Investigating Natural Products involved in Avirulence Signalling between Rice and the Fungal Rice Blast Pathogen Magnaporthe oryzae

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Filamentous fungi produce numerous small molecule natural products which often possess interesting biological properties. This project focusses on a particular (but as yet unknown) natural product produced by the fungal rice pathogen Magnaporthe oryzae. This organism causes rice blast disease and rice seedling wilt and causes global annual losses in rice yield estimated to be equivalent to the annual consumption of rice by 60 million people. It has been observed that avirulent strains of M. grisea possess a biosynthetic gene cluster (known as the ace1 cluster) which appears to be responsible for the biosynthesis of a small molecule signal compound. When this compound is detected by resistant plants, the plants can mount an effective defence against the invading fungal pathogen. Knowledge of the structure of the avirulence signal molecule could allow the development of new classes of agrochemicals which stimulate plants to mount their own native defences against fungal pathogens. However, the structure of the avirulence signal molecule is unknown. The ace1 gene cluster consists of 15 genes which are expressed only during the event of invasion of the plant cuticle by the fungal pathogen, specifically during penetration by the (single cell) fungal apressorium. This means that the amount of signalling compound is vanishingly low and the time period over which it is produced is very short (ca 12 h). We aim to determine the structure of the avirulence signalling metabolite by expressing genes from the ace1 cluster in a heterologous fungal host using constitutive promoters to ensure high levels of protein production and consequent metabolite biosynthesis. Compounds thus-produced will be purified, their structures elucidated using NMR and MS, and tested in various biological screens. In preliminary work we have investigated a limited set of the ace1 genes. The core biosynthetic gene, ace1 itself, encodes a highly reducing polyketide synthase (PKS) fused to a single module of a non-ribosomal peptide synthetase (NRPS). We have extensive expertise in investigating PKS-NRPS systems in fungi using heterologous expression. The enoyl reductase (ER) domain of the Ace1 PKS is known to be inoperative and it is usual in parallel systems that the ER domain is replaced by a trans-acting ER supplied by another protein. However while coexpression of ace1 with an ER-encoding gene (rap1) from the cluster did result in the production of a new compound, but this was shown to be biologically inactive. In this project we will systematically express combinations of the ace1 gene cluster to determine the 'correct' compound. We will also investigate the biosynthesis of the related compound pyrichalasin H from M. grisea with the aim of using its gene cluster to build new chimeric clusters for the production of new related bio-active compounds. New compounds will be tested for avirulence properties by academic partners in France.