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Functional characterization of cellulose and chitin synthase genes in Oomycetes
KTH, School of Biotechnology (BIO), Glycoscience.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Funktionell karaktärisering av cellulosa- och kitinsyntasgener i oomyceter (Swedish)
Abstract [en]

Some species of Oomycetes are well studied pathogens that cause considerable economical losses in the agriculture and aquaculture industries. Currently, there are no chemicals available that are environmentally friendly and at the same time efficient Oomycete inhibitors. The cell wall of Oomycetes consists of b-(1à3) and b-(1à6)-glucans, cellulose and in some species minute amounts of chitin. The biosynthesis of cellulose and chitin in Oomycetes is poorly understood. However, cell wall synthesis represents a potential target for new Oomycete inhibitors. In this work, cellulose and chitin synthase genes and gene products were analyzed in the plant pathogen Phytophthora infestans and in the fish pathogen Saprolegnia monoica.

 

A new Oomycete CesA gene family was identified, containing four subclasses of genes designated as CesA1 to 4. The gene products of CesA1, 2 and 4 contain pleckstrin homology (PH) domains located at the N-terminus, which is unique to the Oomycete CesAs. Our results show that the SmCesA2 PH domain binds to phosphoinositides, F-actin and microtubules in vitro and can co-localize with F-actin in vivo. Functional characterization of the CesA genes by gene silencing in P. infestans led to decreased cellulose content in the cell wall. The cellulose synthase inhibitors DCB and Congo Red inhibited the growth of the mycelium of S. monoica and had an up-regulating effect on SmCesA gene expression. Zoospores from P. infestans treated with DCB were unable to infect potato leaves. In addition, two full-length chitin synthase genes (Chs) were analyzed from S. monoica.  Expression of SmChs2 in yeast yielded an active recombinant protein. The biochemical characterization of the in vitro product of SmChs2 confirmed that the protein is responsible for chitin formation. The chitin synthase inhibitor nikkomycin Z inhibited the SmChs2 both in vivo and in vitro.

 

Altogether these results show that at least some of the CesA1-4 genes are involved in cellulose biosynthesis and that synthesis of cellulose is crucial for infection of potato by P. infestans. The PH domain is involved in the interaction of CesA with the cytoskeleton. In addition, we firmly demonstrate that the SmChs2 gene encodes a catalytically active chitin synthase.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology , 2011. , 86 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2011:13
Keyword [en]
cellulose biosynthesis; chitin biosynthesis; cellulose synthase gene; chitin synthase gene; Oomycetes; Phytophthora infestans; Saprolegnia monoica; pleckstrin homology domain
National Category
Microbiology
Identifiers
URN: urn:nbn:se:kth:diva-34012ISBN: 978-91-7415-971-4OAI: oai:DiVA.org:kth-34012DiVA: diva2:418553
Public defence
2011-06-14, FD5, AlbaNova University centre, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
QC 20110531Available from: 2011-05-31 Created: 2011-05-23 Last updated: 2011-05-31Bibliographically approved
List of papers
1.
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2. Identification of the cellulose synthase genes from the Oomycete Saprolegnia monoica and effect of cellulose synthesis inhibitors on gene expression and enzyme activity
Open this publication in new window or tab >>Identification of the cellulose synthase genes from the Oomycete Saprolegnia monoica and effect of cellulose synthesis inhibitors on gene expression and enzyme activity
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2009 (English)In: Fungal Genetics and Biology, ISSN 1087-1845, E-ISSN 1096-0937, Vol. 46, no 10, 759-767 p.Article in journal (Refereed) Published
Abstract [en]

Cellulose biosynthesis is a vital but yet poorly understood biochemical process in Oomycetes. Here, we report the identification and characterization of the cellulose synthase genes (CesA) from Saprolegnia monoica. Southern blot experiments revealed the occurrence of three CesA homologues in this species and phylogenetic analyses confirmed that Oomycete CesAs form a clade of their own. All gene products contained the D,D,D,QXXRW signature of most processive glycosyltransferases, including cellulose synthases. However, their N-terminal ends exhibited Oomycete-specific domains, i.e. Pleckstrin Homology domains, or conserved domains of an unknown function together with additional putative transmembrane domains. Mycelial growth was inhibited in the presence of the cellulose biosynthesis inhibitors 2,6-dichlorobenzonitrile or Congo Red. This inhibition was accompanied by a higher expression of all CesA genes in the mycelium and increased in vitro glucan synthase activities. Altogether, our data strongly suggest a direct involvement of the identified CesA genes in cellulose biosynthesis.

