Molecular analysis of secondary metabolite formation in Alternaria alternata with a special focus on perylene quinones (PQs)

链格孢属真菌属于黑霉菌，可侵染和侵染番茄、谷类等多种农作物。交链孢醇(AOH)及其衍生物等几种次生代谢物对人类和动物的健康有严重影响，因此被归类为真菌毒素。戊二酚酮(PQS)，如交链孢毒素I-III(ATX)是链格孢霉毒素的另一个例子。互隔交链孢霉基因组包含许多次生代谢物基因簇，目前尚不清楚它们与哪个分子有关。拟议的项目旨在描述次生代谢物基因，特别是PQS交替毒素I-III的基因及其调控。在初步实验中，发现PQ和黑色素的生物合成共享最初的聚酮合成。似乎只有几个产生PQ所必需的基因与聚酮合成酶基因pksA聚在一起。为了确定其他PQ基因，将遵循反向遗传学方法，因为ATXs的生物合成类似于Cercospora beticola的Cercospora beticola的生物合成，其中一些基因是已知的。此外，保存在数据库中的交链孢属基因组序列将被分析以寻找进一步的候选基因。在一些分离株中，基因可能是聚集在一起的。此外，还将对在高PQ生物合成条件下生长的交链孢子进行RNAseq分析，并与低PQ合成条件下的RNAseq进行比较。将产生相应候选基因的突变菌株，并对PQ和中间体进行分析。这将使我们能够提出互隔交链孢子中PQS的生物合成途径。作为第二个目标，我们将研究PQ基因的表达调控。PQS在致病力中的作用将在番茄上进行测试。最后，将对剩余的五个基因簇进行进一步研究，以至少确定它们负责生物合成的相应分子。

Fungi of the genus Alternaria belong to the “black molds” and may infect and colonize a variety of agricultural crops such as tomatoes and cereals. Several secondary metabolites, such as alternariol (AOH) and its derivatives can have serious effects on human and animal health and are therefore classified as mycotoxins. Perylenequinones (PQs), such as altertoxin I-III (ATX) are other examples of Alternaria toxins. The genome of A. alternata contains a number of secondary metabolite gene clusters, where it is unknown for which molecule they are responsible. The proposed project aims at the characterization of secondary metabolite genes, especially for the PQs altertoxins I-III, and their regulation. In preliminary experiments it was found that PQ and melanin biosynthesis share the initial polyketide synthesis. It appears that only a few genes necessary for PQ production are clustered together with the polyketide synthase gene, pksA. In order to identify other PQ genes, a reverse genetics approach will be followed, because ATXs biosynthesis resembles cercosporin biosynthesis in Cercospora beticola, where some genes are known. In addition, the Alternaria genome sequences deposited in the databases will be analyzed for further candidate genes. It is possible that in some isolates the genes are clustered. Furthermore, RNAseq analyses of A. alternata grown under conditions of high PQ biosynthesis will be performed and compared with conditions of low PQ production. Mutant strains of the corresponding candidate genes will be generated and analyzed for PQ and the intermediates. This will allow us to propose the biosynthetic pathway for PQs in A. alternata. As a second objective, we will study the regulation of PQ gene expression. The role of PQs in virulence will be tested with tomatoes. Finally, five remaining gene clusters will be studied to an extend to at least identify the corresponding molecule for which biosynthesis they are responsible.