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

动物细胞通过质膜与邻近细胞分离，而植物细胞则不同，植物细胞之间由细胞膜和坚硬的纤维素壁分开，这限制了细胞之间的直接交流。胞间连丝，连接单个细胞的细胞质小孔，克服了这个问题。原认为胞间连丝的功能直径非常小，只允许小的糖和激素在细胞间传递。然而，最近的发现表明，植物可能交换大分子，如蛋白质和核酸，以达到信号传递的目的。为了允许大分子通过，胞间连丝被一种严格控制的细胞机制加宽（“门控”）；大多数细胞蛋白质和核酸必须保留，而只有特定的子集可以在细胞之间“传输”。由于完整的胞间连丝难以分离，调节“门控”的因素以及通过胞间连丝运输的分子的性质仍然知之甚少。本研究将解决这些问题。植物病毒已经进化到能够通过胞间连丝移动它们的基因组，并且通过编码特定的“移动”蛋白质来实现这一点，这些蛋白质可以阻断胞间连丝并在细胞之间“连接”病毒RNA。一个流行的模型是，在进化过程中，病毒获得（或“劫持”）了植物自身用来运输大分子的基本成分。因此，病毒为胞间连丝的调节以及影响其功能的蛋白质的性质提供了重要的线索。在这个项目中，我们将鉴定出能够诱导胞间连丝“门控”的新型植物蛋白。为了做到这一点，我们将使用一种经过两种方式改变的转基因病毒：1)通过去除其“运动”蛋白，它将无法通过胞间连丝移动；2)它将被改造成表达多个随机基因序列，与绿色荧光蛋白（GFP）融合，从而允许在细胞之间跟踪编码的蛋白质。如果新的植物序列“打开”胞间连丝和/或在细胞之间移动，GFP标签将在最初感染细胞外的细胞中可见。这种基于病毒的基因筛选是高通量的，可以在一天内检查数百种蛋白质，并将导致发现新的植物蛋白质，这些蛋白质像病毒运动蛋白一样，能够改变胞间连丝的功能。

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.