scholarly journals LPMO-oxidized cellulose oligosaccharides evoke immunity in Arabidopsis conferring resistance towards necrotrophic fungus B. cinerea

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Marco Zarattini ◽  
Massimiliano Corso ◽  
Marco Antonio Kadowaki ◽  
Antonielle Monclaro ◽  
Silvia Magri ◽  
...  
Keyword(s):  
Cell Wall ◽  
Crucial Role ◽  
Pairwise Comparison ◽  
Leucine Rich Repeat ◽  
Redox Enzymes ◽  
Transcriptional Reprogramming ◽  
Specific Effects ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Plant Pathogen Interactions

AbstractLytic Polysaccharide Monooxygenases (LPMOs) are powerful redox enzymes able to oxidatively cleave recalcitrant polysaccharides. Widely conserved across biological kingdoms, LPMOs of the AA9 family are deployed by phytopathogens to deconstruct cellulose polymers. In response, plants have evolved sophisticated mechanisms to sense cell wall damage and thus self-triggering Damage Triggered Immunity responses. Here, we show that Arabidopsis plants exposed to LPMO products triggered the innate immunity ultimately leading to increased resistance to the necrotrophic fungus Botrytis cinerea. We demonstrated that plants undergo a deep transcriptional reprogramming upon elicitation with AA9 derived cellulose- or cello-oligosaccharides (AA9_COS). To decipher the specific effects of native and oxidized LPMO-generated AA9_COS, a pairwise comparison with cellobiose, the smallest non-oxidized unit constituting cellulose, is presented. Moreover, we identified two leucine-rich repeat receptor-like kinases, namely STRESS INDUCED FACTOR 2 and 4, playing a crucial role in signaling the AA9_COS-dependent responses such as camalexin production. Furthermore, increased levels of ethylene, jasmonic and salicylic acid hormones, along with deposition of callose in the cell wall was observed. Collectively, our data reveal that LPMOs might play a crucial role in plant-pathogen interactions.

scholarly journals LPMO-oxidized cellulose oligosaccharides evoke immunity in Arabidopsis conferring resistance towards necrotrophic fungus B. cinerea

2021 ◽  
Author(s):  
Marco Zarattini ◽  
Massimiliano Corso ◽  
Marco Antonio Kadowaki ◽  
Antonielle Monclaro ◽  
Silvia Magri ◽  
...  
Keyword(s):  
Cell Wall ◽  
Crucial Role ◽  
Plant Cell Wall ◽  
Pathogenic Fungus ◽  
Pairwise Comparison ◽  
Transcriptional Reprogramming ◽  
Specific Effects ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Plant Pathogen Interactions

AbstractLytic Polysaccharide Monooxygenases (LPMOs) are powerful redox enzymes able to oxidatively cleave cellulose polymers. Widely conserved across biological kingdoms, LPMOs of the AA9 family are deployed by phytopathogens during necrotrophic attack of plant cell wall. In response, plants have evolved sophisticated mechanisms to sense cell wall damage and thus self-triggering Damage Triggered Immunity (DTI) responses. Here, we show that Arabidopsis plants exposed to LPMO products responds by activating the innate immunity ultimately leading to increased resistance to pathogenic fungus Botrytis cinerea. We demonstrated with microarray hybridization that plants undergo a deep transcriptional reprogramming upon elicitation with AA9 derived cellulose- or cello-oligosaccharides (AA9_COS). To decipher the specific effects of native and oxidized LPMO-generated cello-oligosaccharides, a pairwise comparison with cellobiose, the smallest non-oxidized unit constituting cellulose, is presented. Moreover, we identified two leucine-rich repeat receptor-like kinases, namely STRESS INDUCED FACTOR 2 and 4, playing a crucial role in signaling the AA9_COS-dependent responses such as camalexin production. We observed an increased production of ethylene, jasmonic and salicylic acid hormones, and finally deposition of callose in cell wall. Collectively, our data reveal that LPMOs might play a crucial role in plant-pathogen interactions.

