The dynamics of secretory vesicles in living hyphae of the pathogen Ustilago maydis.

Filamentous fungi are an evolutionarily successful group of organisms of enormous ecological importance as symbionts in mycorrhizal interactions with plants and decomposer of plant debris. They serve in industrial production of proteins and as pathogens pose a threat to public health and agriculture. The basic unit of a filamentous fungus is the hypha; this usually consists of a chain of elongated cells that grow by expansion at the tip, a process called tip growth. This mode of growth allows the invasion of tissue and substrate. Tip growth requires a continuous supply of newly synthesised membranes, proteins and cell wall precursors to the hyphal tip in a process named secretion. The carriers for these supplies are secretory transport vesicles that are taken to the tip where they fuse with the plasma membrane. It is important in the context of this grant application to note that our current view of secretion is that post-Golgi vesicles travel uni-directionally to the hyphal tip where they fuse with the plasma membrane, thereby delivering membranes and proteins. The mechanism by which secretory vesicles are delivered to the hyphal tip is not clear. In analogy to other polarised cells, such as animal neurons, it is assumed that microtubules and kinesin motors mediate long-distance transport of secretory vesicles, whereas short range motility at the plasma membrane is mediated by another system, consisting of myosins and filamentous actin (F-actin). Indeed, recent studies on numerous filamentous fungi, including the plant pathogen Ustilago maydis, demonstrated that microtubules, kinesins, F-actin and myosins are essential for hyphal growth. However, most conclusions from these data are speculative. This due to the fact that motility of secretory cargo was never visualised and no systematic studies on the whole repertoire of motors in a cell have been undertaken. In this project we will address this challenge. We have developed a microscopic setup that allows us to visualise the motility of individual chitin synthase-containing vesicles. We will label numerous other secretory proteins, including secreted enzymes, and will monitor their delivery to the growing hyphal tip. In co-localisation studies using red and green fluorescent proteins we will determine whether cargo travels in the same or different transport vesicles, thereby elucidating the pathways of secretion. We will determine the cytoskeletal elements that underlie vesicle motility and address the role of all kinesin and myosin motors in secretion by making use of existing mutant protein constructs. Finally, we will further investigate the reason for the bi-directional motility of chitosomes. This behaviour is unexpected and we will investigate whether it is a general feature of secretory vesicles. Subsequently, we will use photoactivatable fluorescent proteins to characterise this motility in order to get an insight into the reason for this phenomenon. In summary, we will combine molecular genetics and life cell imaging to: (1) determine the pathways of secretion in the hyphal cell, (2) address the role of 10 kinesins and 4 myosins in motility of secretory vesicles, (3) characterise the bi-directional motility of secretory vesicles in order to get to an understanding the logic behind this behaviour. The expected outcome of this project will be novel insights into the secretory pathway in filamentous fungi. We will provide a comprehensive understanding of the pathways by which cargo reaches the hyphal tip and of the role of kinesins and myosins in delivery of secretory vesicles. If it is discovered that bi-directional motility of secretory vesicles is a general feature, the current paradigm for secretion will have to be modified. This project will therefore be of fundamental interest to all aspects of fungal research, but it will be of particular importance in understanding fungal pathogenicity and industrial production of recombinant proteins.

