Project No. 2350
Primary Supervisor
Prof Vincent O’Connor – University of Southampton
Co-Supervisor(s)
Dr James Dillon – University of Southampton
Marcus Guest – Syngenta (Industry partner)
Summary
An important goal for the future development of crop protection strategies is selective targeting of pest species over non-pest species.
Biologics based on protein architectures offer a potential route. The development of antibody technologies that disrupt molecules is an attractive approach. Conventional antibodies are oligomeric proteins with complex biogenesis. However, the relatively large size of antibodies restricts tissue access and penetration to target sites. In this proposal, we will investigate nanobodies: single chain simple polypeptide equivalents of the antibody variable heavy chain (1). These have epitope specificity to selectively target molecules and execute functional perturbation. The nanobodies have simplified recombinant expression and provide interfering technologies readily introduced into biological systems by transgenic approaches. We propose experiments designed to translate nanobody technology by taking advantage of the experimental tractability of C. elegans.
1. Advantaged by C.elegans. C. elegans are bacterivores advantaging feeding based approaches whether free recombinant nanobodies or bacteria expressing nanobodies initiate reagent delivery (2,3). We will use established phenotypes that have well known molecular determinants whose disruption is scored in quantitative behavioural assays. These phenotypes mirror disruptions played out in established chemical mitigation of pests.Furthermore, C. elegans allows the genetic manipulation of the system to express candidate target molecules with artificial tags. Commercial nanobodies against such tags can interrogate the potential of novel targets or pathways.
2. Chemical Biology to support nanobody investigation. Fully in-vitro approaches can identify candidate nanobodies and encoding cDNAs. Commercial nanobodies to artificial tags in engineered targets will be used to initiate work. The known topology and critical domains that control function or protein-protein interaction will optimize the screening of candidate epitopes for function perturbing nanobodies (5).
3. Investigating and modulating nanobody distribution. An important goal is validation of the nanobodies as biologics that can be used in crop protection.The worm provides an opportunity to probe how accessible targets are in context of a complex organism. We will test nanobodies engineered to express epitope recognition with and without a cell and tissue penetrating tags (6). These short stretches of engineered sequence have found excellent value in allowing cell impermeable entities to gain passage across cellular boundaries. We will compare penetration of nanobodies or modified nanobodies into the organism by feeding with native or modified entity (7). Development in the model C. elegans will highlight reagents and protocols for further testing in more genetically tractable pest species.
References
1. Wagne T and Rothbauer U (2021) Dev. 144:2694–2701.
2. Michele Perni et al., (2017) Sci Rep. 7: 15045
3. Corsi AK. (2006) Anal Biochem. 359(1):1-17.
4. Dimov I and Maduro MF. Cell and Tissue Research (2019) 377:383-396.
5. Götzke H et al. Nature Communications 10:4403
6. de Oliveira ECL et al., (2022) Front Cell Infect Microbiol. 12:838259.
7. Herce HD et al., (2017) Nat Chem. 9:762-771.