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

丝状真菌产生许多小分子天然产物，这些产物往往具有有趣的生物学特性。这个项目的重点是稻瘟病菌产生的一种特殊的(但尚不清楚的)天然产物。这种生物会引起稻瘟病和水稻秧苗枯萎，并造成全球稻米产量的每年损失，估计相当于6000万人每年的稻米消费量。已观察到的稻瘟病菌无毒菌株具有一个生物合成基因簇(称为ACE1簇)，该基因簇似乎与小分子信号化合物的生物合成有关。当这种化合物被抗性植物检测到时，植物可以对入侵的真菌病原体建立有效的防御。了解无毒信号分子的结构可以开发新的农用化学品，刺激植物建立自己对真菌病原体的天然防御。然而，无毒信号分子的结构尚不清楚。ACE1基因簇由15个基因组成，这些基因仅在真菌侵染植物角质层时表达，特别是在(单细胞)真菌顶胞穿透过程中表达。这意味着信令化合物的量非常低，并且产生的时间非常短(约12小时)。我们的目标是通过使用结构性启动子在异源真菌宿主中表达来自ACE1簇的基因来确定无毒信号代谢物的结构，以确保高水平的蛋白质生产和随后的代谢物生物合成。由此产生的化合物将被提纯，其结构使用核磁共振和质谱仪进行鉴定，并在各种生物筛选中进行测试。在前期工作中，我们研究了一组有限的ACE1基因。核心生物合成基因ACE1本身编码一个高度还原的聚酮合成酶(PKS)，与非核糖体肽合成酶(NRPS)的单个模块融合在一起。我们在利用异源表达研究真菌中的PKS-NRPS系统方面拥有广泛的专业知识。已知ACE1PKS的Enoyl Reductase(ER)结构域是不起作用的，在平行系统中，ER结构域通常被另一种蛋白质提供的反式作用ER所取代。然而，虽然ACE1与来自该簇的ER编码基因(Rap1)的共表达确实导致了一种新化合物的产生，但这被证明是没有生物学活性的。在这个项目中，我们将系统地表达ACE1基因簇的组合，以确定“正确的”化合物。我们还将研究稻瘟病菌相关化合物Prichalasin H的生物合成，目的是利用其基因簇构建新的嵌合簇，用于生产新的相关生物活性化合物。法国的学术合作伙伴将对新化合物的无毒特性进行测试。

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.