Damage signalling by endo-parasites: A key role for extracellular ATP and calcium channels

如果细胞受伤，他们需要向邻居发出信号，所有细胞都可能受到威胁，他们必须做好应对准备。所使用的信号被称为“损害/危险相关分子模式”(DAMPS)。其中之一就是细胞外三磷酸腺苷(EATP)。在细胞内部，三磷酸腺苷被用作维持细胞存活的能量来源，但如果细胞周围的膜被打破，它就会泄漏出来。一旦它在细胞外，它就可以被邻近的细胞感知到，然后这些细胞就可以通过适应做出反应，包括启动免疫反应。我们现在知道，植物也可能利用eATP作为湿气，帮助它们对伤害做出反应，并保护自己免受有害微生物和害虫的伤害。它还可能参与创伤后组织的再生。我们必须解决的问题是eATP途径是如何工作的。我们知道，在模式植物拟南芥中，eATP与细胞外膜中的蛋白质受体结合，这会导致钙离子的增加，而钙离子是启动防御反应的信号。我们现在发现，一种蛋白质(CNGC19)构成了将钙输送到细胞内的通道，似乎是eATP引起的根中钙增加的主要原因。这让我们有机会弄清楚eATP受体是如何打开CNGC通道让钙进入细胞的。由于受体可以将磷酸盐添加到蛋白质中来改变其活性，我们将首先测试纯化的受体是否能与CNGC蛋白相互作用，然后测试CNGC蛋白是否能被受体磷酸化。然后，我们应该能够找到CNGC蛋白被磷酸化的单个氨基酸构建块，使我们能够产生无法磷酸化的突变体。作为蛋白质信号通路是否起作用的一个很好的测试，这些蛋白质将在另一种细胞类型中制造，我们可以用来测量加入eATP时进入细胞的钙。受体和通道应该让钙进入，而不是受体和不能被磷酸化的通道。为了支持这些研究，我们将测量缺乏受体和通道组合的拟南芥突变体的根中钙的增加，并找出这一信号通路在哪些细胞中起作用。我们将添加eATP，但另一项测试是杀死单个细胞来模拟伤害。这可以用激光来完成，这可以模拟由入侵的包囊线虫造成的单细胞损伤。它们穿过根部的外层细胞层，进入内部取食部位。线虫在世界范围内造成了重大的经济损失，我们将通过测试eATP受体和CNGC是否可能是根部对线虫攻击的抗性反应的一部分来结束这一项目。

If cells are wounded they need to signal to their neighbours that all of them are potentially under threat and that they must gear up to respond. The signals used are termed "Damage/Danger Associated Molecular Patterns" (DAMPs). One of these is extracellular ATP (eATP). Inside the cell, ATP is used as an energy source to keep cells alive but if the membrane surrounding the cell is breached then it leaks out. Once it's extracellular it can be sensed by neighbouring cells which can then respond by adapting, including launching an immune response. We now know that plants might also use eATP as a DAMP, helping them to respond to wounding and defend themselves against damaging microorganisms and pests. It might also be involved in the regeneration of tissues that can occur after wounding. The problem that we have to solve is how the eATP pathway works. We know that in the model plant Arabidopsis, eATP binds to protein receptors in the cell's outer membrane and that this causes an increase in calcium that acts as the signal to launch a defensive response. We have now found that a protein (CNGC19) that makes up a channel to transport calcium into the cell seems responsible for most of the calcium increase in the root that is caused by eATP. This gives us the opportunity to work out how the eATP receptors make the CNGC channels open to let calcium into the cell. As the receptors can add phosphate to proteins to change their activity, we will start by testing whether purified receptor and CNGC proteins can interact and then test whether the CNGC proteins can be phosphorylated by the receptors. We should then be able to find the individual amino acid building blocks of the CNGC protein that are phosphorylated, enabling us to generate mutants that cannot be phosphorylated. As a good test of whether a protein signalling pathway works, these proteins will be made in another cell type that we can use to measure the calcium that comes into the cell when eATP is added. Receptors and channel should let calcium enter but not receptors and the channel that can't be phosphorylated. To support these studies we will measure the calcium increase in roots of Arabidopsis mutants that lack combinations of receptors and channels and find out which cells this signalling pathway works in. We will add eATP but an additional test is to kill an individual cell to simulate a wounding. This can be done with a laser and this simulates the single cell wounding inflicted by invading cyst nematodes. These cut through outer cell layers of the root to gain access to inner feeding sites. Nematodes inflict significant economic damage worldwide and we shall end this project by testing whether the eATP receptors and CNGCs could be part of the root's resistance responses to their attack.