HMA domain proteins as conserved targets of pathogens that exploit plasmodesmata

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.

Disease causing pathogens use a variety of strategies to invade plant tissues, deploying an arsenal of molecules into host cells and tissues to manipulate the structure, function, and metabolism of the host to the advantage of the microbe. Microbial proteins that are delivered to and act in host cells are referred to as effectors, and exhibit diverse sequences, structures, targets, and functions. A variety of effectors from diverse pathogens target host HMA domain proteins that are located at plasmodesmata, suggesting that both offer critical gains to infection. However, research on this topic is obstructed by many species having immune receptors with integrated HMA domains that act as decoys to effector binding and activate strong immune responses.

This proposal aims to exploit the discovery that the Arabidopsis pathogen Colletotrichum higginisianum produces an effector (ChEC108) that targets a plasmodesmata-located HMA domain protein. As Arabidopsis doesn't encode immune receptors with integrated HMA domains this offers an opportunity to investigate the role of effector-HMA domain interactions in infection and thus dissect the reasons for why this is conserved across diverse species against the backdrop of possible immune activation.

We will compare effector-HMA domain complex structures across species using structural biology, characterising the newly identified interaction between ChEC108 and the host HMA. We will use structure to predict how to enhance and/or perturb the interaction and analyse the function of the endogenous and manipulated effector-HMA interactions during infection using biochemical and cell biological approaches. We will use mutants of both Arabidopsis and fungus to study the role of the effector and target, and their interactions in immunity and infection. Ultimately, we will collate this information to explore how novel HMA domains can be engineered into immune receptors to generate novel recognition and resistance.