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，这些产物通常具有有趣的生物学特性。这个项目的重点是水稻真菌病原体Magnaporthe oryzae产生的一种特殊的（但尚不清楚的）天然产物。这种生物体引起稻瘟病和水稻幼苗枯萎病，并造成全球每年水稻产量的损失，估计相当于6 000万人每年消耗的大米。据观察，无毒菌株稻瘟杆菌具有一个生物合成基因簇（称为ace1簇），它似乎负责小分子信号化合物的生物合成。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进行有效的防御。对无毒信号分子结构的了解可以促进新型农用化学品的开发，这些化学品可以刺激植物对真菌病原体建立自身的天然防御。然而，毒性信号分子的结构尚不清楚。ace1基因簇由15个基因组成，这些基因仅在真菌病原体入侵植物角质层时表达，特别是在（单细胞）真菌前压胞渗透时表达。这意味着信号化合物的量非常低，而且产生的时间非常短（约12小时）。我们的目标是通过使用组成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纯化，它们的结构将用核磁共振和质谱进行阐明，并在各种生物筛选中进行测试。在初步工作中，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），融合到一个非核糖体肽合成酶（NRPS）的单个模块中。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Ace1 PKS的烯酰还原酶（ER）结构域不起作用，并且通常在平行系统中，ER结构域被另一蛋白质提供的反式ER所取代。然而，虽然ace1与来自该簇的er编码基因（rap1）共表达确实产生了一种新的化合物，但这被证明是无生物活性的。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pyrichalasin H的生物合成，利用其基因簇构建新的嵌合簇，以生产新的相关生物活性化合物。法国的学术合作伙伴将测试新化合物的无毒特性。

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.