Functional characterization of the pleckstrin homology domain of a cellulose synthase from the oomycete Saprolegnia monoica.

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.

Chitin synthases from Saprolegnia are involved in tip growth and represent a potential target for anti-oomycete drugs.

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.

What do we really know about cellulose biosynthesis in higher plants?

Cellulose biosynthesis is one of the most important biochemical processes in plant biology. Despite the considerable progress made during the last decade, numerous fundamental questions related to this key process in plant development are outstanding. Numerous models have been proposed through the years to explain the detailed molecular events of cellulose biosynthesis. Almost all models integrate solid experimental data with hypotheses on several of the steps involved in the process. Speculative models are most useful to stimulate further research investigations and bring new exciting ideas to the field. However, it is important to keep their hypothetical nature in mind and be aware of the risk that some undemonstrated hypotheses may progressively become admitted. In this review, we discuss the different steps required for cellulose formation and crystallization, and highlight the most important specific aspects that are supported by solid experimental data.

Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans.

Phytophthora infestans is the most destructive pathogen of potato and a model organism for the oomycetes, a distinct lineage of fungus-like eukaryotes that are related to organisms such as brown algae and diatoms. As the agent of the Irish potato famine in the mid-nineteenth century, P. infestans has had a tremendous effect on human history, resulting in famine and population displacement. To this day, it affects world agriculture by causing the most destructive disease of potato, the fourth largest food crop and a critical alternative to the major cereal crops for feeding the world's population. Current annual worldwide potato crop losses due to late blight are conservatively estimated at $6.7 billion. Management of this devastating pathogen is challenged by its remarkable speed of adaptation to control strategies such as genetically resistant cultivars. Here we report the sequence of the P. infestans genome, which at approximately 240 megabases (Mb) is by far the largest and most complex genome sequenced so far in the chromalveolates. Its expansion results from a proliferation of repetitive DNA accounting for approximately 74% of the genome. Comparison with two other Phytophthora genomes showed rapid turnover and extensive expansion of specific families of secreted disease effector proteins, including many genes that are induced during infection or are predicted to have activities that alter host physiology. These fast-evolving effector genes are localized to highly dynamic and expanded regions of the P. infestans genome. This probably plays a crucial part in the rapid adaptability of the pathogen to host plants and underpins its evolutionary potential.

Identification of the cellulose synthase genes from the Oomycete Saprolegnia monoica and effect of cellulose synthesis inhibitors on gene expression and enzyme activity.

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.

Cellulose synthesis in Phytophthora infestans is required for normal appressorium formation and successful infection of potato.

Cellulose, the important structural compound of cell walls, provides strength and rigidity to cells of numerous organisms. Here, we functionally characterize four cellulose synthase genes (CesA) in the oomycete plant pathogen Phytophthora infestans, the causal agent of potato (Solanum tuberosum) late blight. Three members of this new protein family contain Pleckstrin homology domains and form a distinct phylogenetic group most closely related to the cellulose synthases of cyanobacteria. Expression of all four genes is coordinately upregulated during pre- and early infection stages of potato. Inhibition of cellulose synthesis by 2,6-dichlorobenzonitrile leads to a dramatic reduction in the number of normal germ tubes with appressoria, severe disruption of the cell wall in the preinfection structures, and a complete loss of pathogenicity. Silencing of the entire gene family in P. infestans with RNA interference leads to a similar disruption of the cell wall surrounding appressoria and an inability to form typical functional appressoria. In addition, the cellulose content of the cell walls of the silenced lines is >50% lower than in the walls of the nonsilenced lines. Our data demonstrate that the isolated genes are involved in cellulose biosynthesis and that cellulose synthesis is essential for infection by P. infestans.