β-N-Acetylglucosaminidase MthNAG from Myceliophthora thermophila C1, a thermostable enzyme for production of N-acetylglucosamine from chitin
Krolicka, Malgorzata ; Hinz, Sandra W.A. ; Koetsier, Martijn J. ; Eggink, Gerrit ; Broek, Lambertus A.M. van den; Boeriu, Carmen G. - \ 2018
Applied Microbiology and Biotechnology 102 (2018)17. - ISSN 0175-7598 - p. 7441 - 7454.
Chitin - Chitosan - Myceliophthora thermophila C1 - N-Acetylglucosamine - β-N-Acetylglucosaminidase
Thermostable enzymes are a promising alternative for chemical catalysts currently used for the production of N-acetylglucosamine (GlcNAc) from chitin. In this study, a novel thermostable β-N-acetylglucosaminidase MthNAG was cloned and purified from the thermophilic fungus Myceliophthora thermophila C1. MthNAG is a protein with a molecular weight of 71 kDa as determined with MALDI-TOF-MS. MthNAG has the highest activity at 50 °C and pH 4.5. The enzyme shows high thermostability above the optimum temperature: at 55 °C (144 h, 75% activity), 60 °C (48 h, 85% activity; half-life 82 h), and 70 °C (24 h, 33% activity; half-life 18 h). MthNAG releases GlcNAc from chitin oligosaccharides (GlcNAc)2–5, p-nitrophenol derivatives of chitin oligosaccharides (GlcNAc)1–3-pNP, and the polymeric substrates swollen chitin and soluble chitosan. The highest activity was detected towards (GlcNAc)2. MthNAG released GlcNAc from the non-reducing end of the substrate. We found that MthNAG and Chitinase Chi1 from M. thermophila C1 synergistically degraded swollen chitin and released GlcNAc in concentration of approximately 130 times higher than when only MthNAG was used. Therefore, chitinase Chi1 and MthNAG have great potential in the industrial production of GlcNAc.
Colletotrichum higginsianum extracellular LysM proteins play dual roles in appressorial function and suppression of chitin-triggered plant immunity
Takahara, Hiroyuki ; Hacquard, Stéphane ; Kombrink, Anja ; Hughes, H.B. ; Halder, Vivek ; Robin, Guillaume P. ; Hiruma, Kei ; Neumann, Ulla ; Shinya, Tomonori ; Kombrink, Erich ; Shibuya, Naoto ; Thomma, Bart P.H.J. ; O'Connell, Richard J. - \ 2016
New Phytologist 211 (2016)4. - ISSN 0028-646X - p. 1323 - 1337.
Arabidopsis - Appressoria - Biotrophy - Chitin - Colletotrichum - Effector - LysM - Virulence
The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large repertoire of candidate-secreted effectors containing LysM domains, but the role of such proteins in the pathogenicity of any Colletotrichum species is unknown. Here, we characterized the function of two effectors, ChELP1 and ChELP2, which are transcriptionally activated during the initial intracellular biotrophic phase of infection. Using immunocytochemistry, we found that ChELP2 is concentrated on the surface of bulbous biotrophic hyphae at the interface with living host cells but is absent from filamentous necrotrophic hyphae. We show that recombinant ChELP1 and ChELP2 bind chitin and chitin oligomers in vitro with high affinity and specificity and that both proteins suppress the chitin-triggered activation of two immune-related plant mitogen-activated protein kinases in the host Arabidopsis. Using RNAi-mediated gene silencing, we found that ChELP1 and ChELP2 are essential for fungal virulence and appressorium-mediated penetration of both Arabidopsis epidermal cells and cellophane membranes in vitro. The findings suggest a dual role for these LysM proteins as effectors for suppressing chitin-triggered immunity and as proteins required for appressorium function.
Convergent evolution of filamentous microbes towards evasion of glycan-triggered immunity
Rovenich, Hanna ; Zuccaro, Alga ; Thomma, Bart P.H.J. - \ 2016
New Phytologist 212 (2016)4. - ISSN 0028-646X - p. 896 - 901.
Chitin - Effector - Glycan recognition - Microbial cell wall - β-glucan
I. II. III. IV. V. VI. References Summary: All filamentous microbes produce and release a wide range of glycans, which are essential determinants of microbe-microbe and microbe-host interactions. Major cell wall constituents, such as chitin and β-glucans, are elicitors of host immune responses. The widespread capacity for glycan perception in plants has driven the evolution of various strategies that help filamentous microbes to evade detection. Common strategies include structural and chemical modifications of cell wall components as well as the secretion of effector proteins that suppress chitin- and β-glucan-triggered immune responses. Thus, the necessity to avoid glycan-triggered immunity represents a driving force in the convergent evolution of filamentous microbes towards its suppression.
SnTox1, a Parastagonospora nodorum necrotrophic effector, is a dual-function protein that facilitates infection while protecting from wheat-produced chitinases
Liu, Zhaohui ; Gao, Yuanyuan ; Kim, Yong Min ; Faris, Justin D. ; Shelver, Weilin L. ; Wit, Pierre J.G.M. de; Xu, Steven S. ; Friesen, Timothy L. - \ 2016
New Phytologist 211 (2016)3. - ISSN 0028-646X - p. 1052 - 1064.
Parastagonosopora nodorum - Chitin - Host-selective toxin - Necrotroph - Necrotrophic effector - Programmed cell death (PCD) - Wheat (Triticum aestivum) chitinases
SnTox1 induces programmed cell death and the up-regulation of pathogenesis-related genes including chitinases. Additionally, SnTox1 has structural homology to several plant chitin-binding proteins. Therefore, we evaluated SnTox1 for chitin binding and localization. We transformed an avirulent strain of Parastagonospora nodorum as well as three nonpathogens of wheat (Triticum aestivum), including a necrotrophic pathogen of barley, a hemibiotrophic pathogen of sugar beet and a saprotroph, to evaluate the role of SnTox1 in infection and in protection from wheat chitinases. SnTox1 bound chitin and an SnTox1-green fluorescent fusion protein localized to the mycelial cell wall. Purified SnTox1 induced necrosis in the absence of the pathogen when sprayed on the leaf surface and appeared to remain on the leaf surface while inducing both epidermal and mesophyll cell death. SnTox1 protected the different fungi from chitinase degradation. SnTox1 was sufficient to change the host range of a necrotrophic pathogen but not a hemibiotroph or saprotroph. Collectively, this work shows that SnTox1 probably interacts with a receptor on the outside of the cell to induce cell death to acquire nutrients, but SnTox1 accomplishes a second role in that it protects against one aspect of the defense response, namely the effects of wheat chitinases.