HMA domain proteins as conserved targets of pathogens that exploit plasmodesmata

微生物病原体通过一系列感染策略侵入其宿主，这些感染策略允许病原体生长和繁殖。感染可以包括改变宿主细胞和组织以适应入侵者的物理过程，以及蛋白质和小分子交换以阻碍和操纵其他生物体的分子战。在分子水平上，病原体配备了一系列蛋白质和小分子，这些蛋白质和小分子可以被递送到宿主细胞中，靶向特定的生理过程以控制细胞功能。被递送到宿主细胞中的微生物蛋白质被称为效应物，虽然它们在靶向免疫抑制和资源分配中的功能之间存在共同的主题，但它们具有对给定微生物特异性的各种各样的分子靶标。来自不同界域的病原体效应子靶向宿主胞间连丝，即细胞间的细胞质连接。胞间连丝为一些病原体提供了在细胞之间通过并通过宿主组织传播的途径，并且充当分子可以通过其传递到它们在感染中部署的部位的导管;效应物可以从感染的细胞传递到未感染的细胞，并且营养物可以从宿主来源自由地传递到感染部位。正如所料，宿主细胞通常试图关闭它们的胞间连丝作为一种防御机制。然而，靶向胞间连丝的几种效应物可以阻止这种反应并维持宿主细胞之间的连接。因此，胞间连丝已经成为宿主和病原体之间的关键战场。已经有几个观察来自病毒和真菌病原体的效应物靶向位于胞间连丝的重金属相关（HMA）结构域蛋白。这种不同的病原体靶向位于细胞间桥的同一类蛋白质，表明HMA结构域蛋白在感染期间提供了显着的收益。此外，在许多植物物种中，HMA结构域被整合到免疫受体序列中，其中它们充当诱饵以结合相关效应物并激活免疫受体，从而引发细胞死亡和随后的抗性。不幸的是，虽然免疫受体劫持的效应HMA结构域的相互作用点的意义的协会，它也阻碍了研究的作用的效应和HMA的目标，因为它成为掩盖免疫receptor activation.We最近表明，拟南芥真菌病原体Colletotrichum higginsianum产生一个效应，靶向一个位于胞间连丝HMA结构域蛋白在主机。拟南芥不产生具有整合的HMA结构域的免疫受体，这使我们能够研究这种相互作用在感染中的作用和机制。这也将使我们能够问这些效应器如何以及为什么靶向胞间连丝。作为C. higginsianum效应子不仅作用于胞间连丝，而且还能在细胞间移动，并能修饰胞间连丝，使大分子蛋白在细胞间更频繁地移动，这表明效应子的功能之一是增加宿主细胞间分子交换的能力，本研究将利用刺盘孢菌-炭疽菌相互作用来确定效应子和宿主靶标在感染中各自发挥的作用。我们将使用结构生物学来比较来自不同物种的效应器和靶HMA之间的相互作用，并确定发生这种情况的机制之间的任何保守性。我们还将利用任何保守性来确定我们是否可以将来自水稻的免疫受体中的HMA结构域与来自被炭疽菌靶向的拟南芥的HMA结构域交换，从而设计识别炭疽菌效应子并赋予新抗性的免疫受体。

Microbial pathogens invade their hosts via a range of infection strategies that allow the pathogen to grow and reproduce. Infection can include physical processes that transform host cells and tissues to accommodate the invader, and molecular warfare in which proteins and small molecules are exchanged to impede and manipulate the other organism. At the molecular level, pathogens are armed with a repertoire of proteins and small molecules that can be delivered into host cells, targeting specific physiological processes to control cellular function. Microbial proteins that are delivered into host cells are referred to as effectors and while there are common themes amongst their function in targeting immune suppression and resource distribution, they have a wide variety of molecular targets, specific to a given microbe. Pathogen effectors from different kingdoms target host plasmodesmata, the cytoplasmic connections between cells. Plasmodesmata offer a pathway for some pathogens to pass between cells and spread through host tissues, as well as acting as conduits by which molecules can pass to sites where they are deployed in infection; effectors can pass from infected cells into uninfected cells and nutrients can pass freely from host sources to the site of infection. As might be expected, host cells usually try to close their plasmodesmata as a defence mechanism. However, several effectors that target plasmodesmata can prevent this response and maintain connectivity between host cells. Thus, plasmodesmata have emerged as a critical battleground between host and pathogen.There have been several observations of effectors from viral and fungal pathogens that target heavy metal associated (HMA) domain proteins located at plasmodesmata. That such diverse pathogens target the same class of proteins located at intercellular bridges suggests that HMA domain proteins offer significant gains during infection. Further, in many plant species HMA domains are integrated into immune receptor sequences where they act as decoys to bind the relevant effector and activate the immune receptor, triggering cell death and consequent resistance. Unfortunately, while immune receptor hijacking of effector-HMA domain interactions points to the significance of the association, it also impedes research into the role of the effector and the HMA target as it becomes masked by immune receptor activation.We recently showed that the Arabidopsis fungal pathogen Colletotrichum higginsianum produces an effector that targets a plasmodesmata-located HMA domain protein in the host. Arabidopsis does not produce immune receptors with integrated HMA domains, allowing us to investigate the role and mechanism of this interaction in infection. This will also allow us to ask how and why these effectors target plasmodesmata. As the C. higginsianum effector not only targets plasmodesmata but moves cell to cell and modifies plasmodesmata to allow large proteins to move between cells more frequently, it suggests that one effector function is to increase the capacity for molecular exchange between host cells.This proposal will use the Arabidopsis-Colletotrichum interaction to determine what function the effector and host target each play in infection. We will use structural biology to compare the interactions between the effector and target HMAs from diverse species and identify any conservation between the mechanisms by which this occurs. We will also exploit any conservation to determine if we can exchange the HMA domain in immune receptors from rice with the HMA domain from Arabidopsis targeted by Colletotrichum, and thus engineer an immune receptor that recognises the Colletotrichum effector and confers novel resistance.