Precision guidance: Mechanisms driving targeted secretion in response to invasive microbes

Plant cells are surrounded by a plasma membrane (PM) that spans tens of thousands of square microns. Pattern recognition receptors in the PM detect microbial molecules and molecules associated with cell wall damage that may indicate the presence of a pathogen. This information is transduced to activate defensive processes including the production of new proteins and compounds that are then transported back to the site of contact. Recently it was discovered that molecular patterns are sufficient to guide the precise delivery of cargoes effectively allowing the plant cell to change the content of the PM and the cell wall at the site of phytopathogen contact hours before a phytopathogen physically penetrates the host cell wall. This proposal aims to answer the fundamental question of how these cargoes are delivered to only a few specific square microns of PM in the vicinity of the pathogen.

Our preliminary data show that the induction of exocytosis (the process of transporting and delivering material to the PM and cell exterior) by fungal phytopathogen-associated molecules is an excellent system for monitoring plant exocytosis in 'real time'. We have established an assay using Total Internal Reflection Fluorescence (TIRF) microscopy that allows controlled stimulation of exocytosis and visualisation of the interaction of vesicles (small 'packets' of membrane and internal cargo) with the PM. We have shown that PEN3 and other proteins of interest are clustered into stable 'nano domains': sub-domains of the plasma membrane at the nano-metre scale. These nano domains are too small to be fully resolved by standard light microscopy. We also observe mobile vesicles 'touring' PM nano domains. This proposal seeks to understand how these behaviours lead to the emergence of specialised PM zones precisely beneath the site of microbial contact.

To do this we will combine two areas of expertise: plant cell biology and optical physics. Our team includes a laboratory responsible for several innovations in the field of 'super' resolution microscopy. These super resolution techniques, dSTORM and PALM can increase optical resolution to an order of magnitude greater than standard fluorescence microscopy. This will enable accurate assessment of protein co-localisation and interactions with PM nano domains.

In the first part of our study we will use TIRF microscopy and super resolution microscopy to ask whether the 'touring' behaviours of vesicles and their pausing at nano domains results in the transfer of cargo to (or from) nano domains. We will also ask whether the cytoskeleton is anchored to nano domains in order to promote vesicle touring/pausing behaviour. In the second part we will isolate the unknown cargoes of the vesicles we are observing. This will identify novel anti-microbial factors and previously uncharacterised proteins that contribute to vesicle targeting. Finally, we will perform a functional analysis of a protein complex known as the exocyst in parallel with candidate proteins emerging from the vesicle isolation experiments. The exocyst acts as a vesicle-tethering complex in animal and fungal systems but has fundamental behavioural differences in plants. We will therefore test the hypothesis that the exocyst is part of the molecular machinery that tethers vesicles to specific sites at the PM responding to microbes.

By the completion of this inter disciplinary project we will have identified the mechanism by which cargoes are deposited selectively at PM sites in contact with microbes. We will have tested the role of both conserved and novel factors that we have isolated as candidate components of this targeting machinery, giving fundamental insights into targeted exocytosis in plants. Moreover, we will have used our tools to establish a means to deliver enzyme cargoes to the plant pathogen interface. This will underpin future efforts to apply biotechnology solutions to preventing plant disease.

Contact between a plant cell and a phytopathogen stimulates the production of new proteins and compounds that are specifically exocytosed at the site of the stimulus. Among these cargoes are PEN3, an ABC transporter protein that is critical for defence against a wide range of phytopathogens and Formin4, a membrane-integrated cytoskeletal interface protein. Our preliminary data and the recent work of others show that microbial-targeted secretion is activated by the application of molecules associated with phytopathogens. This phenomenon is therefore an excellent model for understanding the fundamental principles of plant exocytosis as it can be stimulated reliably and monitored with relative ease at the plant epidermis. Understanding this pathway is of critical interest as it is suppressed during compatible phytopathogen interactions and has the potential to be exploited by biotechnology strategies to deliver synthetic cargoes to the plant-pathogen interface.

This is an interdisciplinary project that combines expertise in optics and 'super' resolution microscopy with plant cell biology. This is necessary because we have observed that exocytosis at phytopathogen interaction may occur at isolated structures that are at the resolution-limit of standard fluorescence microscopy. These 'nano domains' (sub-domains of the plasma membrane at the nano-metre scale) are an emerging common feature of multiple processes at the plant plasma membrane (PM). In order to understand the spatiotemporal relationships between PM, exocytic vesicles and the cytoskeleton it is therefore necessary to apply specialised technologies such as dSTORM and PALM to resolve these domains and their potential interaction partners. We will use these technologies in combination with molecular genetics and proteomics to identify cargoes transported in this phytopathogen-response pathway and dissect the molecular mechanism governing precision delivery to phytopathogens.

The key question addressed by this proposal is: how does a plant cell direct cargoes to specific sites at the cell surface that are in contact with microbes? This is performed with precision and speed in response to the earliest molecular signals of microbial presence. We will focus on understanding a specific intracellular transport pathway that we have shown responds to a broad range of fungal phytopathogens. It is anticipated that results from this research project will deliver impact and benefits in three key areas: crop protection and enhancement, broader societal benefits and capacity building.

Crop Protection and the Agrichemical Industry
The outcomes of this research will have impact in the long-term for both crop protection and enhancement through an improved understanding of pathogenic and beneficial symbiotic relationships between plants and fungi. Strains of pathogenic fungi from many species currently challenge the genetic defences of high-yield crops established during the Green Revolution. The molecular basis of these adaptations is not currently understood. Our research programme will address this and enable advances using the molecular cell biology of Arabidopsis. Improving understanding of the hyphal interaction could lead to developments in crop protection by the agrichemical industry. Therefore potential exists for the creation of valuable intellectual property (IP) and engagement with relevant industrial partners as this research programme progresses. This will include biotechnology businesses that are members of the national Crop Improvement Research Club (CIRC) and contacts of the Food Security Land Research Alliance. While our current research tackles the fundamental nature of the fungal interaction, findings are anticipated to provide results that will be of high value to the UK biotechnology sector in the long term.

Broader Societal Benefits
Our proposed research will impact upon the food security agenda enabling future reductions in crop disease as well as enhancements through beneficial fungi. Food security is fundamentally important to society and of great interest to both the public and policy makers as concerns about feeding a growing population and climate change are increasing. Understanding how fungi manipulate plant cell biology will improve our capabilities to protect wheat and other crops and it is anticipated that this will be of interest to the wider public. We will therefore use this project as a springboard to engage the public and undertake several activities (see pathways to impact) that will communicate these beneficial impacts of plant science.

Capacity Building
By providing excellent training and support to the PDRA employed to undertake this project we will be helping to secure the science base of the future. Outstanding researchers will be key to overcoming the food security challenges anticipated over the next 50 years and the grounding the PDRA will receive by participating in Exeter's Researcher Development programme will ensure their personal and professional development is tailored to future opportunities in both the public and private sector. The PDRA will engage with both industrial and public beneficiaries as part of the project and will be encouraged to enter the STEM ambassador programme to support outreach activities. The PDRA will complete the project with a unique blend of research skills including protein interaction analysis, quantitative live-cell imaging and advanced super-resolution microscopy. This is a valuable skills package that will benefit future BBSRC funded research.