A novel screen to identify components of the plant macromolecular trafficking pathway

Unlike animal cells, which are separated from their neighbouring cells by their plasma membranes, plant cells are separated from each other by both a membrane and a rigid cellulose wall, limiting direct communication between cells. Plasmodesmata, small cytoplasmic pores that connect individual cells, overcome this problem. The functional diameter of plasmodesmata was originally thought to be very small, allowing only small sugars and hormones to pass between cells. However, recent discoveries suggest that plants may exchange macromolecules such as proteins and nucleic acids for signalling purposes. To allow for the passage of macromolecules, the plasmodesmata are widened ('gated') by a cellular machinery that is tightly controlled; most cellular proteins and nucleic acids must be retained while a only a specific subset can 'traffic' from cell to cell. As intact plasmodesmata are diffiult to isolate, the factors that regulate 'gating', and the nature of the molecules that are trafficked through plasmodesmata, remain poorly understood. This study will address these questions. Plant viruses have evolved to move their genomes through plasmodesmata, and do so by encoding specifc 'movement' proteins that gate the plasmodesmata and 'thread' the viral RNA between cells. A popular model is that during the course of evolution viruses acquired (or 'hijacked') essential components that the plant itself uses to traffic macromolecules. Viruses thus provide important clues as to the regulation of plasmodesmata, and the nature of proteins that influence their function. In this project we will identify novel plant proteins that are able to induce the 'gating' of plasmodesmata. To do this, we will use a genetically modified virus that has been altered in two ways: 1) it will be unable to move through plasmodesmata by removal of its 'movement' protein, and 2) it will be engineered to express multiple, random gene sequences fused to green fluorescent protein (GFP), allowing the encoded proteins to be tracked between cells. If the novel plant sequences 'gate' plasmodesmata and/or move between cells, the GFP tag will be visible in cells outside the initially infected cell. This viral-based genetic screen is high-throughput, allowing several hundred proteins to be examined in a single day, and will lead to the discovery of novel plant proteins, which like viral movement proteins, are able to modify the functions of plasmodesmata.

We have developed a transitory genetic screen to identify novel plant sequences that modify the size exclusion limit of plasmodesmata and/or traffic from cell-to-cell through plasmodesmata. The screen is based on a modified tobacco mosaic virus (TMV) vector that lacks its viral movement protein (TMV.deltaMP), and is therefore restricted to single cells. Co-expression of a putative plant cDNA that 'gates' plasmodesmata alongside GFP, or as a translational fusion to GFP, permits the fluorescent protein to move through plasmodesmata, resulting in the formation of a 'leaky-cell' phenotype. The cDNA sequence is recovered by agroinfiltration of the infection site with a binary vector expressing the full-length viral MP. This rescues virus movement allowing the novel cDNA to be isolated from the multicellular infection site. Positive 'gating' controls will include viral MPs and non- cell-autonomous homeodomain proteins. A cDNA library constructed from the phloem is expected to provide a rich source of non- cell-autonomous proteins. The screen will allow novel proteins of the plant macromolecular trafficking pathway to be isolated and characterised.