Assaying allosteric modulators of plant immune receptors for innovative crop protection
Lead Research Organisation:
Newcastle University
Department Name: Sch of Natural & Environmental Sciences
Abstract
This project will address fundamental questions regarding allosteric modulation of tomato PSKR1 receptors, as we will be able to accurately quantify receptor-ligand interactions through development and validation of robust and facile to use cell-free assay, based on microscale thermophoresis (MST). MST detection enables characterisation of interactions for a wide range of biomolecules, even for challenging samples such as whole cell lysates.
The compelling goal of this proposal lies in the application of a fluorophore-tagged PSKR1 receptor into MST immobilisation-free technology without a requirement of protein purification. We will transiently express tomato PSKR1 in tobacco leaves to ensure native microenvironment and post-translational modifications (e.g. glycosylations), and subsequently test, validate and optimise the assay conditions for binding peptides and small molecules alike.
Through this approach, we will develop a new technology underpinning research in plant immune system applied to crop protection and enabling target-focused discovery of novel protective agents. We will establish new ways to study the function of plant PSK receptors in health and disease, and involvement of plant immune receptors in host-pathogen interactions at unprecedented level of detail. We will understand how interfering with specific allosteric binding site, identified in our pilot data, impacts interactions between PSKR1 and pathogen-derived peptides showed to be involved in the disease, and how these sites can be utilised in boosting plant immune responses against the pathogens.
Technology and proof-of-concept data generated in this proposal will be used to support the larger funding bid that we plan with our project partner and collaborators, and will also provide a route for commercialisation through target validation of PSKR1 and discovery of novel chemical entities that will be jointly developed into novel crop protective agents.
Proposed research, spanning biomolecular interactions, biochemistry, cell, molecular and plant biology will have an impact on in the areas of crop protection, plant immunology, training of early career scientists, and cross-sectoral engagement between academics from diverse related disciplines, industrial partners, and growers.
In conclusion, this multidisciplinary proposal will yield (i) the assay, quantifying interactions between PSKR1 and ligands; (ii) proof-of-concept data on host-pathogen interactions via PSKR1 receptors; (iii) development of allosteric modulator leads to enable development of new agents for crop protection, to underpin future grant applications, and to establish a new intellectual property.
The compelling goal of this proposal lies in the application of a fluorophore-tagged PSKR1 receptor into MST immobilisation-free technology without a requirement of protein purification. We will transiently express tomato PSKR1 in tobacco leaves to ensure native microenvironment and post-translational modifications (e.g. glycosylations), and subsequently test, validate and optimise the assay conditions for binding peptides and small molecules alike.
Through this approach, we will develop a new technology underpinning research in plant immune system applied to crop protection and enabling target-focused discovery of novel protective agents. We will establish new ways to study the function of plant PSK receptors in health and disease, and involvement of plant immune receptors in host-pathogen interactions at unprecedented level of detail. We will understand how interfering with specific allosteric binding site, identified in our pilot data, impacts interactions between PSKR1 and pathogen-derived peptides showed to be involved in the disease, and how these sites can be utilised in boosting plant immune responses against the pathogens.
Technology and proof-of-concept data generated in this proposal will be used to support the larger funding bid that we plan with our project partner and collaborators, and will also provide a route for commercialisation through target validation of PSKR1 and discovery of novel chemical entities that will be jointly developed into novel crop protective agents.
Proposed research, spanning biomolecular interactions, biochemistry, cell, molecular and plant biology will have an impact on in the areas of crop protection, plant immunology, training of early career scientists, and cross-sectoral engagement between academics from diverse related disciplines, industrial partners, and growers.
In conclusion, this multidisciplinary proposal will yield (i) the assay, quantifying interactions between PSKR1 and ligands; (ii) proof-of-concept data on host-pathogen interactions via PSKR1 receptors; (iii) development of allosteric modulator leads to enable development of new agents for crop protection, to underpin future grant applications, and to establish a new intellectual property.
Technical Summary
In this project, we will build upon our exploration of the conformational landscape of tomato PSKR1 receptor and identification of a new allosteric binding site, and we will assess PSKR1 interactions with ligands using a biophysical assay to be developed and validated.
To overcome known issues with recombinant protein production, purification, and cell-based assays, we will develop a robust purification-free assay with microscale thermophoresis (MST) readout, which relies on GFP-fused protein construct transiently expressed in planta. The MST technology and assay design is our format of choice for a number of reasons: (i) a full characterisation of stoichiometry and thermodynamics; (ii) very low reagent volume which minimises sample requirement; (iii) a wide range of affinities determined (sub-nanomolar to millimolar affinity constants); (iv) applicability to ternary complexes studies, crucial for assessing allosteric regulators; (v) no requirement for immobilisation; (vi) suitability for cell lysates, with no requirement for purification; (vii) suitability for high-throughput scale, as the software allows rapid data fitting and analysis of multiple samples; (viii) value: the assay, once established and validated would be highly cost-effective. This diversity is unmatched by any other biophysical assay techniques currently available.
Following established protocols, we will transiently express tomato PSKR1 in tobacco leaves to ensure native microenvironment and post-translational modifications (e.g. glycosylations), and subsequently test, validate and optimise the MST assay conditions, suitable for binding peptides and small molecules alike. Native PSK peptide will be used as a positive control.
After the optimisation and validation, the assay will be utilised to (i) test interactions between a panel of disease-linked peptides derived from B. cinerea, and (b) small molecules identified as putative allosteric modulators in our initial virtual screen.
To overcome known issues with recombinant protein production, purification, and cell-based assays, we will develop a robust purification-free assay with microscale thermophoresis (MST) readout, which relies on GFP-fused protein construct transiently expressed in planta. The MST technology and assay design is our format of choice for a number of reasons: (i) a full characterisation of stoichiometry and thermodynamics; (ii) very low reagent volume which minimises sample requirement; (iii) a wide range of affinities determined (sub-nanomolar to millimolar affinity constants); (iv) applicability to ternary complexes studies, crucial for assessing allosteric regulators; (v) no requirement for immobilisation; (vi) suitability for cell lysates, with no requirement for purification; (vii) suitability for high-throughput scale, as the software allows rapid data fitting and analysis of multiple samples; (viii) value: the assay, once established and validated would be highly cost-effective. This diversity is unmatched by any other biophysical assay techniques currently available.
Following established protocols, we will transiently express tomato PSKR1 in tobacco leaves to ensure native microenvironment and post-translational modifications (e.g. glycosylations), and subsequently test, validate and optimise the MST assay conditions, suitable for binding peptides and small molecules alike. Native PSK peptide will be used as a positive control.
After the optimisation and validation, the assay will be utilised to (i) test interactions between a panel of disease-linked peptides derived from B. cinerea, and (b) small molecules identified as putative allosteric modulators in our initial virtual screen.