Elucidating the OXI1 stress signalling network

In common with us, plants utilise chemical compounds formed from oxygen, not for breathing but for an altogether different purpose. These highly-reactive compounds are know as 'reactive oxygen species' (ROS) and are produced for instance when a white blood cell finds a bacterium in the bloodstream. This initiates a series of events which allows the blood cell to kill the bacterium, and thus prevent spread of infection. The ROS are not themselves used to kill the bacterium, rather they 'turn on' processes in the blood cell which help it to kill the bacterium. Understandably there is a lot of excitement in the scientific world regarding this type of system, as an understanding of it could help with combating disease (some diseases such as rheumatoid arthritis occurs when cells in joints do not perform this function correctly). Recently it was found that the use of ROS in this way is not limited to human cells, and in fact plants utilise this mechanism also. Perhaps not surprisingly, one such instance is when plants are themselves attacked by bacteria and fungi. Plants do not have blood cells, so instead, produce chemicals which will kill the pathogen, and prevent spread of infection. We were interested in finding what the steps were leading from ROS to the management of plant disease in this way. A protein, called OXI1, was identified and was found to be involved in one such step. If this protein was removed from plants, they were no longer able to defend themselves properly from microbes attacking them. It was found that OXI1 worked by 'labelling' other proteins by adding a chemical 'tag' to them, namely phosphate. This is a common trick in cells, used to either make a protein work, or to stop in working i.e. to control its function. The research being proposed here seeks to identify exactly which proteins OXI1 'tags' and which ones it binds to do its work. In this way, the series of steps from ROS to combating plant disease can be discovered, and utilised to human benefit i.e. breeding crops with increased resistance to pests e.g. mildew.

Reactive oxygen species (ROS) generated in oxidative bursts are able to function as signalling molecules mediating diverse processes in eukaryotic organisms such as coping with stress and regulation of developmental programs. Relatively little is known about signalling components downstream of ROS that mediate any of these processes. MRK's lab has recently shown that an Arabidopsis thaliana AGC-class protein kinase (OXI1), is induced in response to a wide range of H2O2-generating stimuli and is necessary for at least two very different oxidative burst-mediated processes: basal resistance to microbial pathogens and root hair growth (Rentel et al. (2004). Nature 427, 858-861.). Thus, OXI1 is an essential part of the signal transduction pathway(s) linking oxidative bursts to diverse downstream responses. As OXI1 is involved in at least two different, and important, signalling pathways, it is a key target for research into these pathways and the crosstalk between them which forms a signalling network. The first step in this long-term goal is the elucidation of the other components in the OXI1 signalling network. Therefore this is the focus of the research proposed here. This goal will be achieved through a combinatorial approach. Protein interactors of OXI1 will be identified by a combination of in vivo TAP-tagging and yeast 2 hybrid analysis (part 1 of research proposal). Direct and indirect substrates of OXI1 kinase activity will be identified by quantitative phosphoproteomic analysis (part 2 of research proposal). Proteins isolated by TAP-tagging and phosphoproteomic analysis will be identified by mass spectrometery (part 2 of research proposal). Finally confirmation of interaction of proteins with OXI1 in vivo will be achieved, also by TAP-tagging, and determination of specific protein substrates for OXI1 kinase activity will be tested by in vitro kinase assays (part 3 of research proposal).