Putting the squeeze on PDs - reticulons, plasmodesmata and viral trafficking in plants

Plant viruses are a serious economic pest, causing significant crop losses worldwide due to an ability to control their movement effectively. All plant viruses are extremely efficient at moving extensively within plants, causing necrosis and yield losses wherever they replicate to high levels. To move between plant cells, all viruses exploit plasmodesmata, specialised pores in the walls between adjacent cells. During the course of evolution viruses adopted very efficient strategies for modifying plasmodesmata so that their genetic material can be passed from one cell to the next. All plant viruses encode 'movement proteins', specialised proteins that interact with plasmodesmata, causing them to dilate and allow the viral genome to traffic through the pore. Through the center of the pore lies an axial membranous structure known as the desmotubule, and it is with this structure that movement proteins are thought to interact.

The desmotubule is an enigmatic structure, unique to plants, and at only 15 nm in diameter is one of the most tightly constricted membrane structures known in nature. We hypothesise that a recently discovered family of proteins, known as the reticulons, function to remodel the plant endoplasmic reticulum into desmotubules, thus giving rise to plasmodesmata each time plant cells divide. We suggest, further, that the reticulons of the desmotubule function as the central target of viral movement proteins that bind to reticulons, causing an increase in the transport properties of plasmodesmata.

It is argued that understanding the mechanism by which viruses pass through plasmodesmata is central to the development of strategies for eliminating virus movement in plants, thus controlling infections at the entry stage. This project will 'tag' reticulon proteins to examine the ways in which they give rise to the formation of plasmodesmata. It will also use genetic strategies to overproduce or eliminate reticulons during plant-cell division to examine the effects on the formation of plasmodesmata. It is likely that reticulons interact with other important proteins within plasmodesmata, and specific tagged reticulons will be used as biochemical 'bait' to identify these unknown proteins.

The project will also attempt to make 'artificial desmotubules' by constricting membranes into fine tubes biochemically. These 'artificial desmotubules' will be used to study how viral movement proteins interact with desmotubules and to study how proteins are able to pass through native plasmodesmata. The project will involve a unique combination of state-of-the-art microscopy, genetics, virology and cell biology to understand how plants alone are able to make plasmodesmata. The results will have far reaching implications for controlling the movement of substances between plant cells, specifically the transport of infectious genetic material exemplified by viruses. An overall goal, through increased understanding of plasmodesmatal structure and function, is to develop effective strategies for the control of virus movement in crop species.

Plasmodesmata, the small pores that interconnect plant cells, are enigmatic structures central to plant development, physiology and pathology. Many substances, including small solutes, signalling molecules and proteins can move through plasmodesmata, but the control of movement is poorly understood. Plant viruses exclusively use plasmodesmata to move between plant cells during infection and all plant viruses encode specialised movement proteins that interact with plasmodesmata to allow passage of the viral genome. During infection the plasmodesmata become transiently 'gated' by an unknown interaction. Plasmodesmata contain a central structure, the desmotubule, that is comprised of tightly appressed ER membranes. At only 15 nm in diameter, the desmotubule is one of the most tightly constricted membrane structures known in nature, and is a prime target for viral movement proteins during infection.

We have recently obtained data to suggest that the reticulons, a specific class of membrane tubulating proteins, may be involved in the formation of desmotubules during cell plate formation. We hypothesise that reticulons function to remodel the cortical ER at the end of cell division when plasmodesmata are being laid down between adjoining cells, and that these proteins link the desmotubule to oether unknown proteins within the plasmodesmal channel.

This project will use a unique combination of virology, biochemistry, cell biology and state-of-the-art imaging to study how reticulons are involved in the biogeneisis of plasmodesmata, and how they interact with viral movement proteins. The overall goal, through increased understanding of plasmodsmal development and structure, is to develop strategies for limiting virus spread in plants.

This project will examine how plant cells construct plasmodesmata, the small communication channels between cells, and will study the mechanisms by which plant viruses interact with these pores to facilitate their spread in the plant. Plant viruses are a serious worldwide threat, yet little is known of the mechanism of cell-cell passage. This is a basic project and the main audience for the work is expected to be the scientific community. However, the spread of viruses is a topical area in the media and we will endeavour to publicise our work through a number of distinct routes. These include:

1. Publication of articles in the popular press. The strong imaging component of this work lends itself to the production of new images of virus movement that can be used by the media. One of us (KO) has previous experience in dealing with popular press and with documentary programmes, such as 'Horizon'. KO has also been approached by the BBC with a view to acting as a consultant on a newly commissioned series on plant function. Images form the current proposal are likely to find their way rapidly into mainstream reports on virus infection. We will also publish in BBSRC literature and in popular journals such as New Scientist and The Scientist. Through the respective press offices at Edinburgh and Oxford Brookes, we will release appropriate news articles for dissemination to the popular press.

2. Influence on the agricultural sector. A major goal of the project is to understand the process of virus movement such that strategies may be developed for impeding cell-cell transport of viruses in economically important plants. The viruses being used as 'models' in the study are also important crop pathogens, and the results are likely to be transferable to major crop families such as the Solanaceae (e.g. potato, tomato). KO has strong links with the agricultural sector and we will inform applied crop biologists and breeders of our findings at key national and international symposia.

3. Influence on policy makers. Crop protection in a changing climate is a major issue of current concern. As temperatures increase in the UK, so also will the virus pathogen profiles of the principal crop species. KO has strong links with Scottish Government-funded virologists through institutes such as SASA (Dr. Christophe Lacomme) and The James Hutton Institute (Dr. Lesley Torrance and Prof. Misha Taliansky). Regular meetings with policy-led scientists will ensure that our work is disseminated appropriately across a broad spectrum of plant pathologists and Government representatives.

4. Intellectual property release. Two areas are worth specific note in this area of Impact. (i) The project, through the development of novel imaging methods and approaches, has the potential to generate IP related to developments in microscopy and imaging. In this respect, both PIs will work closely with their respective technology transfer companies, ERI (Edinburgh) and RBDO (Oxford), to ensure a timely protection of IP as the project develops.

5. Outreach activities. Both PIs are involved in regular outreach activities, including the supply of images to the Gatsby Foundation (Schools), presentation of talks and demonstrations at national science festivals (e.g Edinburgh Science Festival), The Royal Society of Edinburgh (KO) and The Royal Microscopy Society (CH).

Our Impact plan will exploit these four areas of engagement to ensure maximum publicity and dissemination of the science conducted in the project.