Chitin pleomorphism in the cellulosic cell wall fungus Saprolegnia

Gay, L.; Bulone, Vincent; Grandgirard, Virginie; Fèvre, Michel; Chanzy, H.

doi:10.1111/j.1574-6968.1992.tb05732.x

Cited by 7 publications

(8 citation statements)

References 15 publications

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“…Saprolegnia monoica is the only oomycete in which both crystalline chitin and putative chitin synthase sequences have been identified [11] , [15] . Isolated membranes and detergent extracts of plasma membranes from this species exhibit chitin synthase activity in vitro [11] , [17] but there is no evidence that the detected activity corresponds to the sequences identified [15] . Preliminary work in S. monoica using the chitin synthase inhibitor polyoxin D suggested that chitin may be involved in hyphal growth [11] .…”

Section: Introductionmentioning

confidence: 85%

“…Alternatively, SmCHS1 may be involved in chitin biosynthesis during non-mycelial developmental stages, which would be consistent with the observed low level of expression in the mycelium. It is noteworthy that the assay used here was optimized for the measurement of GlcNAc incorporation into insoluble chitin polymers or into chito-oligosaccharides of a degree of polymerization higher than 6 [11] , [17] . Thus, another possibility explaining the absence of in vitro activity with the recombinant SmCHS1 is that the protein may form chito-oligosaccharides of a low degree of polymerization that could possibly be involved in priming chitin biosynthesis.…”

Section: Discussionmentioning

confidence: 99%

“…Chitin synthase activity was measured as described earlier [11] , [17] . Briefly, 50 µl of enzyme preparation was mixed with 150 µl of reaction buffer consisting of 10 mM Tris-HCl pH 7.4, 10 mM MgCl 2 , 20 mM N -acetyl- d -glucosamine, 1.25 µg trypsin/ml, 0.5 mM UDP- N -acetyl- d -glucosamine and 434 nM UDP- N -acetyl- d -[U- 14 C]glucosamine (318 mCi/mmol; Amersham).…”

Section: Methodsmentioning

confidence: 99%

“…The extent of hydrolysis was determined by comparison with controls in which the hydrolytic enzyme had been replaced by the corresponding incubation buffer. For electron microscopy observation of the chitin synthesized in vitro by the recombinant chitin synthases expressed in Pichia and solubilized with CHAPS, a drop of the reaction mixture obtained after incubation in the same conditions as for a chitin synthase assay, but in the presence of nonradioactive UDP-GlcNAc, was deposited on carbon-coated grids and observed after negative staining with 2% uranyl acetate as described earlier [17] . Control experiments were performed by using a similar protein fraction prepared from the wild-type Pichia cells.…”

Section: Methodsmentioning

confidence: 99%

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Chitin Synthases from Saprolegnia Are Involved in Tip Growth and Represent a Potential Target for Anti-Oomycete Drugs

et al. 2010

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Oomycetes represent some of the most devastating plant and animal pathogens. Typical examples are Phytophthora infestans, which causes potato and tomato late blight, and Saprolegnia parasitica, responsible for fish diseases. Despite the economical and environmental importance of oomycete diseases, their control is difficult, particularly in the aquaculture industry. Carbohydrate synthases are vital for hyphal growth and represent interesting targets for tackling the pathogens. The existence of 2 different chitin synthase genes (SmChs1 and SmChs2) in Saprolegnia monoica was demonstrated using bioinformatics and molecular biology approaches. The function of SmCHS2 was unequivocally demonstrated by showing its catalytic activity in vitro after expression in Pichia pastoris. The recombinant SmCHS1 protein did not exhibit any activity in vitro, suggesting that it requires other partners or effectors to be active, or that it is involved in a different process than chitin biosynthesis. Both proteins contained N-terminal Microtubule Interacting and Trafficking domains, which have never been reported in any other known carbohydrate synthases. These domains are involved in protein recycling by endocytosis. Enzyme kinetics revealed that Saprolegnia chitin synthases are competitively inhibited by nikkomycin Z and quantitative PCR showed that their expression is higher in presence of the inhibitor. The use of nikkomycin Z combined with microscopy showed that chitin synthases are active essentially at the hyphal tips, which burst in the presence of the inhibitor, leading to cell death. S. parasitica was more sensitive to nikkomycin Z than S. monoica. In conclusion, chitin synthases with species-specific characteristics are involved in tip growth in Saprolegnia species and chitin is vital for the micro-organisms despite its very low abundance in the cell walls. Chitin is most likely synthesized transiently at the apex of the cells before cellulose, the major cell wall component in oomycetes. Our results provide important fundamental information on cell wall biogenesis in economically important species, and demonstrate the potential of targeting oomycete chitin synthases for disease control.

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Section: Introductionmentioning

confidence: 85%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Chitin Synthases from Saprolegnia Are Involved in Tip Growth and Represent a Potential Target for Anti-Oomycete Drugs

et al. 2010

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“…The major cell wall components in oomycetes are cellulose, β-(1 → 3)-and β-(1 → 6)-glucans, but some species also produce small amounts of chitin Mélida et al, 2013). In Saprolegnia monoica's hyphal cells, chitin occurs in the form of granular structures whereas regenerating protoplasts were shown to form microfibrils of chitin, suggesting that the environment in which chitin is deposited, i.e., the presence or absence of other cell wall components, influences its morphology and, most likely also, its physical properties Gay et al, 1992). The presence of chitin in oomycetes was used to group their cell walls in three main types based on the abundance of N-acetylglucosamine (GlcNAc), the monomer of chitin.…”

Section: Introductionmentioning

confidence: 99%

Identification and Characterization of the Chitin Synthase Genes From the Fish Pathogen Saprolegnia parasitica

2019

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Saprolegnia parasitica is a pathogenic oomycete responsible for severe fish infections. Despite its low abundance in the cell wall of S. parasitica, chitin is essential for hyphal growth as the inhibition of its biosynthesis leads to highly reduced growth. Here we identified and characterized chitin synthases (CHS) from S. parasitica as potential targets for anti-oomycete drugs. Bioinformatics analyses allowed the identification of six different putative Chs genes in the genome of the pathogen. The total number of genes was confirmed by Southern blot analysis and their expression levels were determined by quantitative PCR. Four of the six Chs genes were expressed in the mycelium, while the two others exhibited undetectable levels of expression. The mycelium was highly sensitive to the addition of nikkomycin Z (NZ) in the culture medium, which led to a decreased amount of chitin in the cell wall by up to 40% in the conditions tested, and to the formation of abnormal branching structures in the hyphae. The presence of NZ increased the expression level of one of the genes, Chs3, suggesting that the corresponding product is compensating the disruption of chitin biosynthesis in the hyphae. In addition, the activity of isolated CHS was strongly inhibited by NZ in vitro. Altogether our data indicate the importance of CHS for the vegetative growth of S. parasitica and demonstrate that these enzymes represent promising targets for the control of diseases caused by oomycetes.

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