The role of RIPP proteins in plant pathogenic fungi.

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Recently a small secreted toxin called Ustiloxin has been characterised from Aspergillus sp. It is thefirst Ribosomally synthesised Post-translationally modified Protein (RiPP) of its class in fungi, where asmall peptide sequence (present in multiple tandem copies) is excised from a larger peptide thencyclised and extensively modified, indeed the modifications make it impossible to distinguish suchproducts from NRPS-derived natural products, the only other fungal RiPPs to date are the amatoxinsfrom Amanita sp (fly agaric, deathcap & etc), fungi famous for having wide ranging biological activities.The Wheat-pathogenic fungus Zymoseptoria tritici has a similar gene cluster to ustiloxin within itsgenome, and RNAseq data shows abundant expression during the biotrophic growth stages, peakingat the transition to necrotrophic growth of this major wheat pathogen. This is the sort of expressionprofile that might be expected from an effector molecule, perhaps being used to minimise hostdefence reactions during early stages of infection. Other studies have also shown this locus to bevariable between isolates, suggesting that it is under diversifying selection as might be expected ifinvolved in host-pathogen interactions where the hostplant is undergoing frequent genetic change(due to the activity of plant breeders). The gene for the core protein is embedded in a co-expressedgene cluster with genes for various tailoring functions also present so that the chemical structure ofthe final mature compound cannot be readily predicted - other than its core peptide sequence and alikely cyclisation event involving an internal tyrosine residue.The project will aim to identify the final mature metabolite made by the fungus, to characterise itsrole (if any) during disease and to then seek to explore the wider implications of this class ofmetabolite in various host-pathogen interactions.The project will involve genomic and genetic analysis of Z. tritici and related fungi, using genedisruption techniques to explore the role of the gene cluster in disease in these fungi. It will alsoinvolve heterologous expression of the genes as a synthetic biology approach to explore thebiosynthetic pathway for this compound, coupled with analytical chemistry to determine the structureand conformation of the metabolites.