Keyword
Cellulose synthase genes, Cell wall biosynthesis, Congo Red, 2, 6-Dichlorobenzonitrile (DCB), Oomycetes, Saprolegnia monoica, fungus saprolegnia, acetobacter-xylinum, catalytic subunit, chitin, synthase, plasma-membrane, wall chemistry, cell-walls, congo red, biosynthesis, dna
Identifiers
urn:nbn:se:kth:diva-18715 (URN)10.1016/j.fgb.2009.07.001 (DOI)000269368000005 ()2-s2.0-68549136474 (ScopusID)
Note
QC 20100525Available from: 2010-08-05 Created: 2010-08-05 Last updated: 2011-05-31Bibliographically approved
3. Functional characterization of the pleckstrin homology domain of a cellulose synthase from the Oomycete Saprolegnia monoica
Open this publication in new window or tab >>Functional characterization of the pleckstrin homology domain of a cellulose synthase from the Oomycete Saprolegnia monoica
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2012 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 417, no 4, 1248-1253 p.Article in journal (Refereed) Published
Abstract [en]

Some oomycetes, for instance Saprolegnia parasitica, are severe fish pathogens that cause important economic losses worldwide. Cellulose biosynthesis is a vital process for this class of microorganisms, but the corresponding molecular mechanisms are poorly understood. Of all cellulose synthesizing enzymes known, only some oomycete cellulose synthases contain a pleckstrin homology (PH) domain. Some human PH domains bind specifically to phosphoinositides, but most PH domains bind phospholipids in a non-specific manner. In addition, some PH domains interact with various proteins. Here we have investigated the function of the PH domain of cellulose synthase 2 from the oomycete Saprolegnia monoica (SmCesA2), a species closely related to S. parasitica. The SmCesA2 PH domain is similar to the C-terminal PH domain of the human protein TAPP1. It binds in vitro to phosphoinositides, F-actin and microtubules, and co-localizes with F-actin in vivo. Our results suggest a role of the SmCesA2 PH domain in the regulation, trafficking and/or targeting of the cell wall synthesizing enzyme.

Place, publisher, year, edition, pages
Academic Press, 2012
Keyword
Pleckstrin homology domain; Cellulose synthase; Cell wall biosynthesis; Oomycetes; Phosphoinositides; F-actin
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-34284 (URN)10.1016/j.bbrc.2011.12.118 (DOI)000300196100024 ()2-s2.0-84856215112 (ScopusID)
Note
Updated from manuscript to article in journal. QC 20120306Available from: 2011-05-31 Created: 2011-05-31 Last updated: 2015-10-14Bibliographically approved
4. Chitin Synthases from Saprolegnia Are Involved in Tip Growth and Represent a Potential Target for Anti-Oomycete Drugs
Open this publication in new window or tab >>Chitin Synthases from Saprolegnia Are Involved in Tip Growth and Represent a Potential Target for Anti-Oomycete Drugs
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2010 (English)In: PLOS PATHOG, ISSN 1553-7366, Vol. 6, no 8, e1001070- p.Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Microbiology
Identifiers
urn:nbn:se:kth:diva-26805 (URN)10.1371/journal.ppat.1001070 (DOI)000281399900041 ()2-s2.0-77958127571 (ScopusID)
Note
QC 20101209Available from: 2010-12-09 Created: 2010-11-29 Last updated: 2011-05-31Bibliographically approved

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