To survive, plants must maintain an upright stem in the face of wind, while their roots must forage for water and nutrients despite soil hardness and compaction. These mechanical stresses account for approximately 10% of annual European crop yield loss from wind lodging and 10-50% yield loss can arise from soil compaction. This project aims to identify new components in the pathways plants use to sense such mechanical stress and signal that they must adapt, providing a platform to improve crop lodging and root growth for agriculture. The overall objective is to test whether the DORN1 protein in the model plant Arabidopsis co-ordinates mechanical signalling with that by extracellular ATP. DORN1 is the first discovered plant cell membrane receptor for extracellular ATP and it activates the wound response of the plant. We hypothesise that it can signal not only that a cell has been wounded but also that it is under less severe mechanical stress. We envisage that wall deformation from mechanical stress causes the DORN1 protein to detach from the cell wall and this allows it to bind extracellular ATP to signal. In Objective 1 we will test this by generating mutants of DORN1 that are compromised in their ability to bind the wall and then examining whether this affects signalling by extracellular ATP. We will look at how the signalling messengers downstream of DORN1 are affected and examine whether gene activation is compromised. To understand further how DORN1 works, we will identify the proteins that it interacts with. This could reveal how it links to the wall and also what the first protein messengers are in the DORN1 signalling pathway. In Objective 2 we aim to identify the channel proteins that are activated in DORN1 signalling and allow calcium into the cell to act as a signal. One or more of these could be revealed as interacting proteins of DORN1 in Objective 1. We will measure channel activity as the electrical current that flows when the channel opens and lets calcium pass into the cell. We will compare wild type against mutants for candidate calcium channels, concentrating on the stem epidermis and root cap as key sites of sensing and signalling mechanical stress. Objective 3 aims to put DORN1 and downstream calcium channel activity into the context of the plant response to mechanical stress. We will determine the effect of DORN1 and channel mutation on stem posture as wind stress is imposed and analyse the effect on gene activation. We will also examine how losing DORN1 and calcium channel function affects the ability of the root to generate a calcium signal, penetrate hard substrate and navigate obstacles. By comparing the mutants to the wild type it will be possible to deduce the importance of the DORN1 signalling pathway to the mechanical stress responses of the plant.