No description available.

Oomycetes have long been considered as a separate class within the kingdom Fungi, but they are in fact closer to brown algae. They are currently classified in the Stramenopile eukaryotic kingdom, which includes heterokont algae and water molds. The major cell wall polysaccharides in Oomycetes are b-(1à3) and b-(1à6)-glucans, as well as cellulose, which has never been reported in any fungal species. Chitin - the major cell wall polysaccharide in fungi - occurs in minor amounts in the walls of some Oomycetes. Some Oomycete species are pathogens of great economical importance. For example, species of the genus Phytophthora are well studied plant pathogens that cause considerable economical losses in agriculture. Saprolegniosis, a fish disease caused by species from the genus Saprolegnia, is a major problem in the aquaculture industry and represents a threat to populations of salmonids in natural habitats. Currently, there are no chemicals available that are at the same time efficient Oomycete inhibitors, environmentally friendly and safe for human consumption of treated fishes. The biosynthesis of cellulose in Oomycetes is poorly understood, even though this biochemical pathway represents a potential target for new Oomycete inhibitors. In this work, cellulose biosynthesis was investigated in two selected Oomycetes, the plant pathogen Phytophthora infestans and the fish pathogen Saprolegnia monoica. A new Oomycete CesA gene family was identified. It contains four homologues designated as CesA1, CesA2, CesA3 and CesA4. The gene products of CesA1, 2 and 4 contain Pleckstrin Homology domains located at the N-terminus. This represents a novel feature, unique to the Oomycete CesA genes. CesA3 is the dominantly expressed CesA homologue in the mycelium of both S. monoica and P. infestans, while CesA1 and CesA2 are up-regulated in virulent life stages of P. infestans. CesA4 was expressed only in minute amounts in all investigated types of cells. Gene silencing by RNA interference of the whole CesA gene family in P. infestans lead to decreased amounts of cellulose in the cell wall. The inhibitors of cellulose synthesis DCB and Congo Red had an up-regulating effect on SmCesA gene expression, which was accompanied by an increased b-glucan synthase activity in vitro. In addition, these inhibitors slowed down the growth of the mycelium from S. monoica. Zoospores from P. infestans treated with DCB were unable to infect potato leaves and showed aberrant cell wall morphologies similar to those obtained by silencing the CesA gene family. Altogether these results show that at least some of the CesA1-4 genes are involved in cellulose biosynthesis and that the synthesis of cellulose is crucial for infection of potato by P. infestans. / QC 20101110

No description available.

La saprolégniose est une maladie fongique causée par le champignon aquatique Saprolegnia sp. qui affecte les poissons sauvages et ceux provenant des piscicultures. L’apparition de touffes cotonneuses semblables à de la ouate de couleur blanche à grise est souvent la première indication de l’infection. Ce saprophyte ubiquitaire se nourrit habituellement des œufs de poissons morts, mais peut se propager rapidement aux œufs sains causant la mort de ces derniers. La saprolégniose est souvent une infection secondaire, mais des souches virulentes peuvent facilement se développer sur les salmonidés ayant subi un stress ou une mauvaise manipulation. De grandes pertes économiques associées à la saprolégniose sont rapportées chaque année à travers le monde surtout dans l’industrie de la pisciculture. Jusqu’en 2002, le contrôle de la saprolégniose pouvait se faire par l’utilisation du vert de malachite, un colorant organique ayant une grande activité antifongique. Malheureusement, cette molécule a été bannie à cause de ses propriétés cancérigènes. Aucun composé aussi efficace n’est actuellement disponible pour traiter les infections de la saprolégniose. Des molécules ou extraits naturels ayant un potentiel antifongique ont donc été testés à l’aide de deux techniques (par graines de chanvre et par cylindre d’agar). Les molécules d’un extrait de propolis (cire de ruches d’abeilles) démontrant de l’activité anti-Saprolegnia ont été identifiées. De plus, une bactérie, Pseudomonas aeruginosa, pouvant être retrouvée dans le même environnement que Saprolegnia sp. a démontré un effet antagoniste au champignon. Une molécule de signalisation intercellulaire produite par P. aeruginosa, 4-hydroxy-2-heptylquinoline (HHQ), a été identifiée comme responsable de l’effet antagoniste contre Saprolegnia sp. / Saprolegniosis, a fungal disease which affects wild and farm fish, is caused by the water mold Saprolegnia sp. Visible cotton-like white or grey patches on fish skin are often the first sign of infection. This ubiquitous saprophyte infects dead fish eggs and then spreads to healthy eggs resulting in major losses due to mortalities. Saprolegniosis is often a secondary infection; however some virulent strains are known to cause primary infection on injured or stressed salmonids. Important economic losses in aquaculture are reported every year worldwide due to saprolegniosis. Infection by Saprolegnia sp. was well controlled with the use of an organic dye with great antifungal efficacy; malachite green. Unfortunately, the use of malachite green was banned in 2002 worldwide due to its carcinogenic and toxicological effects. Up until now, no new treatment as effective as malachite green has been discovered. Natural extracts or molecules with potential antifungal properties have also been tested using two techniques (with hemp seeds and with agar cylinders). Molecules from propolis extracts (beeswax), active against Saprolegnia sp. have been identified. Furthermore, culture supernatant of the bacteria Pseudomonas aeruginosa was found to be able to inhibit the growth of Saprolegnia. The intercellular signal molecule produced by P. aeruginosa 4-hydroxy-2-heptylquinoline (HHQ) was identified as the active compound against Saprolegnia.

