scholarly journals Characterisation of the enzyme transport path between shipworms and their bacterial symbionts

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Giovanna Pesante ◽  
Federico Sabbadin ◽  
Luisa Elias ◽  
Clare Steele-King ◽  
J. Reuben Shipway ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Glycosyl Hydrolases ◽  
Cell Wall Degrading Enzymes ◽  
Bacterial Enzymes ◽  
Symbiotic Interaction ◽  
Lytic Polysaccharide Monooxygenase ◽  
Biochemical Characterisation ◽  
Food Groove ◽  
Wood Digestion

Abstract Background Shipworms are marine xylophagus bivalve molluscs, which can live on a diet solely of wood due to their ability to produce plant cell wall-degrading enzymes. Bacterial carbohydrate-active enzymes (CAZymes), synthesised by endosymbionts living in specialised shipworm cells called bacteriocytes and located in the animal’s gills, play an important role in wood digestion in shipworms. However, the main site of lignocellulose digestion within these wood-boring molluscs, which contains both endogenous lignocellulolytic enzymes and prokaryotic enzymes, is the caecum, and the mechanism by which bacterial enzymes reach the distant caecum lumen has remained so far mysterious. Here, we provide a characterisation of the path through which bacterial CAZymes produced in the gills of the shipworm Lyrodus pedicellatus reach the distant caecum to contribute to the digestion of wood. Results Through a combination of transcriptomics, proteomics, X-ray microtomography, electron microscopy studies and in vitro biochemical characterisation, we show that wood-digesting enzymes produced by symbiotic bacteria are localised not only in the gills, but also in the lumen of the food groove, a stream of mucus secreted by gill cells that carries food particles trapped by filter feeding to the mouth. Bacterial CAZymes are also present in the crystalline style and in the caecum of their shipworm host, suggesting a unique pathway by which enzymes involved in a symbiotic interaction are transported to their site of action. Finally, we characterise in vitro four new bacterial glycosyl hydrolases and a lytic polysaccharide monooxygenase identified in our transcriptomic and proteomic analyses as some of the major bacterial enzymes involved in this unusual biological system. Conclusion Based on our data, we propose that bacteria and their enzymes are transported from the gills along the food groove to the shipworm’s mouth and digestive tract, where they aid in wood digestion.

scholarly journals In vitro biosynthesis of poly-β-1,4-glucan derivatives using a pro-miscuous glycosyltransferase

2020 ◽  
Author(s):  
Gregory S. Bulmer ◽  
Ashley P. Mattey ◽  
Fabio Parmeggiani ◽  
Ryan Williams ◽  
Helene Ledru ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Chemical Probes ◽  
Cell Wall Degrading Enzymes ◽  
Lytic Polysaccharide Monooxygenases ◽  
Natural Cellulose ◽  
Polysaccharide Monooxygenases ◽  
In Vitro Biosynthesis ◽  
New Applications ◽  
Biosynthetic Machinery

AbstractThe β-1,4-glucose linkage of cellulose is the most abundant polymeric linkage on earth and as such is of considerable interest in biology and biotechnology. It remains challenging to synthesize this linkage in vitro due to a lack of suitable biocatalysts; the natural cellulose biosynthetic machinery is a membrane-associated complex with processive activity that cannot be easily manipulated to synthesize tailor-made oligosaccharides and their derivatives. Here we identify a promiscuous activity of a soluble recombinant biocatalyst, Neisseria meningitidis glycosyltransferase LgtB, suitable for the polymerization of glucose from UDP-glucose via the generation of β-1,4-glycosidic linkages. We employed LgtB to synthesize natural and derivatized cello-oligosaccharides and we demonstrate how LgtB can be incorporated in biocatalytic cascades and chemo-enzymatic strategies to synthesize cello-oligosaccharides with tailored functionalities. We also show how the resulting glycan structures can be applied as chemical probes to report on activity and selectivity of plant cell wall degrading enzymes, including lytic polysaccharide monooxygenases. We anticipate that this biocatalytic approach to derivatized cello-oligosaccharides via glucose polymerization will open up new applications in biology and nanobiotechnology.

