Oomycete plant pathogens, such as Phytophthora species, are major sources of crop loss in agriculture. Their control relies heavily on application of chemicals. Phytophthora infestans is the most notorious oomycete, causing late blight disease of potato and tomato. It is also a model species for understanding oomycete pathogenesis. Little is known about pathogen nutrition and metabolism during infection, which may be important determinants of pathogenicity. During infection, the majority of pathogen biomass is located primarily in the plant apoplast. Previously we have shown that P. infestans utilises diverse nutrients from the apoplast, and that amino acids glutamate and aspartate are essential for infection development. Nutrients taken up by P. infestans can potentially enter pathogen metabolic pathways at multiple points, but the pathway(s) important to the pathogen during infection remains to be determined.
Aims: This project aims to examine metabolism in Phytophthora pathogens by determining the fate of carbon and nitrogen through metabolic pathways in P. infestans and identify essential components of P. infestans metabolism.
Experimental Approach: The fate of specific nutrients in P. infestans metabolism will require experimental analysis of P. infestans biomass composition to establish biomass constraints: metabolites, nucleic acids, proteins, lipids, phospholipids and cell wall constituents will be measured. P. infestans will be incubated with stable isotope labelled carbon sources (e.g. [13C6]glucose or [13C]amino acids) or nitrogen sources (e.g. [15N]glutamine or [15N]urea). Major biosynthetic end products will be determined using GC-MS and metabolic fluxes deduced using flux analysis software. The resulting data will be analysed in the context of existing genomic and transcriptomic data to confirm the presence of the appropriate pathways and their expression through the pathogen lifecycle.
Subsequently, the project may be developed in a variety of directions such as:
(i) Focus on a specific aspect of P. infestans metabolism, to determine changes in metabolite abundance through the pathogen lifecycle, and the effects of perturbation by known pharmacological inhibitors or crop protection chemistries.
(ii) Select genes for expression knockdown using RNA silencing to determine if they are essential for pathogen survival.
(iii) Comparison of P. infestans metabolic pathway activities with additional, broad host range Phytophthora species such as P. capsici, to identify common attributes.
(iv) Develop a strategy for testing Phytophthora metabolic activity during infection of plants.
Outcomes: The project builds on recent knowledge of plant metabolites utilized for P. infestans growth and will deliver the first detailed understanding of the metabolic pathways used by P. infestans and related pathogens to generate the energy and metabolites for growth and completion of the lifecycle. Resolution of the metabolic pathways preferred by P. infestans to complete its infection lifecycle, and the impact of the host environment on metabolic activity may provide an insight into the suitability of plant hosts for colonization by Phytophthoras and tools to help chemical validation of pathways and targets for the development of new control chemicals.
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Ah-Fong AM, Kim KS, Judelson HS. RNA-seq of life stages of the oomycete Phytophthora infestans reveals dynamic changes in metabolic, signal transduction, and pathogenesis genes and a major role for calcium signaling in development. BMC Genomics. 2017; 18(1):198. doi: 10.1186/s12864-017-3585-x.
Whisson SC, Boevink PC, Wang S, Birch PR. The cell biology of late blight disease. Curr Opin Microbiol. 2016; 34:127-135. doi: 10.1016/j.mib.2016.09.002.