La saprolégniose est une maladie fongique causée par le champignon aquatique Saprolegnia sp. qui affecte les poissons sauvages et ceux provenant des piscicultures. L’apparition de touffes cotonneuses semblables à de la ouate de couleur blanche à grise est souvent la première indication de l’infection. Ce saprophyte ubiquitaire se nourrit habituellement des œufs de poissons morts, mais peut se propager rapidement aux œufs sains causant la mort de ces derniers. La saprolégniose est souvent une infection secondaire, mais des souches virulentes peuvent facilement se développer sur les salmonidés ayant subi un stress ou une mauvaise manipulation. De grandes pertes économiques associées à la saprolégniose sont rapportées chaque année à travers le monde surtout dans l’industrie de la pisciculture. Jusqu’en 2002, le contrôle de la saprolégniose pouvait se faire par l’utilisation du vert de malachite, un colorant organique ayant une grande activité antifongique. Malheureusement, cette molécule a été bannie à cause de ses propriétés cancérigènes. Aucun composé aussi efficace n’est actuellement disponible pour traiter les infections de la saprolégniose. Des molécules ou extraits naturels ayant un potentiel antifongique ont donc été testés à l’aide de deux techniques (par graines de chanvre et par cylindre d’agar). Les molécules d’un extrait de propolis (cire de ruches d’abeilles) démontrant de l’activité anti-Saprolegnia ont été identifiées. De plus, une bactérie, Pseudomonas aeruginosa, pouvant être retrouvée dans le même environnement que Saprolegnia sp. a démontré un effet antagoniste au champignon. Une molécule de signalisation intercellulaire produite par P. aeruginosa, 4-hydroxy-2-heptylquinoline (HHQ), a été identifiée comme responsable de l’effet antagoniste contre Saprolegnia sp. / Saprolegniosis, a fungal disease which affects wild and farm fish, is caused by the water mold Saprolegnia sp. Visible cotton-like white or grey patches on fish skin are often the first sign of infection. This ubiquitous saprophyte infects dead fish eggs and then spreads to healthy eggs resulting in major losses due to mortalities. Saprolegniosis is often a secondary infection; however some virulent strains are known to cause primary infection on injured or stressed salmonids. Important economic losses in aquaculture are reported every year worldwide due to saprolegniosis. Infection by Saprolegnia sp. was well controlled with the use of an organic dye with great antifungal efficacy; malachite green. Unfortunately, the use of malachite green was banned in 2002 worldwide due to its carcinogenic and toxicological effects. Up until now, no new treatment as effective as malachite green has been discovered. Natural extracts or molecules with potential antifungal properties have also been tested using two techniques (with hemp seeds and with agar cylinders). Molecules from propolis extracts (beeswax), active against Saprolegnia sp. have been identified. Furthermore, culture supernatant of the bacteria Pseudomonas aeruginosa was found to be able to inhibit the growth of Saprolegnia. The intercellular signal molecule produced by P. aeruginosa 4-hydroxy-2-heptylquinoline (HHQ) was identified as the active compound against Saprolegnia.

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

No description available.

No description available.

Les maladies constituent présentement la cause la plus importante de perte économique en aquaculture moderne. Chez certaines espèces, notamment les salmonidés (Oncorhynchus sp. et Salmo sp.), on rapporte des pertes annuelles atteignant cinquante pour cent de la production. À l’heure actuelle, les infections fongiques occupent le second rang derrière les maladies bactériennes en fonction de leur importance économique. Ces poissons sont particulièrement vulnérables à une infection fongique causée par Saprolegnia sp. qui infecte habituellement les oeufs morts. Le saprophyte ubiquitaire se propage ensuite aux oeufs sains et aux individus matures. Malheureusement, le traitement efficace de cette infection, souvent primaire et parfois secondaire, est de plus en plus difficile en raison de nouvelles réglementations restrictives entourant le vert de malachite. Jadis, ce colorant constituait le fongicide le plus efficace dans la lutte contre la saprolégniose, mais son potentiel cancérigène en limite maintenant l’utilisation. Jusqu'à présent, aucun traitement disponible n’est aussi efficace que le vert de malachite pour le contrôle de la saprolégniose. Récemment, nous sommes parvenus à isoler trois bactéries capables d’inhiber la croissance de Saprolegnia sp. in vitro. Ces trois Pseudomonas fluorescens proviennent d’une pisciculture dans laquelle survenaient des cas d’infections à Saprolegnia parasitica. En poussant la caractérisation de l’activité grâce à des analyses de chromatographie liquide haute performance et de spectrométrie de masse, nous avons réussi à isoler et à identifier la molécule responsable. L’acide phénazine-1-carboxylique (PCA), sécrété par deux de nos trois souches, cause l’inhibition de la croissance de Saprolegnia. / Disease is the single largest cause of economic losses in aquaculture, and fungal infections are second only to bacterial diseases in economic importance. Fifty percent per year losses due to fungal infections have been reported in a number of species including salmonids, (Oncorhynchus sp., Salmo sp.) which are particularly susceptible to Saprolegnia sp. The ubiquitous saprophyte commonly infects dead fish eggs and spreads to healthy eggs and fry resulting in a deadly, usually secondary, infection. The ability to effectively treat fungal infections has become increasingly difficult with the accrual of restrictions on the use of the most effective fungicide available, namely malachite green due to concerns regarding its carcinogenicity. Hitherto, no new treatment as effective as malachite green has been available to fish farmers. Recently, we have isolated three Pseudomonas fluorescens bacterial strains, from a Saprolegnia parasitica-infected fish farm, adept at the inhibition of the growth of this oomycete in vitro. The inhibitory activity was found to be present in the culture supernatant of the three strains. Further characterization by high performance liquid chromatography and mass spectrometry has been performed to identify the nature of the inhibition. Phenazine-1-carboxylic acid (PCA), produced by two of the three isolates, was found to be able of inhibiting the growth of Saprolegnia. The causal factor producing inhibition for the third isolate remains a mystery.

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>