In the filamentous fungus Ustilago maydis microtubules and F-actin are essential for extended growth. Hyphae contain both microtubules and long actin cable (Fuchs et al. 2005, Mol Biol Cell, 16:2746), suggesting that both are involved in secretion. Indeed, class V myosin cooperates with kinesins in polarised growth and pathogenicity (Weber et al. 2003, 15:2826; Schuchardt et al. 2005, Mol Biol Cell, 16:5191). However, to date no secretory cargo of any motor is known in filamentous fungi. We recently succeeded in visualising the motility of chitosomes carrying the class V chitin synthase Mcs1, which is essential for pathogenicity of U. maydis (Weber et al. Plant Cell 2006, 18:225). Surprisingly, we found that secretory chitosomes move bi-directionally and unpublished evidence exist that this long-range motility is mediated by actin and myosin V (Treitschke et al., submitted). These findings raise the possibility that: (1) secretion in fungi is not a uni-directional process and (2) long-distance transport of secretory cargo involves both microtubules/kinesins and F-actin/myosins. Both of these possibilities challenge the current concepts of secretion in filamentous growth of fungi. In this project we address secretion in U. maydis in more detail. We will investigate the motility of 12 secreted proteins, ranging from exo-enzymes to cell wall synthesising enzymes. We will generate GFP or mCherry fusion proteins and analyse their motility using ultra-sensitive laser-based epi-fluorescence in a qualitative and quantitative way. Co-localisation studies will elucidate which cargo co-migrates in the same transport vesicle. Subsequently, we will make use of numerous existing molecular tools, including dominant-negative mutant alleles of all kinesins and myosins and triple-tags of photoactivatable GFP and mCherry, to further characterise the role of the cytoskeleton in secretion and the dynamics of bi-directional chitosome motility in hyphae.

1. Exploitation and application Knowledge gained from this study promises to help the identification of novel targets for fungicide development as well as in the industrial production of recombinant proteins. Specifically, we intend to integrate the following commercialisation activities into our study: i. At the start of the project, the PI and project staff will meet Research & Knowledge Transfer's IP specialist in order to agree a strategy to protect intellectual property and consider potential commercialisation opportunities. ii. We will agree milestones, which will trigger further meetings in order to ensure that exploitation and application issues will be considered throughout the grant lifecycle. iii. During each meeting we will determine whether the project is sufficiently advanced to establish or investigate commercial partnerships. We have identified two initial potential partners - Professor Steinberg has active and established relationships with both organisations and they each could provide opportunities to commercialise the work. 2. Communications and Engagement In addition to our standard communication and engagement activities (publishing in appropriate journals, conference attendance, and presenting scientific seminars) we intend to conduct the following activities: i. Promotion of careers in the biosciences. During September 2009, Professor Steinberg participated in our 'Britain needs Bioscientists' one day conference. The event attracted 150 year 12 and 13 students from ten schools from Dorset, Devon and Cornwall. In addition, he visited schools and gave talks and organized an open day for excellent pupils. We intend to participate in this event again and also continue our outreach activities by conducting further school visits. ii. Molecular Fungal Cell Biology website. Prof. Steinberg's group has an established website (http://www.gerosteinberg.com/). The site currently hosts a range of teaching resources and we will enhance this website by including further information based on this study. This project will be integrated into the existing website using an experienced web developer. iii. Public Understanding of Science. The School of Biosciences is committed to the public understanding of science. We will prepare and submit articles to popular science magazines in order to develop public interest and awareness of this exciting topic. The School has been very successful in promoting science to the wider public and we will, with support from our press office, ensure that results will be disseminated widely. 3. Capability The PI will have overall responsibility for ensuring the delivery of this impact plan. The appointed postdoctoral research assistant will be expected to contribute substantially to these activities and will be given appropriate training. i. PDRA training. We will ensure that the PDRA selected is provided with the opportunity to develop their awareness of, and skills in, knowledge exchange so that we can contribute to training the next generation of scientists. Specifically, we will: - Provide knowledge-exchange mentors (the PI and a member of the university's Research & Knowledge Transfer section) who will meet regularly with the PDRA to provide training support and conduct a yearly review; - Organise a secondment for the PDRA to work with Research & Knowledge Transfer; - Encourage the PDRA to engage in other activities such as participating in a Biotechnology Young Entrepreneurs' Scheme event. - Expect the PDRA to participate in all the IP and commercialization meetings during the project. - Expect the PDRA to author an article for the popular scientific press and conduct school visits. ii. Support for impact activities. The University of Exeter has extensive expertise in promoting impact activities. The project team will be supported by staff from the School of Biosciences and from the university's Research & Knowledge Transfer section.