scholarly journals Fenton-type chemistry by a copper enzyme: molecular mechanism of polysaccharide oxidative cleavage

2016 ◽  
Author(s):  
Bastien Bissaro ◽  
Asmund K Rohr ◽  
Morten Skaugen ◽  
Zarah Forsberg ◽  
Svein J Horn ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Reaction Kinetics ◽  
Mode Of Action ◽  
Process Conditions ◽  
Enzyme Assays ◽  
Enzymatic Conversion ◽  
Redox Enzymes ◽  
Anaerobic Conditions ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases

The discovery of Lytic Polysaccharide Monooxygenases (LPMOs) has been instrumental for the development of economically sustainable lignocellulose biorefineries. Despite the obvious importance of these exceptionally powerful redox enzymes, their mode of action remains enigmatic and their activity and stability under process conditions are hard to control. By using enzyme assays, mass spectrometry and experiments with labeled oxygen atoms, we show that H2O2, and not O2 as previously thought, is the co-substrate of LPMOs. By controlling H2O2 supply, stable reaction kinetics and high enzymatic rates are achieved, the LPMOs work under anaerobic conditions, and the need for adding stoichiometric amounts of reductants is alleviated. These results offer completely new perspectives regarding the mode of action of these unique mono-copper enzymes, the enzymatic conversion of biomass in Nature, and industrial biorefining.

scholarly journals Secreted pectin monooxygenases drive plant infection by pathogenic oomycetes

Science ◽  
2021 ◽  
Vol 373 (6556) ◽  
pp. 774-779
Author(s):  
Federico Sabbadin ◽  
Saioa Urresti ◽  
Bernard Henrissat ◽  
Anna O. Avrova ◽  
Lydia R. J. Welsh ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Crop Protection ◽  
Model Organism ◽  
Plant Infection ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Encoding Gene ◽  
Plant Pathogen Interactions ◽  
Encoding Genes ◽  
Secreted Enzymes

The oomycete Phytophthora infestans is a damaging crop pathogen and a model organism to study plant-pathogen interactions. We report the discovery of a family of copper-dependent lytic polysaccharide monooxygenases (LPMOs) in plant pathogenic oomycetes and its role in plant infection by P. infestans. We show that LPMO-encoding genes are up-regulated early during infection and that the secreted enzymes oxidatively cleave the backbone of pectin, a charged polysaccharide in the plant cell wall. The crystal structure of the most abundant of these LPMOs sheds light on its ability to recognize and degrade pectin, and silencing the encoding gene in P. infestans inhibits infection of potato, indicating a role in host penetration. The identification of LPMOs as virulence factors in pathogenic oomycetes opens up opportunities in crop protection and food security.

scholarly journals Specific Xylan Activity Revealed for AA9 Lytic Polysaccharide Monooxygenases of the Thermophilic Fungus Malbranchea cinnamomea by Functional Characterization

2019 ◽  
Vol 85 (23) ◽  
Author(s):  
Silvia Hüttner ◽  
Anikó Várnai ◽  
Dejan M. Petrović ◽  
Cao Xuan Bach ◽  
Dang Thi Kim Anh ◽  
...  
Keyword(s):  
Cell Wall ◽  
Plant Cell ◽  
Plant Cell Wall ◽  
Functional Characterization ◽  
Cellulose Microfibrils ◽  
Cell Wall Polysaccharide ◽  
Poor Growth ◽  
Content Type ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases

