. Through the comparison of the abundance and integration of the peak signals in the spectra under different transmittances, it was determined that when the milling time was 6 h, the quality of the NMR spectra was the best. The optimum conditions of characterizing tobacco structure were DMSO-d6/HMPA-d18 solution and 6 h milling time. Under these conditions, complete representation of the structure of lignocellulose and simplified process could be achieved.

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

2000 ◽  
Vol 182 (5) ◽  
pp. 1304-1312 ◽  
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.

TAMI-37. STEAROYL-COA DESATURASE 1 IS ESSENTIAL FOR THE GROWTH OF IDH MUTANT GLIOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi206-vi206
Author(s):  
Tomohiro Yamasaki ◽  
Lumin Zhang ◽  
Tyrone Dowdy ◽  
Adrian Lita ◽  
Mark Gilbert ◽  
...  
Keyword(s):  
Glioma Cell ◽  
De Novo ◽  
Glioma Cells ◽  
Model Systems ◽  
De Novo Lipogenesis ◽  
Wild Type ◽  
Knock Out ◽  
Stearoyl Coa Desaturase ◽  
Scd1 Expression

Abstract BACKGROUND Increased de novo lipogenesis is a hallmark of cancer metabolism. In this study, we interrogated the role of de novo lipogenesis in IDH1 mutated glioma’s growth and identified the key enzyme, Stearoyl-CoA desaturase 1 (SCD1) that provides this growth advantage. MATERIALS ANDMETHODS We prepared genetically engineered glioma cell lines (U251 wild-type: U251WT and U251 IDHR132H mutant: U251RH) and normal human astrocytes (empty vector induced-NHA: NHAEV and IDHR132H mutant: NHARH). Lipid metabolic analysis was conducted by using LC-MS and Raman imaging microscopy. SCD1 expression was investigated by The Cancer Genome Atlas (TCGA) data analysis and Western-blotting method. Knock-out of SCD1 was conducted by using CRISPR/Cas9 and shRNA. RESULTS Previously, we showed that IDH1 mut glioma cells have increased monounsaturated fatty acids (MUFAs). TCGA data revealed IDH mut glioma shows significantly higher SCD1 mRNA expression than wild-type glioma. Our model systems of IDH1 mut (U251RH, NHARH) showed increased expression of this enzyme compared with their wild-type counterpart. Moreover, addition of D-2HG to U251WT increased SCD1 expression. Herein, we showed that inhibition of SCD1 with CAY10566 decreased relative cell number and sphere forming capacity in a dose-dependent manner. Furthermore, addition of MUFAs were able to rescue the SCD1 inhibitor induced-cell death and sphere forming capacity. Knock out of SCD1 revealed decreased cell proliferation and sphere forming ability. Decreasing lipid content from the media did not alter the growth of these cells, suggesting that glioma cells rely on de novo lipid synthesis rather than scavenging them from the microenvironment. CONCLUSION Overexpression of IDH mutant gene altered lipid composition in U251 cells to enrich MUFA levels and we confirmed that D-2HG caused SCD1 upregulation in U251WT. We demonstrated the glioma cell growth requires SCD1 expression and the results of the present study may provide novel insights into the role of SCD1 in IDH mut gliomas growth.

scholarly journals Hypolignification: A Decisive Factor in the Development of Hyperhydricity

Plants ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 2625
Author(s):  
Nurashikin Kemat ◽  
Richard G. F. Visser ◽  
Frans A. Krens
Keyword(s):  
Cell Wall ◽  
Lignin Content ◽  
Coumaric Acid ◽  
Wild Type ◽  
Higher Plant ◽  
Gene Coding ◽  
Phenylpropanoid Biosynthesis ◽  
Total Lignin ◽  
Total Lignin Content

One of the characteristics of hyperhydric plants is the reduction of cell wall lignification (hypolignification), but how this is related to the observed abnormalities of hyperhydricity (HH), is still unclear. Lignin is hydrophobic, and we speculate that a reduction in lignin levels leads to more capillary action of the cell wall and consequently to more water in the apoplast. p-coumaric acid is the hydroxyl derivative of cinnamic acid and a precursor for lignin and flavonoids in higher plant. In the present study, we examined the role of lignin in the development of HH in Arabidopsis thaliana by checking the wild-types (Ler and Col-0) and mutants affected in phenylpropanoid biosynthesis, in the gene coding for cinnamate 4-hydroxylase, C4H (ref3-1 and ref3-3). Exogenously applied p-coumaric acid decreased the symptoms of HH in both wild-type and less-lignin mutants. Moreover, the results revealed that exogenously applied p-coumaric acid inhibited root growth and increased the total lignin content in both wild-type and less-lignin mutants. These effects appeared to diminish the symptoms of HH and suggest an important role for lignin in HH.

scholarly journals Role of cytoskeleton in plant cell wall formation

2020 ◽  
Vol 50 (2) ◽  
pp. 176-186
Author(s):  
Yi MAN ◽  
RuiLi LI ◽  
YuFen BU ◽  
Na SUN ◽  
YanPing JING ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Cell Wall Formation ◽  
Wall Formation

scholarly journals Sheep Digestive Physiology and Constituents of Feeds

2021 ◽  
Author(s):  
Samir Medjekal ◽  
Mouloud Ghadbane
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Early Stage ◽  
Growth Period ◽  
Cell Plate ◽  
Soluble Carbohydrates ◽  
Winter Period ◽  
The Common

Sheep have a gastrointestinal tract similar to that of other ruminants. Their stomach is made up of four digestive organs: the rumen, the reticulum, the omasum and the abomasum. The rumen plays a role in storing ingested foods, which are fermented by a complex anaerobic rumen microbiota population with different types of interactions, positive or negative, that can occur between their microbial populations. Sheep feeding is largely based on the use of natural or cultivated fodder, which is exploited in green by grazing during the growth period of the grass and in the form of fodder preserved during the winter period. Ruminant foods are essentially of plant origin, and their constituents belong to two types of structures: intracellular constituents and cell wall components. Cellular carbohydrates play a role of metabolites or energy reserves; soluble carbohydrates account for less than 10% dry matter (DM) of foods. The plant cell wall is multi-layered and consists of primary wall and secondary wall. Fundamentally, the walls are deposited at an early stage of growth. A central blade forms the common boundary layer between two adjacent cells and occupies the location of the cell plate. Most of the plant cell walls consist of polysaccharides (cellulose, hemicellulose and pectic substances) and lignin, these constituents being highly polymerized, as well as proteins and tannins.

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