Directional control of extreme polar growth in filamentous fungi

A key aspect of growth and development is the control of cell shape. The basis of this is the ability for a cell to establish and maintain polarity, each cell having a defined orientation. An extreme example are neurons, which are able to extend in a linear fashion over the long distances necessary for their role in signal transduction throughout an organism. Similarly filamentous fungi produce linear hyphae, which extend with branching, to form a network. The processes by which fungal cells and other Eukaryotes polarise to form a front are highly conserved, as such fungi represent a good model for understanding the fundamental biological processes that determine polarity.

In addition to providing a good model organism for other cells, the study of fungi is important in its own right. Fungi are the major cause of plant disease and food spoilage, being responsible for the loss of 10-20% of crop production worldwide. They are also important clinical pathogens, causing more deaths than either malaria or tuberculosis. However, fungi are of major benefit in industrial biotechnology, food production, as symbionts increasing crop yields and are fundamental to our ecosystem through biomass degradation and nutrient cycling. Hyphal growth is the means by which fungi explore and enter host tissue, and secretory enzymes can be released from hyphal tips during growth. As such understanding how hyphal growth is controlled has potential impact all the areas outlined above.

Polarity in all Eukaryotic cells is controlled by the same set of proteins, the Rho-GTPases. The action of these proteins is very versatile; in neuronal cells, for example, they create one front during axon formation, and multiple fronts during dendritic spine formation. The same set of proteins can orchestrate varying numbers of fronts for varying periods of time because of the differences between the interaction networks they are embedded within. In this project we will look at one such case, present at the hyphal tip, to understand how these very important Rho-GTPases are tuned to direct the extreme polarised growth. When fungi are grown in favourable conditions they tend have straight hyphae. A set of proteins has been found that is necessary for the straight hyphal morphology, termed 'cell-end-marker proteins'. The function of the cell-end-marker proteins and the Rho-GTPases, in directing growth, overlap. However, it is not yet known how the two sets of proteins interact and how the cell-end-marker proteins tune the Rho-GTPases to ensure persistent unidirectional growth.

Cell polarity is fundamental to most biological systems and processes. In the case of filamentous fungi the highly polar growth of hyphae underpins many aspects of fungal biology, including development, colonisation of habitats, invasion of hosts, fungal interactions, reproduction and the secretion of proteins and chemicals. Currently, work on polar growth has focused primarily on the Cdc42 polar module which is conserved across eukaryotes. Less well characterised is the fungal specific TeaR/Mod5 polar module. In filamentous fungi both modules are present but to date no research has focused on how they work together. Another central and broadly significant question is how these modules can be modulated to produce distinct readouts. For example, transitioning from a single point of polar growth to multiple points, as is seen in neuronal development. A specific example of this is tip splitting which we will explore. Similarly, by understanding the attributes of specific polarity mechanisms, their biological consequences can provide new insights into important processes, as we have shown previously with respect to Cdc42 characteristics underpinning chemotropism in yeast.

Central to our work will be the creation of a 3D model describing growth as a direct consequence of protein dynamics and membrane insertion at the hyphal tip. This model will then be used as a tool to test and develop hypotheses, being refined by incorporating new data as it emerges. To facilitate phenotypic analysis we will use mathematical tools to measure hyphal morphologies, allowing quantitative characterisation and comparison of real and in-silico morphologies. Laboratory based work will include the identification and characterisation of proteins that interact with the polarity modules and a detailed assessment of module components with respect to localisation and dynamics within the cell and how these relate to phenotype.

This project has potential impacts in health, agriculture & biotechnology & indirect impacts as a model for mammalian or plant cells. The technology, methodologies, computational tools, strains & data developed & produced during the course of this will be made freely available to the scientific community. Potential areas for impact are described below.
1.) Agriculture
1.1) Pathogenicity
Each year pathogenic fungi destroy crops that could feed 600 million people. The main defence is the use of fungicides. Good practice involves the rotation of fungicides but options are very limited. Excessive use leads to resistance which also impacts on their clinical use. Current fungicides focus on compromising cell wall maintenance or interfere with energy production. An under explored target is to disrupt polarity & filamentous growth. Filamentous growth is the route by which; pathogenic fungi enter host cells, secret virulence factors, obtain nutrients & invade new tissue. Developing fungicides to target filamentous growth could be a valuable strategy.
1.2) Symbiosis
Nitrogen, phosphorous, potassium & magnesium are important nutrients for plant fitness & crop production. Most worked soils lack sufficient nutrients. To improve nutrient levels large amounts of fertilizer are added, with commensurate environmental implications. Mycorrhizal fungi live symbiotically with the roots of numerous plants. They improve nutrient & water uptake, release nutrients to the plants via degradation of organic matter & improve disease resistance. As the plant/ fungal interaction is via fungal hyphae, a better underst & ing of hyphal growth could contribute to the development of more efficient symbiotic strains.
3) Health
3.1) Fungal infection
300 million people, worldwide, suffer from fungal infections every year, with a morbidity rate of 1.35 million. This death toll is comparable to diseases such as malaria & tuberculosis (1.2 & 1.4 million deaths/ year, respectively, www.gaffi.org). Invasive fungal infections are difficult to treat because of the conservation between fungal & host cells. By understanding better the mechanisms of polar growth it will be possible to inform production of drugs to be used.
3.2) Fungi as model organism
Mathematical modelling is a tool already proven to benefit drug development & it is commonly used in the drugs industry. Polarity & morphology defects are implicated in many diseases. For example, Alzheimer's is characterised by a build-up of amyloid plaques in the brain plasma of sufferers. These plaques result in an abundance of active Rho-GTPase within neuronal cells causing morphological defects in, & loss of, dendritic spines. The mathematical tools developed in this project can be used to investigate the morphological effects of drug-induced changes to underlying cell biological mechanisms controlling dendritic spine plasticity, for example.
4) Biotechnology
Filamentous fungi are of major importance in industrial biotechnology. They are used extensively in the production of bulk chemicals (eg 98% of citric acid production) & specialist high value products (eg antibiotics & statins), enzyme production both as a source & production organism (eg biomass degradation), heterologous protein production (eg pharmaceuticals) & directly in the food industry for the production of various fermented products. Hyphal morphology is of direct relevance: The morphology of the fungus is critical when developing high volume production in fermenters. Protein & metabolite secretion is often linked to the morphology, most secretion takes place at the hyphal tip. Therefore an understanding of how the tip is controlled & can be manipulated is of significant potential.
4) Education & outreach
The group will host & maintain a project website which will be linked from all members' University webpages. Material to promote biology, mathematics, & computer science to year 9, 10 & 11, students.