ABSTRACT The thermophilic biomass-degrader Malbranchea cinnamomea exhibits poor growth on cellulose but excellent growth on hemicelluloses as the sole carbon source. This is surprising considering that its genome encodes eight lytic polysaccharide monooxygenases (LPMOs) from auxiliary activity family 9 (AA9), enzymes known for their high potential in accelerating cellulose depolymerization. We characterized four of the eight (M. cinnamomea AA9s) McAA9s, namely, McAA9A, McAA9B, McAA9F, and McAA9H, to gain a deeper understanding about their roles in the fungus. The characterized McAA9s were active on hemicelluloses, including xylan, glucomannan, and xyloglucan, and furthermore, in accordance with transcriptomics data, differed in substrate specificity. Of the McAA9s, McAA9H is unique, as it preferentially cleaves residual xylan in phosphoric acid-swollen cellulose (PASC). Moreover, when exposed to cellulose-xylan blends, McAA9H shows a preference for xylan and for releasing (oxidized) xylooligosaccharides. The cellulose dependence of the xylan activity suggests that a flat conformation, with rigidity similar to that of cellulose microfibrils, is a prerequisite for productive interaction between xylan and the catalytic surface of the LPMO. McAA9H showed a similar trend on xyloglucan, underpinning the suggestion that LPMO activity on hemicelluloses strongly depends on the polymers’ physicochemical context and conformation. Our results support the notion that LPMO multiplicity in fungal genomes relates to the large variety of copolymeric polysaccharide arrangements occurring in the plant cell wall. IMPORTANCE The Malbranchea cinnamomea LPMOs (McAA9s) showed activity on a broad range of soluble and insoluble substrates, suggesting their involvement in various steps of biomass degradation besides cellulose decomposition. Our results indicate that the fungal AA9 family is more diverse than originally thought and able to degrade almost any kind of plant cell wall polysaccharide. The discovery of an AA9 that preferentially cleaves xylan enhances our understanding of the physiological roles of LPMOs and enables the use of xylan-specific LPMOs in future applications.

scholarly journals Exploitation of Enzymes for the Production of Biofuels: Electrochemical Determination of Kinetic Parameters of LPMOs

2021 ◽  
Vol 11 (11) ◽  
pp. 4715
Author(s):  
Dimitrios Zouraris ◽  
Anthi Karnaouri ◽  
Raphaela Xydou ◽  
Evangelos Topakas ◽  
Antonis Karantonis
Keyword(s):  
Kinetic Parameters ◽  
Filamentous Fungus ◽  
Electrode Surface ◽  
Plant Biomass ◽  
Electrochemical Determination ◽  
Redox Enzymes ◽  
Electrode Surfaces ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) consist of a class of enzymes that boost the release of oxidised products from plant biomass, in an approach that is more eco-friendly than the traditional ones, employing harsh chemicals. Since LPMOs are redox enzymes, they could possibly be exploited by immobilisation on electrode surfaces. Such an approach requires knowledge of kinetic and thermodynamic information for the interaction of the enzyme with the electrode surface. In this work, a novel methodology is applied for the determination of such parameters for an LPMO from the filamentous fungus Thermothelomyces thermophila, MtLPMO9H.

scholarly journals Novel molecular biological tools for the efficient expression of fungal lytic polysaccharide monooxygenases in Pichia pastoris

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Lukas Rieder ◽  
Katharina Ebner ◽  
Anton Glieder ◽  
Morten Sørlie
Keyword(s):  
Pichia Pastoris ◽  
Biomass Conversion ◽  
Single Step ◽  
Additional Advantage ◽  
Lytic Polysaccharide Monooxygenases ◽  
Expression Host ◽  
Shake Flask Cultivation ◽  
Polysaccharide Monooxygenases ◽  
Efficient Expression ◽  
High Yields

Abstract Background Lytic polysaccharide monooxygenases (LPMOs) are attracting large attention due their ability to degrade recalcitrant polysaccharides in biomass conversion and to perform powerful redox chemistry. Results We have established a universal Pichia pastoris platform for the expression of fungal LPMOs using state-of-the-art recombination cloning and modern molecular biological tools to achieve high yields from shake-flask cultivation and simple tag-less single-step purification. Yields are very favorable with up to 42 mg per liter medium for four different LPMOs spanning three different families. Moreover, we report for the first time of a yeast-originating signal peptide from the dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1 (OST1) form S. cerevisiae efficiently secreting and successfully processes the N-terminus of LPMOs yielding in fully functional enzymes. Conclusion The work demonstrates that the industrially most relevant expression host P. pastoris can be used to express fungal LPMOs from different families in high yields and inherent purity. The presented protocols are standardized and require little equipment with an additional advantage with short cultivation periods.

