The role of endocytosis for pathogenic development of the corn smut fungus Ustilago maydis

It has long been established that fungal growth and pathogenic development are supported by exocytosis. However, the role of endocytosis during these growth processes has not been elucidated yet. It was the aim of the present study to use the temperature-sensitive mutant of the endocytic t-SNARE Yup1, to analyse the importance of endocytosis for fungal growth and pathogenic development in Ustilago maydis. U. maydis is a basidiomycete fungus that infects maize plants and causes the formation of tumors in the corncob and other parts of the plant. Pathogenic development is initiated by pheromone recognition of compatible haploid partner cells. Yup1ts cells are defective in pheromone recognition and do not initiate the subsequent mating reaction. Key player for pheromone recognition is the G-protein coupled pheromone receptor Pra1. In the absence of pheromone, biologically active Pra1-GFP is constitutively endocytosed from the plasma membrane and degraded in the vacuole. After pheromone ligand binding, growth of conjugation hyphae is initiated and Pra1-GFP localises to the tip of conjugation hyphae while in yup1ts cells, Pra1-GFP accumulates in small endocytic vesicles, which are no longer able to fuse with early endosomes. These findings suggest a role for Yup1 on early endosomes at the intersection of incoming endocytic vesicles and the degradation pathway as well as a putative recycling pathway. Therefore recycling and degradation of Pra1 are inhibited in yup1ts cells. In an independent experiment it was found that wild-type Pra1-GFP is recycled. Thus it is thought that Pra1-GFP is depleted from the plasma membrane due to malfunction of Yup1. The recycling deficiency of Pra1-GFP in yup1ts results in the defect in pheromone perception. Interestingly, increased expression of Pra1-GFP can rescue this perception defect. However, even the restored partner recognition could not resolve the subsequent mating defects identified for cell-cell fusion, while filamentous growth, formation of appressoria and plant infection which follow cell-cell fusion, are only affected to a small extend. Interestingly, the tumors that are formed in the maize plant after Ustilago infection are empty, indicating a lack of teliospore formation in the absence of yup1-mediated endocytosis. Independently, germination of teliospores is reduced and the promycelium shows morphological alterations in yup1ts cells. Endocytosed cargos responsible for these phenotypes have not been determined so far. First indications point to an involvement of chitin synthases. One of their representatives, the myosin chitin synthase 1 (Mcs1) was shown to be endocytosed in a yup1-mediated fashion. The presented results are supported by the analysis of the U. maydis genome, which reveals the presence of components commonly discussed to mediate endocytosis. Taken together the results clearly proof the existence and the importance of endocytosis for growth and virulence in the filamentous fungus U. maydis