scholarly journals The infection cushion: a fungal “weapon” of plant-biomass destruction

2020 ◽  
Author(s):  
Mathias Choquer ◽  
Christine Rascle ◽  
Isabelle R Gonçalves ◽  
Amélie de Vallée ◽  
Cécile Ribot ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Ornamental Plants ◽  
Sugar Transport ◽  
Differential Staining ◽  
List Type ◽  
In Planta ◽  
Cell Wall Degrading Enzymes

SummaryGrey mold disease affects fruits, vegetables and ornamental plants around the world, causing considerable losses every year. Its causing agent, the necrotrophic fungus Botrytis cinerea, produces infection cushions (IC) that are compound appressorial structures dedicated to the penetration of the plant tissues.A microarray analysis was performed to identify genes up-regulated in mature IC. The expression data were supported by RT-qPCR analysis performed in vitro and in planta, proteomic analysis of the IC secretome and mutagenesis of two candidate genes.1,231 up-regulated genes and 79 up-accumulated proteins were identified. They highlight a secretion of ROS, secondary metabolites including phytotoxins, and proteins involved in virulence: proteases, plant cell wall degrading enzymes and necrosis inducers. The role in pathogenesis was confirmed for two up-regulated fasciclin genes. DHN-melanin pathway and chitin deacetylases genes are up-regulated and the conversion of chitin into chitosan was confirmed by differential staining of the IC cell wall. In addition, up-regulation of sugar transport and sugar catabolism encoding genes was found.These results support a role for the B. cinerea IC in plant penetration and suggest other unexpected roles for this fungal organ, in camouflage, necrotrophy or nutrition of the pathogen.

scholarly journals Structure and activity of ChiX: a peptidoglycan hydrolase required for chitinase secretion by Serratia marcescens

2018 ◽  
Vol 475 (2) ◽  
pp. 415-428 ◽  
Author(s):  
Richard A. Owen ◽  
Paul K. Fyfe ◽  
Adam Lodge ◽  
Jacob Biboy ◽  
Waldemar Vollmer ◽  
...  
Keyword(s):  
Serratia Marcescens ◽  
Mutant Strain ◽  
In Vitro Assays ◽  
Lytic Polysaccharide Monooxygenase ◽  
Biochemical Characterisation ◽  
Cross Links ◽  
Bacterial Peptidoglycan ◽  
Variant Protein ◽  
Structure And Activity

The Gram-negative bacterium Serratia marcescens secretes many proteins that are involved in extracellular chitin degradation. This so-called chitinolytic machinery includes three types of chitinase enzymes and a lytic polysaccharide monooxygenase. An operon has been identified in S. marcescens, chiWXYZ, that is thought to be involved in the secretion of the chitinolytic machinery. Genetic evidence points to the ChiX protein being a key player in the secretion mechanism, since deletion of the chiX gene in S. marcescens led to a mutant strain blocked for secretion of all members of the chitinolytic machinery. In this work, a detailed structural and biochemical characterisation of ChiX is presented. The high-resolution crystal structure of ChiX reveals the protein to be a member of the LAS family of peptidases. ChiX is shown to be a zinc-containing metalloenzyme, and in vitro assays demonstrate that ChiX is an l-Ala d-Glu endopeptidase that cleaves the cross-links in bacterial peptidoglycan. This catalytic activity is shown to be intimately linked with the secretion of the chitinolytic machinery, since substitution of the ChiX Asp-120 residue results in a variant protein that is both unable to digest peptidoglycan and cannot rescue the phenoytype of a chiX mutant strain.

scholarly journals The dual function receptor kinase, OsWAKL21.2, is involved in elaboration of lipaseA/esterase induced immune responses in rice

2019 ◽  
Author(s):  
Kamal Kumar Malukani ◽  
Ashish Ranjan ◽  
Hota Shiva Jyothi ◽  
Hitendra Kumar Patel ◽  
Ramesh V. Sonti
Keyword(s):  
Cell Wall ◽  
Immune Responses ◽  
Plant Pathogens ◽  
Plant Cell Wall ◽  
Ectopic Expression ◽  
Dual Function ◽  
Receptor Kinase ◽  
Cell Wall Degrading Enzymes ◽  
Cell Wall Damage

