Investigating the role of chloroplasts in the plant focal immune response

Plants are sessile organisms that lack motile cells, relying primarily on cell autonomous immunity to stave off their many pathogens and pests. Such responses must be rapid and effective in order to prevent the spread of pathogens to uninfected tissue - one such way of improving an immune response is to target defences towards pathogens in a polarised manner within the confines of a single cell. Phytophthora infestans is the causative agent of potato late blight, a disease that alongside a range of socio-economic factors, led to the Great Irish Famine in the mid 1800's. This event caused the death of approximately one million individuals and caused mass emigration from Ireland; on top of this P. infestans remains an economically relevant pathogen, affecting solanaceous crops around the world today. During infection, P. infestans forms intracellular haustoria surrounded by a host-derived extra-haustorial membrane across which effectors are secreted to perturb the host-immune system and nutrients are up-taken. This plant-pathogen interface is vital to the success or failure of a plant immune response and is unsurprisingly targeted by a series of focal immune responses. Recently, the Bozkurt lab showed that chloroplasts are involved in such a response, swarming intracellular haustoria and forming networks of stroma filled tubules (stromules) that, together, reduce the success of pathogen infection. Furthermore, this response relies on CHUP1, a chloroplast outer-envelope protein known to be involved in chloroplast light-avoidance responses.

In a developing field of chloroplast immunity, this work will aim to further investigate the signalling pathways involved in chloroplast mediated immunity, with a specific focus on chloroplast movement and stromule induction. Additionally, the extent to which CHUP1 mediates chloroplast defence responses and how this is achieved through inter-molecular interactions will be investigated. I will use Nicotiana benthamiana as a model plant system, utilising Agrobacterium mediated transient expression in combination with confocal microscopy, biochemical assays, and infection assays to investigate relevant molecular and cellular processes. Using these techniques, two key aims will be addressed:

1) Decipher the signalling pathway leading to pathogen induced chloroplast movement and stromule induction.

Putative components of the signalling pathway, based on unpublished results, will be probed via the use of virus induced gene silencing (VIGs) and transient overexpression. Using this methodology, the pathway involved in signalling chloroplast movement and stromule induction will be traced from initial recognition of the pathogen to major signalling events, through to possible changes in gene expression, and the induction of chloroplast mediate immune responses.

2) Determine the extent to which CHUP1 mediates defence-related chloroplast functions.

The structure-function of CHUP1 in the context of immunity will be investigated via the generation of a series of CHUP1 mutants based on published, putative domains of CHUP1. These mutants will be tested for their ability to complement CHUP1 silencing with regards to immunity, as well as be probed for their interaction with putative interactors via biochemical assays. Combined, I hope to show the domains required for CHUP1 function, and how together they facilitate chloroplast mediated immune responses.

These results of these investigations will determine the direction of late stage research which we hope will help translate to other model pathogen-host systems in economically relevant crops, as well as early branching lineages of plants.