Molecular mechanism and control of a fungal exocytosis pathway in the plant pathogens Ustilago maydis and Mycosphaerella graminicola

Pathogenic fungi are a major threat to human food security. While they are controlled by active pest management, fungi rapidly adjust and develop resistances. Thus, research on fungal specific and pathogenicity-relevant cellular processes is required to identify new targets for fungicide development. Fungi invade the host tissue by expanding at the end of their elongated cells. This process is named 'tip growth' and it involves the polar release of enzymes that participate in the formation of the fungal cell wall. We have recently reported that a particular fungal-specific myosin-chitin synthase-like protein (Mcs1) has a key role in tip growth in the corn smut fungus Ustilago maydis. This protein is also found in many other pathogenic fungi, where it also essential for plant infection. In this project we will join up with an industrial partner, Syngenta Ltd., to identify regulators that control this important Mcs1 protein. We will elucidate the role of a so far unknown Mcs1 protein in the fungus Mycosphaerella graminicola, the causal agent of Septoria tritici blotch on wheat, which is the most devastating wheat pathogen in the UK and Europe. In addition, we will use our existing knowledge and molecular tools in U. maydis to identify new factors that control the function of Mcs1. When considered together the project promises to provide fundamental new insight into a fungal specific pathway, essential for plant infection. This will provide knowledge that will help to develop new fungicides and therefore is of high value to the agricultural biotechnology industry in the UK.

This is an industrial partnership project that will analyze the structural and regulatory environment of the fungal-specific myosin-chitin synthase Mcs1 during exocytosis. We have recently shown that the myosin motor domain (MMD) of Mcs1 in the corn pathogen U. maydis (UmMcs1) is deispensible for delivery of vesicles, but serves local roles at the apical growth site. Unpublished data show that the MMD is required to prolong the tethering time of vesicles at the plasma membrane, and that altered pausing times result in defects in Mcs1 exocytosis and reduced virulence of the fungus. In collaboration with Syngenta Ltd. we have identified a Mcs1-like enzyme in the wheat leaf blotch fungus Mycosphaerella graminicola, named MgMcs1, which also localizes to the apical plasma membrane and is required for cellular morphogenesis. In a first part of the project, the biological function of MgMcs1 will be characterized by generating mutants in the motor domain and by complementation of the null mutant phenotypes in M. graminicola and U. maydis. In a second part, a two-hybrid screen is planned to identify interacting partners of the MMD of MgMcs1 and UmMcs1. This will be done with particular emphasize on a loop that is inserted in the MMD and that shows sequence conservation among Mcs1-proteins. In a third part, existing U. maydis mutants will be characterized that combine hypersensitivity to the chitin synthase inhibitor NikkomycinZ, severely attenuated plant pathology and abnormal GFP-Mcs1 localization. All these attributes are characteristic for mutant defective in Mcs1 exocytosis, suggesting that these mutants might lead to the identification of regulatory factors involved in Mcs1-dependent exocytosis. The characterization of all new factors derived from the two-hybrid screen or the complementation approach in the evolutionary distant U. maydis and M. graminicola will allow a more general approach in understanding this pathogenicity related exocytosis mechanism.

This work is industry-driven, and aims to understand fundamental processes in fungal cell biology which have great potential to be exploited in order to protect globally-important crops from fungal diseases. The human world population is constantly increasing - projections indicate that cereal production will need to increase by 40% by 2030 to help feed mankind. Currently, between 30-50% of all crop yield is lost due to pests and diseases (and can exceed 80% without effective pest management). Fungi are responsible for 7-15% of this loss, with some specific pathogens being particularly important. Mycosphaerella graminicola, the causal agent of Septoria tritici blotch on wheat, can cause up to 40% yield loss and is therefore considered the most devastating wheat pathogen in Western Europe. Whilst yield loss is generally considered moderate (when appropriate management strategies are used), there is a significant risk that rapidly-emerging resistance will provide a significant threat to current yields. Thus, developing new fungicides is a continuous challenge, and this study will provide knowledge critical to the future development of novel fungal pathogen control measures by provision of key molecular tools for two fungal model systems, the corn pathogen U. maydis and the wheat pathogen M. graminicola). This project focuses on how to disrupt the processes by which fungi are able to infect their hosts. Specifically, we intend to provide understanding of a pathogenicity-related process and, alongside, a toolkit for the development of fungal control strategies using Mcs1 enzymes; it is known that fungal-specific Mcs1 has an essential role in the virulence of numerous plant pathogenic fungi. Currently, we do not have any knowledge about the regulatory and structural environment that controls the activity of Mcs1 at sites of exocytosis. This project aims to address this lack of knowledge using economically important models. The proposed work is supported directly by Syngenta through the Industrial Partnership Award scheme which demonstrates its relevance to, and commitment from, industry. Through our partnership with Syngenta and appropriate publication we will provide fundamental and industry-orientated knowledge necessary to investigate the development of Mcs1-mediated fungal control strategies.