Genomic Analysis of Secondary Metabolism in U. maydis
Summary Ustilago maydis is a well established model organism for the study of plant-microbe interactions although its biosynthetic potential has not been totally explored. Therefore, in this work we focused our attention on identifying potential secondary metabolite (SM) gene clusters by mining U....
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Ustilago maydis is a well established model organism for the study of plant-microbe interactions although its biosynthetic potential has not been totally explored. Therefore, in this work we focused our attention on identifying potential secondary metabolite (SM) gene clusters by mining U. maydis genome. The combination of different strategies as manual annotation and bioinformatic approaches allowed us the detection of 4 potential SM gene clusters (A-D). The further selection of cluster A as a subject of this study, was based on its chromosomal location and the analysis of gene expression profiles among members of each cluster. Such analysis was possible due to the construction of an excel table in which all available U. maydis gene expression data from Gene Expression Omnibus were compiled and normalized. Overexpression of the transcription factor Mtf1 in cluster A resulted in the activation of at least 12 genes including three polyketide synthases (pks3, pks4 and pks5), a cytochrome P450 (cyp4) and a versicolorin B synthase (vbs1), among others. Prolonged induction of cluster A triggered the production of a black-greenish pigment mainly composed of 1,3,6,8-tetrahydroxynaphthalene (T4HN), therefore cluster A was named as the melanin-like cluster. This result showed that U. maydis synthesizes melanin using an unusual pathway, since most fungal melanins are derived from DHN, whose precursor is T4HN. Mutants defective for pks3, pks4, pks5 and cyp4 did not accumulate melanin, indicating a crucial role of these genes at the first stages of its biosynthesis. Deletion of cyp4 produced orsellinic acid (OA) and two of its derivatives. Interestingly, a feeding experiment with OA rescued the melanization defect of pks3 and pks4 deletion mutants. Moreover, the simultaneous expression of the pks3 and pks4 genes produced OA, suggesting that both genes are involved in OA biosynthesis, which is then used as a substrate for further chemical conversion into T4HN, a reaction presumably catalyzed by Cyp4 and/or Pks5. Overexpression of pks1, a polyketide synthase gene in U. maydis previously reported to play a role in melanization together with pks2 and lac1, could rescue the phenotype in the strain MB215 pks3 Pcrg::mtf1, suggesting that Pks3 and Pks1 have complementary functions. Maize seedlings infected with single deletion mutants of the melanin-like cluster genes showed no effect on spore coloration and had only a minor effect on virulence, supporting the previous finding that pks1 and pks2 are the major contributors of melanization during sporulation. On the other hand, SG200 pks3, SG200 pks4 and SG200 pks5 showed no significant differences compared to the wild type (SG200) when exposed to hydrogen peroxide, indicating that melanin-like cluster genes may be involved in other kind of stress responses, hence further experiments need to be performed to understand the conditions under which the melanin-like cluster is activated.