scholarly journals Oxidative Power: Tools for Assessing LPMO Activity on Cellulose

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1098
Author(s):  
Federica Calderaro ◽  
Loes E. Bevers ◽  
Marco A. van den Berg
Keyword(s):  
Experimental Design ◽  
Mode Of Action ◽  
Data Interpretation ◽  
Electron Donors ◽  
Cellulolytic Enzymes ◽  
Inhibiting Factors ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Power Tools ◽  
Reliable Methods

Lytic polysaccharide monooxygenases (LPMOs) have sparked a lot of research regarding their fascinating mode-of-action. Particularly, their boosting effect on top of the well-known cellulolytic enzymes in lignocellulosic hydrolysis makes them industrially relevant targets. As more characteristics of LPMO and its key role have been elucidated, the need for fast and reliable methods to assess its activity have become clear. Several aspects such as its co-substrates, electron donors, inhibiting factors, and the inhomogeneity of lignocellulose had to be considered during experimental design and data interpretation, as they can impact and often hamper outcomes. This review provides an overview of the currently available methods to measure LPMO activity, including their potential and limitations, and it is illustrated with practical examples.

Actin has multiple roles in the formation and architecture of zoospores of the oomycetes, Saprolegnia ferax and Achlya bisexualis

1992 ◽  
Vol 102 (3) ◽  
pp. 611-627 ◽  
Author(s):  
I. BRENT HEATH ◽  
RUTH L. HAROLD
Keyword(s):  
Cell Wall ◽  
Germ Tube ◽  
Multiple Roles ◽  
Rhodamine Phalloidin ◽  
Saprolegnia Ferax ◽  
Functional Changes ◽  
Specific Effects ◽  
Changing Patterns ◽  
Microtubular Root ◽  
Achlya Bisexualis

Very similar changing patterns of actin are described with rhodamine-phalloidin labelling during the zoosporic life cycle of the oomycetes, Saprolegnia ferax and Achlya bisexualis. By comparing the changes with previously described ultrastructural and functional changes, we show that actin functions in numerous previously unrecognized processes. Most spectacularly, the directed vesicle expansions of the cytokinetic system involve newly formed actin which outlines the developing zoospores. Disruption of this actin with cytochalasins leads to abnormal cleavage as witnessed by the formation of enlarged and irregular cysts. Prior to cytokinesis, two new types of organelle are synthesized and one, known as K bodies, clusters around the nuclei. These organdies are actin-rich during development and clustering, consistent with actin functioning in their positioning. In the zoospores, actin is concentrated around the water expulsion vacuoles, indicating that they are contractile, and permeates the cytoplasm, probably with a skeletal role. This concept is supported by the first demonstration of actin specifically associated with a microtubular root in the secondary zoospore. Upon encystment there is a dramatic increase in stained actin in the form of peripheral plaques associated with the newly synthesized cell wall. When the cysts germinate, a fibrillar actin cap, comparable to that previously described in hyphal tips, forms in the germ tube apex, but only after cell wall softening to permit germ tube protrusion. This sequence is consistent with the actin cap modulating turgor-driven expansion of the tip as previously discussed for hyphae. In addition to disrupting cleavage-associated actin, cytochalasins show developmental stage, dose and drug (CE≥CD≥CB) specific effects on zoosporulation-related actin, which indicates that, contrary to previous suggestions, rhodamine-phalloidin staining is a useful indicator of actin behaviour in response to cytochalasins. These responses include differential effects on adjoining actin arrays, some of which are transient in the continued presence of the drugs, indicating a mechanism of drug adaptation.

Sign in / Sign up

Export Citation Format

Share Document