New Enzymatic Virulence Factors In Phytophthora Infestans

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.

Plant infection by oomycetes requires penetration of the host cell wall. To accomplish host penetration, pathogens have evolved a range of cell wall degrading enzymes. We recently identified a new family of lytic polysaccharide monooxygenase (LPMO) enzymes in oomycetes. Our studies show that three LPMO encoding genes are highly expressed in Phytophthora infestans during early plant infection. We have characterised a recombinant version of the most abundantly expressed LPMOs and found that it specifically oxidises homogalacturonan (the major pectin in plants). Our preliminary studies show that inactivating the expression of this gene in P. infestans severely impeded the ability of the pathogen to infect plant tissues, indicating a potentially important role in host penetration. Transcriptomic studies reveal a number of other uncharacterised P. infestans genes that are induced in early infection and are potentially targeting the cell wall.

We propose to carry out targeted proteomic studies during the early stages of infection of tomato cells by P. infestans in order to produce a detailed inventory of proteins produced by the pathogen and host during this process. This will provide new insight into the molecular processes and interactions between host and pathogen during wall penetration, and provide us with a list of proteins for further study. We will make a short list of target proteins based on results from loss of gene function experiments, focusing on those that impede infection. We will produce recombinant forms of target proteins in order to characterise biochemical activity and protein structure. We will use molecular localisation studies to characterise the spatio-temporal functioning of the target proteins during infection. We will work with companies, such as Syngenta, in the agrochemical sector to develop strategies for targeting the encoding genes in a crop protection context.

The proposed work fits well with the BBSRC's strategic priorities of sustainably enhancing agricultural production by better countering of diseases or pests (including weeds or parasites) of crops or farmed animals.
The work described in this proposal will benefit the private commercial sector by identifying new virulence factors produced by major crop pathogens, we will provide genes that can serve as targets for precision crop protection strategies using short double stranded nucleic acids for silencing critical pathogen-specific genes. Such approaches are being developed by major players in the agrochemical sector.
New carbohydrate active enzymes, such as the pectin-active LPMOs we recently discovered, may find utility as industrial enzymes. Pectinases are used widely in the food and beverage sector to modify food texture and clarify fruit juices. The York team has a close working relationship with Novozymes (a major producer of industrial enzymes) and will explore this potential with them and other potential industry partners in this sector.
We have assembled an extremely strong group of scientists to maximise the chances of the full promise of the scientific research being realised. In addition, we are committed to seeing that the impacts of the work are maximised. This will, in part, be achieved by working closely with potential industrial partners to ensure that gene targets and enzymes have a route for rapid industrial uptake. In addition, the team has a good record in terms of public outreach and communication.

We will use proven processes to protect IP and publish results in scientific journals and at conferences. We will also use existing UK networks (NIBB, KTNs etc.) to communicate progress through their events and web-based or printed media. When appropriate, discoveries will be disseminated by the University of York and James Hutton Institute to the general media through press releases. To ensure professional management of intellectual property, CNAP and JHI both operate regular IP reviews of all projects. CNAP has an outstanding track record in commercialisation of strategic research through on-going collaborations with companies throughout the biorenewable supply chain.
The programme will provide researchers with wide-ranging skills relevant to the establishment of a vibrant industrial biotechnology and bioenergy research and innovation-led industrial sector in the UK.