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Functional characterization of the pleckstrin homology domain of a cellulose synthase from the Oomycete Saprolegnia monoica
KTH, School of Biotechnology (BIO), Glycoscience.
KTH, School of Biotechnology (BIO), Glycoscience.
KTH, School of Biotechnology (BIO), Glycoscience.
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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. Vol. 417, no 4, 1248-1253 p.
Keyword [en]
Pleckstrin homology domain; Cellulose synthase; Cell wall biosynthesis; Oomycetes; Phosphoinositides; F-actin
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-34284DOI: 10.1016/j.bbrc.2011.12.118ISI: 000300196100024Scopus ID: 2-s2.0-84856215112OAI: oai:DiVA.org:kth-34284DiVA: diva2:420127
Note
Updated from manuscript to article in journal. QC 20120306Available from: 2011-05-31 Created: 2011-05-31 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Functional characterization of cellulose and chitin synthase genes in Oomycetes
Open this publication in new window or tab >>Functional characterization of cellulose and chitin synthase genes in Oomycetes
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Funktionell karaktärisering av cellulosa- och kitinsyntasgener i oomyceter
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
cellulose biosynthesis; chitin biosynthesis; cellulose synthase gene; chitin synthase gene; Oomycetes; Phytophthora infestans; Saprolegnia monoica; pleckstrin homology domain
National Category
Microbiology
Identifiers
urn:nbn:se:kth:diva-34012 (URN)978-91-7415-971-4 (ISBN)
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
2. Characterization of specific domains of the cellulose and chitin synthases from pathogenic oomycetes
Open this publication in new window or tab >>Characterization of specific domains of the cellulose and chitin synthases from pathogenic oomycetes
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Some oomycetes species are severe pathogens of fish or crops. As such, they are responsible for important losses in the aquaculture industry as well as in agriculture. Saprolegnia parasitica is a major concern in aquaculture as there is currently no method available for controlling the diseases caused by this microorganism. The cell wall is an extracellular matrix composed essentially of polysaccharides, whose integrity is required for oomycete viability. Thus, the enzymes involved in the biosynthesis of cell wall components, such as cellulose and chitin synthases, represent ideal targets for disease control. However, the biochemical properties of these enzymes are poorly understood, which limits our capacity to develop specific inhibitors that can be used for blocking the growth of pathogenic oomycetes.

In our work, we have used Saprolegnia monoica as a model species for oomycetes to characterize two types of domains that occur specifically in oomycete carbohydrate synthases: the Pleckstrin Homology (PH) domain of a cellulose synthase and the so-called ‘Microtubule Interacting and Trafficking’ (MIT) domain of chitin synthases. In addition, the chitin synthase activity of the oomycete phytopathogen Aphanomyces euteiches was characterized in vitro using biochemical approaches.

The results from our in vitro investigations revealed that the PH domain of the oomycete cellulose synthase binds to phosphoinositides, microtubules and F-actin. In addition, cell biology approaches were used to demonstrate that the PH domain co-localize with F-actin in vivo. The structure of the MIT domain of chitin synthase (CHS) 1 was solved by NMR. In vitro binding assays performed on recombinant MIT domains from CHS 1 and CHS 2 demonstrated that both proteins strongly interact with phosphatidic acid in vitro. These results were further supported by in silico data where biomimetic membranes composed of different phospholipids were designed for interaction studies. The use of a yeast-two-hybrid approach suggested that the MIT domain of CHS 2 interacts with the delta subunit of Adaptor Protein 3, which is involved in protein trafficking. These data support a role of the MIT domains in the cellular targeting of CHS proteins. Our biochemical data on the characterization of the chitin synthase activity of A. euteiches suggest the existence of two distinct enzymes responsible for the formation of water soluble and insoluble chitosaccharides, which is consistent with the existence of two putative CHS genes in the genome of this species.

Altogether our data support a role of the PH domain of cellulose synthase and MIT domains of CHS in membrane trafficking and cellular location.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2015. viii, 90 p.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:15
Keyword
Cellulose biosynthesis; chitin biosynthesis; cellulose synthase genes; chitin synthase genes; oomycetes; Saprolegnia monoica; Microtubule Interacting and Trafficking (MIT) domain; Pleckstrin Homology (PH) domain
National Category
Biochemistry and Molecular Biology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-175375 (URN)978-91-7595-690-9 (ISBN)
Public defence
2015-10-23, FB53, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

QC 20151014

Available from: 2015-10-14 Created: 2015-10-13 Last updated: 2015-10-14Bibliographically approved

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