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宿主，使病原体生长和繁殖。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，以及交换蛋白质和小分子以阻止和操纵其他生物体的分子战。在分子水平上，病原体拥有一系列蛋白质和小分子，它们可以被运送到宿主细胞中，针对特定的生理过程来控制细胞功能。传递到宿主细胞中的微生物蛋白被称为效应器，虽然它们在靶向免疫抑制和资源分配方面的功能有共同的主题，但它们具有各种各样的分子靶标，特定于给定的微生物。来自不同领域的病原体效应物靶向宿主胞间连丝，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间传递和在宿主组织中传播的途径，同时也充当了分子可以传递到它们在感染中部署的位置的管道；效应器可以从感染细胞进入未感染细胞，营养物质可以自由地从宿主源传递到感染部位。正如预期的那样，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，一些靶向胞间连丝的效应物可以阻止这种反应并维持宿主细胞之间的连通性。因此，胞间连丝已成为宿主与病原体之间的关键战场。已经有一些观察到的病毒和真菌病原体的效应物靶向位于胞间连丝上的重金属相关（HMA）结构域蛋白。这些不同的病原体靶向位于细胞间桥的同一类蛋白质，这表明HMA结构域蛋白在感染期间提供了显著的增益。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，在那里它们作为诱饵结合相关效应物并激活免疫受体，引发细胞死亡和随之而来的抗性。不幸的是，虽然免疫受体劫持效应-HMA结构域相互作用指出了这种关联的重要性，但它也阻碍了对效应物和HMA靶点作用的研究，因为它被免疫受体激活所掩盖。我们最近发现拟南芥真菌病原体Colletotrichum higginsianum在宿主体内产生一种靶向位于胞浆连丝的HMA结构域蛋白的效应物。拟南芥不产生具有整合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。这也将使我们能够询问这些效应物如何以及为什么靶向胞间连丝。由于C. higginsianum效应物不仅靶向胞间连丝，而且在细胞间移动并修饰胞间连丝，使大蛋白更频繁地在细胞间移动，这表明效应物的一个功能是增加宿主细胞间分子交换的能力。本研究将利用拟南芥-炭疽菌相互作用来确定效应体和宿主靶点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体和靶HMAs之间的相互作用，并确定这种相互作用发生的机制之间的任何守恒。我们还将利用任何保护性来确定我们是否可以将来自水稻的免疫受体中的HMA结构域与炭疽杆菌靶向的拟南芥的HMA结构域交换，从而设计出识别炭疽杆菌效应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.