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. / QC 20110531

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. / <p>QC 20151014</p>

Hemicelluloses are synthesized by proteins encoded by genes from the cellulose synthasegene superfamily. One subgroup of this gene family is the cellulose synthase-like B, which islargely uncharacterized and unexplored. The common model organism Nicotianabenthamiana has one such gene in its genome, NbCslB, encoding a membrane protein. Theexpression of this gene has previously been studied in vivo, but in order to study the protein invitro a viable solubilization and purification protocol is required. This study evaluated the useof the detergent n-Dodecyl β-D-maltoside (DDM) for solubilization, followed by purificationusing immobilized metal ion affinity chromatography (IMAC), and thereafter reconstitutionof the protein into proteoliposomes. SDS-PAGE as well as Western blot analyses showed thatthe purification was successful and provided a pure sample of protein. Throughout theanalyses performed, an anti-FLAG antibody was discovered to bind well to the protein, andthereby be especially useful for analysis. An activity assay was performed on the purifiedprotein, to characterize its function and evaluate whether the protein had maintained itsactivity and conformation after the steps of purification and reconstitution. No activity couldbe detected in the enzymatic assay, which indicated that the purification protocol may havebeen too rough on the protein, that the reconstitution was not successful, or that the assayconditions were not optimal. These results can be used as a base for future research, where theprotocols for solubilization, purification, and reconstitution should be further refined in orderto obtain an end result where the purified protein is active. When an active and pure proteinsample is achieved, it will be possible to perform further attempts at characterizing thefunction of the protein using enzymatic activity assays. Additionally, the results showed thatthe choice of antibody can be crucial for proper analysis of this protein. / Hemicellulosa syntetiseras av proteiner vars gener återfinns i genfamiljen cellulosasyntas. Enundergrupp till denna genfamilj är cellulosasyntasliknande B, en grupp som till stor del ärokarakteriserad och outforskad. Den vanliga modellorganismen Nicotiana benthamiana haren sådan gen i sitt genom, NbCslB, som kodar för ett membranprotein. Hur denna genuttrycks har tidigare studerats in vivo, men for att kunna studera proteinet in vitro krävs etthållbart protokoll för solubilisering och rening. Denna studie utvärderade användningen avlösningsmedlet n-Dodecyl β-D-maltoside (DDM) för solubilisering, följt av rening medimmobiliserad metalljon-affinitetskromatografi (IMAC), och efter det rekonstitution avproteinet till proteoliposomer. SDS-PAGE och Western blot analyser visade att reningen varlyckad, och att ett rent proteinprov erhållits. När analyserna genomfördes upptäcktes att enanti-FLAG antikropp band särskilt väl till proteinet, och därmed var mycket användbar vidanalys. En aktivitetsanalys genomfördes med det renade proteinet för att karakterisera dessfunktion och utvärdera huruvida proteinet hade bevarat sin aktivitet och konformation efterrening och rekonstitution. Ingen aktivitet kunde detekteras i den enzymatiskaaktivitetsanalysen, vilket indikerade att reningen eventuellt var för hård mot proteinet,alternativt att rekonstitutionen inte var lyckad, eller att förhållandena för analysen inte varoptimala. Dessa resultat kan användas som en bas för framtida forskning om proteinet, därprotokollen för solubilisering, rening och rekonstitution bör vidareutvecklas för att uppnå ettslutresultat där det renade proteinet är aktivt. När ett aktivt och rent proteinprov uppnåtts ärdet möjligt att genomföra ytterligare försök att karakterisera proteinets funktion medenzymatiska aktivitetsanalyser. Resultaten visade också att valet av antikropp kan varaavgörande för att ordentligt kunna analysera detta protein.

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