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

Alternaria属真菌属于“黑色霉菌”，可以感染和殖民各种农作物，如西红柿和谷物。几种次生代谢物，如异丙醇（AOH）及其衍生物可对人类和动物健康产生严重影响，因此被归类为真菌毒素。二烯丙二烯酮（PQs），如交替毒素I-III （ATX）是交替毒素的另一个例子。互生草的基因组包含许多次级代谢物基因簇，其中它们负责哪个分子尚不清楚。本项目旨在研究次生代谢产物基因，特别是PQs替代毒素I-III的基因特征及其调控。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。似乎只有少量的PQ产生所必需的基因与聚酮合成酶基因pksA聚集在一起。为了鉴定其他PQ基因，将采用反向遗传学方法，因为ATXs的生物合成类似于Cercospora beticola中的cercosporin生物合成，其中一些基因是已知的。此外，还将对数据库中储存的Alternaria基因组序列进行进一步的候选基因分析。在某些分离株中，这些基因可能聚集在一起。此外，我们还将对高PQ生物合成条件下生长的刺草进行RNAseq分析，并与低PQ生物合成条件下的刺草进行比较。将产生相应候选基因的突变株，并对PQ及其中间产物进行分析。这将允许我们提出的PQs的生物合成途径。作为第二个目标，我们将研究PQ基因表达的调控。pq在毒力中的作用将用番茄来测试。最后，将研究剩余的五个基因簇，以至少确定它们负责的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.