Analysis of the mechanism of cytoskeletal reorganisation in plants in response to pathogenic fungi

The actin cytoskeleton is an organised network of protein filaments that controls many cellular processes including the movement of organelles and vesicles within the cell. These filaments consist of chains of individual actin proteins. Importantly, this network is dynamic, as actin proteins can rapidly change from being free monomers in the cell cytoplasm (known as G-actin) to being incorporated into filaments (F-actin) and vice-versa. The assembly and disassembly of actin filaments is under the control of actin binding proteins (ABPs) which are in turn under the control of cell signalling pathways. A change in the environment of the cell or a developmental cue can stimulate signalling networks to cause the re-organisation of actin filaments through localised changes in ABP behaviour. Each ABP has a specific function and their activities are often intertwined in cooperative and/or competitive interactions. Some of these proteins nucleate actin filaments, others modulate monomer or filament dynamics through their binding to G-actin and/or F-actin. The state of the actin network at any given time and in any given space will depend upon the summation of the activities of each of these proteins. Plant pathogens cause damaging diseases of economically important plants, with approximately 6% of the UK wheat harvest currently being lost to disease. The actin cytoskeleton is a key element of plant defence against pathogens. At the earliest stages of pathogen invasion the actin network is stimulated to reorganise so that vesicles containing wall-forming materials can be transported to the site of the infection threat to thicken and reinforce the plant cell wall. If the actin cytoskeleton is broken down the chances of successful infection are greatly increased. This project is designed to understand the molecular events controlling this response of actin and its associated proteins to pathogen attack. How it works is currently unknown, but we do have an indication that ABPs are important and that their activity can protect plants from disease. One of the few disease resistance genes in cereals to a particularly virulent pathogen Puccinia graminis Ug99 is rpg4, and this encodes a small G-actin and F-actin modulating protein called Actin Depolymerising Factor (ADF). ADF is just one of a plethora of ABPs that control the actin network and we have evidence that at least one other group of ABPs is involved; the actin-nucleating formin proteins. Interestingly, formin proteins are controlled in animal and fungal cells by signal transduction pathways that do not exist in plants. Plants have adapted their formins to plant specific needs and we have found that a particular plant formin, (AtFH4), interacts with an enzyme called a respiratory burst oxidase that has an established role in signalling in the defence of plants against pathogens and this programme also aims to understand the functional significance of this interaction. For our experiments we use model systems and the model we use here for plant pathogen attack is Arabidopsis thaliana. An important question is whether this model relates to real-life situations in the UK's most important crop species. Here we will examine whether similar mechanisms of actin reorganisation in plant defence occur in cereals in response to disease, and we will use wheat for this purpose.

The actin cytoskeleton reorganises and guides wall-forming vesicles to the site of pathogen attack to thicken the cell wall and impede pathogen ingression. The mechanism of actin reorganisation is currently unknown be it by F-actin reorganisation and/or de novo nucleation of actin filaments. Actin cytoskeleton dynamics are controlled by a plethora of actin binding proteins working cooperatively and/or competitively in signal transduction pathways that still need to be fully defined. Using advanced light microscopy techniques TIRF/VAEM and CLSM /spinning disk confocal microscopy (recently purchased using a BBSRC REI grant) we will examine the dynamics of actin reorganisation upon pathogen attack (Blumeria graminis). We will use our catalogue of Arabidopsis mutants in actin regulatory proteins, actin signalling proteins and also in known pathogen signalling proteins to probe and understand the mechanism of actin reorganisation. We have established that the plant actin nucleating protein formin (AtFH4) is transcriptionally upregulated in single cells targeted by fungal hyphae. Additionally, we have identified several independent clones of a specific respiratory burst oxidase in a yeast 2-hybrid screen using AtFH4 as bait and confirmed this interaction in vitro. As respiratory burst oxidase is a key component of the pathogen defence response, we will probe the functional significance of the AtFH4 / burst oxidase interaction using colocalisation studies and experiments designed to interfere with this interaction. Additionally, as we are using Arabidopsis as our model and differences exist in the regulation of actin binding proteins in monocots and dicots and in the signalling pathways used within the monocot family in response to the same pathogen, we will determine if similar mechanisms of actin reorganisation occur upon pathogen attack (powdery mildews and diseases of the Puccinia genus) in wheat.

The majority of this work represents fundamental research and will serve to increase human knowledge and understanding. However, work in this programme will underpin the development of new solutions to crop protection contributing to global food security. Previous basic research on the cytoskeleton from the PJH lab has led to a patent on generating herbicide resistance plants, as the target site for certain agronomically important herbicides was identified. This work was in collaboration with Zeneca in the 1990's. Together with this patent the work was published in Nature and Nature Biotechnology in 1998 and 1999 respectively (see Part 1A). The basic research described in this proposal could potentially follow the same industrial route. This is because the actin cytoskeleton is known to play a key role in plant defence against pathogen attack. It is currently known that at least one component of the actin regulatory network can protect barley from pathogen (P.graminis Ug99) invasion. As we have identified another component of the actin regulatory network that is upregulated upon pathogen attack and that can potentially link with known pathogen defence signalling pathways, it is not unreasonable to suggest that this research may result in a patent. Our collaborator on this Program is the research director of the National Institute of Agricultural Botany based in Cambridge and this link will afford the opportunity to translate our studies (for this and future studies) to cereals for potential economic benefit. This is a new link for the PJH group and will allow our research to be tested in wheat. NIAB Research has interests in cereal disease resistance, novel traits and prebreeding targets. Consequently, the research described in the current programme affords an excellent opportunity for both organisations. If any of the research develops into a commercially viable prospect then The Durham University Technology Transfer Office will help take this forward. This Office has been formally established to provide the mechanism to enhance the exploitation of the Intellectual Property generated by high quality research activities. The Office supports staff in the overall University mission, recognising the core teaching, research and 'third-leg' strategies of the University. Support is provided to staff whose research outputs have commercial significance and who (1) Have research results and intellectual property that has commercial potential and may require patent protection; (2) Have know-how that may have commercial value; (3) Are interested in exploring the possibility of setting up a spin-off company; (4) Are looking to identify a commercial partner for a joint business venture based on research output. To this end, we will approach and engage with appropriate agrochemical companies (e.g. BASF, Bayer, Syngenta) and explore the possibilities of chemical intervention of cytoskeletal processes for the improvement of crops against disease. This project will also result in the training of personnel. The postdoc named on this proposal (MJD) will advance his knowledge in plant pathogen defence research and the technician to be appointed will have the opportunity to learn molecular/cell techniques providing a new person with skills that can be utilised in the plant biology sector be it industrial or academic. Communication of results generated will be mainly through publication in refereed journals and presentations at meetings and these activities will be performed by PI (PJH), Co-I (AG) and researcher and Co-I (MJD). The PI (PJH) regularly organises conferences under the umbrella of the Society of Experimental Biology and next year he is running a 1 day meeting on the Cell Biology of Plant Defence to which academics, industrialists, post docs and students are welcome to attend. It is hoped that such meetings will result in future collaborative projects in this important area of research which has a direct impact on the UK economy.