Supervisors: John Jones, Sophie Mantelin
Project description:
Resistance genes provide plants with protection against a range of pathogens. Despite their use in crop breeding programmes, our knowledge of how plant resistance genes function is still sketchy and mainly based on studies in leaves rather than roots. In particular, little is known about how resistance against plant-parasitic nematodes (PPN) works and very few nematode resistance genes have been cloned to date[1].
The H2 resistance gene provides complete resistance against the Pa1 pathotype of the potato cyst nematode (PCN), Globodera pallida, and partial resistance against other pathotypes (Pa2/3) of this species. Recent work at The James Hutton Institute has allowed a candidate H2 gene to be identified, providing an opportunity for a PhD student to undertake detailed functional analysis of the mechanisms of resistance mediated by H2. Proposed work areas will include:
Validation of the H2 candidate gene and analysis of the cell biology of the H2 response to infection. PCN can infect potato as well as tomato. Transgenic plants will be produced that express the H2 candidate in a susceptible background to test its efficiency in both crops. In addition, transgenic plants expressing tagged-versions of H2 will be made for imaging (e.g fluorescent protein fusion or through use of emerging labelling tools such as DNA PAINT[2] that allow detailed analysis of protein spatial expression patterns). We will investigate relocalisation of H2 upon recognition of Pa1 and Pa2/3; this is a critical component of defence signalling for many other resistance genes and will provide information that will be of use for interpretation of data obtained on the proteins interacting with H2.
Analysis of the signalling response induced by H2 in response to Pa1 and Pa2/3. Transcriptomic approaches will be used to investigate which defence signalling pathways are upregulated upon infection in resistant plants. H2 will also be subjected to random mutagenesis and clones will be screened through transient expression in tobacco leaves to identify a constitutively active form of H2 that causes cell-death. This will subsequently allow dissection of the H2-mediated response through expression in mutant tomato lines defective in various signalling components or in plants in which key signalling pathways are impaired through virus-induced gene silencing (VIGS). The autoactive H2 will also be used to screen for putative cell-death suppressors among effector proteins from Pa2/3 nematodes[3].
Identification of signalling partners of H2. In order to understand the detail of how H2 functions requires knowledge of the proteins that it interacts with. Therefore, these proteins will be identified through pull-down of tagged H2 expressed in plants followed by mass spectrometry analysis of proteins captured. A new proximity labelling method based on pupylation is currently being developed by Dr Piers Hemsley at the institute, and we will investigate the use of this system as an alternative approach for identifying H2 interaction partners.
This project will provide training in a wide range of state-of-the-art molecular biology and cell biology tools. In addition, the student will have the opportunity to gain experience in production of transgenic plants and associated techniques.
References:
[1] Davies & Elling (2015) “Resistance genes against plant-parasitic nematodes: a durable control strategy?” Nematology (17), 249-263 [DOI:10.1163/15685411-00002877].
[2] Strauss et al. (2017) “Modified aptamers enable quantitative sub-10-nm cellular DNA-PAINT imaging”. Nature Methods [DOI: 10.1038/s41592-018-0105-0].
[3] Thorpe et al. (2014) “Genomic characterisation of the effector complement of the potato cyst nematode Globodera pallida” BMC Genomics (15), Article 923 [DOI: 10.1186/1471-2164-15-923].
To apply for this project, please go to this link.
