New Enzymatic Virulence Factors In Phytophthora Infestans

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Oomycetes are fungal-like eukaryotic organisms evolutionarily related to diatoms and brown algae, and cause some of the most devastating plant and animal diseases in agriculture and aquaculture. The Phytophthora genus contains over 140 species, including the late blight pathogen Phytophthora infestans, which caused the Irish potato famine that led to death-by-starvation of over one million people in the 19th century. P. infestans infects both potato and tomato crops and is the most damaging oomycete pathogen in agriculture, causing economic losses in excess of $6 billion annually. Plant pathogens need to penetrate the plant cell wall in order to infect their host, and major aspects of the molecular struggle between the host and pathogen occur in the cell wall. Pathogens, such as P. infestans, secrete a range of cell wall degrading enzymes to facilitate host penetration. Many of these pathogen enzymes (such as polygalacturonase and pectin methylesterase) target the pectin components of plant cell walls. Pectins provide the glue that binds plant cells to one another, and degrading pectin allows the pathogen to grow through the middle lamella between plant cells. In response, plants have evolved a range of counter measures, including inhibitors of pathogen enzymes. These anti-pathogen responses are induced in plants following the detection of small molecules derived from breakdown of the host cell wall (such as pectic oligogalacturonides) by pathogen enzymes.We recently discovered a new class of lytic polysaccharide monooxygenase (LPMO) enzymes in oomycetes, which are notably abundant in plant pathogenic species, such as P. infestans. Three LPMO genes in P. infestans are highly expressed very early during plant infection, ands blocking the action of the most abundantly expressed of these impedes the pathogen's ability to infect plant tissues, suggesting it is important for host penetration. Previously characterised classes of LPMOs are oxidative enzymes able to digest the most recalcitrant forms of polysaccharides such as crystalline cellulose or chitin. We have shown that the most expressed P. infestans LPMO oxidatively attacks polygalacturonan, the main pectic polymer in plants. This is the first time LPMOs have been shown to attack pectins, indicating this enzyme has evolved specifically to facilitate host penetration, perhaps helping the pathogen to evade the polygalacturonase- and pectin methyl esterase-inhibiting proteins produced by the host. Previously published work has shown that oxidised oligogalacturonides do not induce pathogen responses in plants, suggesting that LPMO attack may help the pathogen to slip under the host's radar. Gene expression studies indicate a range of other, uncharacterised, secreted enzymes are produced early in infection. We propose to study the roles of these proteins during infection and assess their importance in the ability of the pathogen to infect plants.To achieve our aims we will generate an inventory of proteins found in the host cell wall during early pathogen infection using highly sensitive proteomic approaches. This will provide a comprehensive view of pathogen and host proteins expressed during infection. We will select a subset of these proteins for further study including the LPMO family. The selection of targets for study will be based on their novelty and their importance in pathogenesis based on gene silencing studies. We will study the biochemical activity of recombinant versions of target proteins, characterise their 3-dimensional structure, and study their specific localisation in the cell wall during infection. The results of our studies will provide new understanding of enzymes and proteins produced by pathogens during infection and by host plants in response to this. The pathogen genes that we show to be important for infection (such as the LPMOs) will provide new molecular targets for targeted crop protection strategies.