ABSTRACTUstilago maydis is a phytopathogenic fungus responsible for corn smut disease. Although it is a very well established model organism for the study of plant-microbe interactions, its biosynthetic potential has not been totally explored. By analyzing U. maydis genome, we identified a biosynthetic gene cluster whose activation led to the production of a black melanin pigment. Single deletion mutants of the cluster genes revealed that five encoded enzymes are required for the accumulation of the black pigment, including three polyketide synthases (pks3, pks4 and pks5), a cytochrome P450 monooxygenase (cyp4) and a protein with similarity to versicolorin B-synthase (vbs1). Moreover, metabolic profiles of the mutants defective for pks3 and pks4 indicated that the products of these genes catalyze together the first step in the melanin biosynthetic pathway since none of the mutants accumulated any melanin or intermediate products. Mutants deleted for pks5 produced orsellinic acid (OA) and triacetic acid lactone (TAL), suggesting that both products are produced by Pks3 and Pks4. It might thus demonstrate that Pks5 plays a role in a reaction downstream of that catalyzed by Pks3 and Pks4. OA and TAL were also found in extracts of a cyp4 deletion mutant along with several heterodimers of TAL and Pks5-derived orsellinic aldehyde compounds. According to their phenotypes and the intermediate products isolated from these strains, Cyp4 and Vbs1 seem to be involved in reactions downstream of Pks5. Our findings suggest that U. maydis synthesizes a new melanin based on coumarin and pyran-2-one intermediates, while most fungal melanins are derived from 1,8-dihydroxynaphthalene (DHN) or L-3,4-dihydroxyphenylalanine (L-DOPA). Along with these observations, this work also provides an insight into the mechanisms of polyketide synthases in this filamentous fungus.IMPORTANCEUstilago maydis represents one of the major threats for maize plants since it is responsible for corn smut disease, which generates considerable economical losses around the world. Therefore, contributing to a better understanding of the biochemistry of defense mechanisms used by U. maydis to protect itself against harsh environments, as the synthesis of melanin, could provide improved biological tools for tackling the problem and protect the crops. In addition, the fact that this fungus synthesizes melanin in a very unique way, requiring more than one polyketide synthase for producing this secondary metabolite, gives a different perspective on the complexity of these multimodular enzymes and their evolution in the fungal kingdom.
Pheromone-Induced G2 Arrest in the Phytopathogenic Fungus Ustilago maydis