AbstractPlant pathogens secrete cell wall degrading enzymes (CWDEs) to degrade various components of the plant cell wall. Plants sense this cell wall damage as a mark of infection and induce immune responses. Little is known about the plant functions that are involved in the elaboration of cell wall damage-induced immune responses. Transcriptome analysis revealed that a rice receptor kinase, WALL-ASSOCIATED KINASE-LIKE 21 (OsWAKL21.2), is upregulated following treatment with either Xanthomonas oryzae pv. oryzae (Xoo, a bacterial pathogen) or lipaseA/esterase (LipA: a CWDE of Xoo). Downregulation of OsWAKL21.2 attenuates LipA mediated immune responses. Overexpression of OsWAKL21.2 in rice mimics LipA treatment mediated induction of immune responses and enhanced expression of defence related genes, indicating it could be involved in the perception of LipA induced cell wall damage in rice. OsWAKL21.2 is a dual function kinase having in-vitro kinase and guanylate cyclase (GC) activities. Ectopic expression of OsWAKL21.2 in Arabidopsis also activates plant immune responses. Interestingly, OsWAKL21.2 needs kinase activity to activate rice immune responses while in Arabidopsis it needs GC activity. Our study reveals a novel receptor kinase involved in elaboration of cell wall damage induced rice immune responses that can activate similar immune responses in two different species via two different mechanisms.One sentence SummaryA novel rice receptor WAKL21 that sense cell wall damage caused by Xanthomonas secreted cell wall degrading enzyme to induce immune responses.

scholarly journals The Endo-Arabinanase BcAra1 Is a Novel Host-Specific Virulence Factor of the Necrotic Fungal Phytopathogen Botrytis cinerea

2014 ◽  
Vol 27 (8) ◽  
pp. 781-792 ◽  
Author(s):  
Majse Nafisi ◽  
Maria Stranne ◽  
Lisha Zhang ◽  
Jan A. L. van Kan ◽  
Yumiko Sakuragi
Keyword(s):  
Cell Wall ◽  
Botrytis Cinerea ◽  
Plant Cell ◽  
Plant Pathogens ◽  
Plant Cell Wall ◽  
High Efficiency ◽  
Microbial Infection ◽  
Cell Wall Degrading Enzymes ◽  
Novel Host

The plant cell wall is one of the first physical interfaces encountered by plant pathogens and consists of polysaccharides, of which arabinan is an important constituent. During infection, the necrotrophic plant pathogen Botrytis cinerea secretes a cocktail of plant cell-wall-degrading enzymes, including endo-arabinanase activity, which carries out the breakdown of arabinan. The roles of arabinan and endo-arabinanases during microbial infection were thus far elusive. In this study, the gene Bcara1 encoding for a novel α-1,5-L-endo-arabinanase was identified and the heterologously expressed BcAra1 protein was shown to hydrolyze linear arabinan with high efficiency whereas little or no activity was observed against the other oligo- and polysaccharides tested. The Bcara1 knockout mutants displayed reduced arabinanase activity in vitro and severe retardation in secondary lesion formation during infection of Arabidopsis leaves. These results indicate that BcAra1 is a novel endo-arabinanase and plays an important role during the infection of Arabidopsis. Interestingly, the level of Bcara1 transcript was considerably lower during the infection of Nicotiana benthamiana compared with Arabidopsis and, consequently, the ΔBcara1 mutants showed the wild-type level of virulence on N. benthamiana leaves. These results support the conclusion that the expression of Bcara1 is host dependent and is a key determinant of the disease outcome.

