Determining how phosphorylation drives functional protein interactions

Secondary Supervisor(s): Professor Phill Stansfeld

University of Registration: University of Warwick

BBSRC Research Themes:

• Sustainable Agriculture and Food (Plant and Crop Science)
• Understanding the Rules of Life (Plant Science, Structural Biology)

Project Outline

Phosphorylation is one of the most highly studied posttranslational modifications, due to its fundamental importance in regulating protein function, interactions, subcellular localisation, and turnover. Protein phosphorylation is also one of the central components of signal transduction pathways within cells through cascades of protein kinase activity. The addition of a phosphate group, PO4, at specific amino acid residues on a protein (typically serine threonine, tyrosine) imparts a localised negative charge that is believed to induce conformational changes or alterations to the surface charge of a protein.Conveniently, phosphorylation also provides an effective means for selective charge-based enrichment of peptides, thus enabling large-scale phospho-proteomics experiments. We now have substantial datasets that have identified many thousands of phosphorylation sites on proteins. However, deriving generally applicable mechanistic knowledge from large phospho-proteomic datasets (PhosPhAt #refs) about how phosphorylation at a specific amino acid governs function has been much slower, due to a need to experimentally validate on an individual basis (#refs Kadota, etc). It would be useful to be able to recognise, prioritise and even predict functionally important phosphorylation sites, in many different systems.

In this project we will focus on the FERONIA pathway because this receptor kinase is involved in many aspects of plant growth, development and immunity, several interacting proteins are known and I have a unique preliminary dataset of FERONIA interactions and downstream phosphorylation events. I hypothesise that FERONIA serves as a hub to integrate stress and growth responses from several different stimuli and unrelated pathways and thus will be a valuable pathway to explore in detail and will provide broadly applicable insights on how protein phosphorylation regulates function through protein interactions. We will leverage 3D protein structure prediction to prioritise specific phosphorylation sites (identified in our preliminary data) for experimental validation in the FERONIA pathway. You will use a combination of bioinformatics, 3D modelling, molecular biology and proteomic methods to identify and validate key functional phosphorylation events.

Determining the mechanism of elicitor specific innate immunity in plants

Secondary Supervisor(s): Professor Vardis Ntoukakis

University of Registration: University of Warwick

BBSRC Research Themes:

• Sustainable Agriculture and Food (Plant and Crop Science)
• Understanding the Rules of Life (Plant Science)

Project Outline

Plants are constantly under attack from a diverse array of pathogens, including bacteria, fungi, viruses, and nematodes. To survive these threats, plants have evolved a sophisticated innate immune system that can recognize and defend against a wide range of invaders. Conserved features of microbes, such as flagellin in bacteria or chitin in fungal cell walls are recognised by receptor kinases and trigger a variety of defense responses, including the production of antimicrobial compounds, the strengthening of cell walls, and the activation of programmed cell death. The flagellin and chitin defense pathways are two of the most well-conserved and well-studied pathways in plants. Despite the conservation of signaling components, there is also some redundancy and specificity in the plant innate immune system. This means that there are multiple signaling pathways that can lead to the activation of defense responses, and that different signaling pathways are activated in response to different pathogens. This redundancy provides plants with a robust defense system that can effectively protect them from a wide range of pathogens.

Previous research in the Jones and Ntoukakis groups has identified a number of proteins that are involved in innate immune signalling and plant resistance to pathogens and we now have substantial datasets of proteins that are differentially phosphorylated in response to chitin and/or flagellin in several plant species (Brassica, tomato, maize, and the model plant Arabidopsis thaliana). We have a number of candidate proteins that seem to improve resistance of A. thaliana to pathogens when the protein is knocked out. However, removing a full protein often has other unintended and undesirable side-effects, for example, one of our candidate proteins is involved in cell wall biosynthesis and whilst plants with lower levels of this protein show improved resistance to both bacteria and fungi, the leaves are more brittle and easily damaged.

The objective of this project is to explore precision editing of candidate proteins involved in innate immunity in plants. We know that our proteins of interest are phosphorylated at specific amino acid residues during defence responses. Rather than knocking out the entire protein we wish to explore the impact of the phosphorylation sites specifically. Therefore, you would use gene edited versions of the proteins to create phospho-mimetic or phosphorylation-resistant transgenic plants. We believe that this precision editing approach would help to reduce undesirable off target effects of removing an entire protein. Our research from A. thaliana and into crops such as Brassica olereacea, we have extensive experience working with a double haploid line that makes genetic analysis and mutations more tractable. Your work would include screening transgenic plants against a variety of pathogens and identifying protein-protein interactions or changes to protein localisation that results from the mutations at the phosphorylation site. You will gain an in-depth understanding of both conserved and pathway specific signalling events, the molecular machinery involved in plant innate immunity, a solid foundation in plant molecular biology and protein biochemistry.