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 Loss of bcbrn1 and bcpks13 in Botrytis cinerea Not Only Blocks Melanization But Also Increases Vegetative Growth and Virulence

2015 ◽  
Vol 28 (10) ◽  
pp. 1091-1101 ◽  
Author(s):  
Chenghua Zhang ◽  
Yifan He ◽  
Pinkuan Zhu ◽  
Lu Chen ◽  
Yiwen Wang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Botrytis Cinerea ◽  
Plant Cell Wall ◽  
Hydrolytic Enzyme ◽  
Complete Medium ◽  
Cell Wall Degrading Enzymes ◽  
Mycelium Growth ◽  
Necrotrophic Pathogen ◽  
Melanin Biosynthesis

Botrytis cinerea is a necrotrophic pathogen that causes gray mold disease in a broad range of plants. Dihydroxynaphthalene (DHN) melanin is a major component of the extracellular matrix of B. cinerea, but knowledge of the exact role of melanin biosynthesis in this pathogen is unclear. In this study, we characterize two genes in B. cinerea, bcpks13 and bcbrn1, encoding polyketide synthase and tetrahydroxynaphthalene (THN) reductases, respectively, and both have predicted roles in DHN melanin biosynthesis. The ∆bcpks13 and ∆bcbrn1 mutants show white and orange pigmentation, respectively, and the mutants are also deficient in conidiation in vitro but show enhanced growth rates and virulence on hosts. Moreover, the mutants display elevated acidification of the complete medium (CM), probably due to oxalic acid secretion and secretion of cell wall–degrading enzymes, and preferably utilize plant cell-wall components as carbon sources for mycelium growth in vitro. In contrast, overexpression of bcbrn1 (OE::bcbrn1 strain) results in attenuated hydrolytic enzyme secretion, acidification ability, and virulence. Taken together, these results indicate that bcpks13 and bcbrn1 participate in diverse cellular and developmental processes, such as melanization and conidiation in B. cinerea in vitro, but they negatively regulate the virulence of this pathogen.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

EFFECT OF STRUCTURAL AND CHEMICAL FACTORS OF FORAGES ON POTENTIALLY DIGESTIBLE FIBER, INTAKE, AND TRUE DIGESTIBILITY BY RUMINANTS

1988 ◽  
Vol 68 (3) ◽  
pp. 787-799 ◽  
Author(s):  
V. GIRARD ◽  
G. DUPUIS
Keyword(s):  
Cell Wall ◽  
Dry Matter ◽  
Plant Cell Wall ◽  
Neutral Detergent Fiber ◽  
Optimum Number ◽  
Apparent Digestibility ◽  
Cool Season ◽  
Dry Matter Digestibility ◽  
True Digestibility

In view of the large variation found in plant cell wall digestibilities with ruminants, an attempt was made to group 124 feeds into different lignification classes (clusters) on the basis of chemical characteristics. Each feed cluster was described using a structural coefficient [Formula: see text] that related the potentially digestible fiber (PDF, %) to the ratio between lignin and cell wall volume. The optimum number of clusters was determined iteratively by performing a regression of the apparent digestibility of dry matter at maintenance level (DDM1, %) against the PDF and cell soluble (SOL, %) contents of feeds. The [Formula: see text] coefficients varied from 0.05 (grains, N = 13) to 1.85 (corn silage, N = 3) and increased with the maturity of the grasses from 0.88 (legumes, vegetative cool season grasses, N = 26) to 1.33 (mature, cool season grasses, N = 19). Predicted PDF were closely correlated (r > 0.9, P < 0.01) to in vitro cell wall disappearances (IVCWD). Apparently digestible cell wall in four grasses and four legumes increased linearly with 96-h IVCWD and standard error (SE) was similar to the SE of predicted apparent digestible SOL from SOL concentrations. Assuming that similarity between SE could be also observed in larger samples, PDF and SOL were used in summative equations to predict apparent dry matter digestibility. DDM1 discounted for intake (DDM1 – 4, %) was regressed against SOL and PDF concentrations of 87 feeds:[Formula: see text]with ds and df, the true digestibilities of SOL and PDF. Estimates of ds and df were 0.98 and 0.95 for a zero-production (maintenance) level of intake, and 0.91 and 0.79 for an intake level four times maintenance. Since the true digestibility of the PDF component was only 4% – 13% lower than that of the cell soluble component, the concentration of PDF in cell wall was the major determinant in the variation in apparent digestibility of forages. Key words: lignin, neutral detergent fiber, true digestibility, cluster analysis, feeds

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