Primary supervisor: Dr Graeme Kettles & Dr Megan McDonald, School of Biosciences
Non-academic partner: Syngenta
Project Title: Identification of essential components for protein secretion in the phytopathogen Zymoseptoria tritici
Z.tritici is a sustained threat to wheat production due to its ability to overcome chemical control and plant genetic resistance. This fungus relies on its secretory machinery to manipulate wheat plants throughout its life cycle: the secretion of adhesins and digestive enzymes during early colonisation, and the secretion of virulence proteins (effectors) throughout infection. Work using model yeasts has identified many components critical for protein secretion in fungi, accompanied by markers/reporters for investigating the secretory apparatus. However, these resources are presently lacking for Z. tritici. This project will develop a range of markers and reporter systems for monitoring secretion, and use forward genetic screens to identify Z. tritici components essential for secretion. This will provide new fungal targets for chemical inhibition of secretion against this priority pathogen.
The project has the following objectives:(1) To develop new marker/reporter systems for investigating protein secretion in Z. tritici. (2) To identify Z. tritici genes that are essential for a functional secretory pathway, as novel targets for chemical inhibition.
This will be achieved by work in three areas:
- Develop Z. tritici secretion pathway markers and a high-throughput secretory reporter assay
The student will develop fluorescent markers for all stages of the secretory pathway e.g. endoplasmic reticulum, Golgi, early/late endosomes. Secondly, we will develop a high-throughput functional assay to measure the integrity of the Z. tritici secretory system. This will be achieved by generating secreted reporters for colorimetric, fluorescent and luminescent analyses. It is anticipated that the CLuc system developed in yeast will be most amenable to high-throughput robotic handling and allows measurement of both quantity and quality of secreted protein.
- Forward genetics to identify essential secretory system components
We will perform forward genetic screens by UV mutagenesis using the reporter strains developed above as the genetic background. Surviving mutants will be screened for loss of reporter activity in a high-throughput 96-well plate-based screen. Whole-genome re-sequencing of mutants of interest, and a control group of mutants with functional secretion, will be performed to identify candidate loci important for protein secretion.
- Functional analysis of secretory genes and contribution to virulence
The importance of candidate genes to secretion will be validated by Agrobacterium-mediated targeted gene deletion or gene knockdown. Mutants generated in this work will be used to confirm the candidate genes’ involvement in the secretory pathway, and will also be used for in planta infection assays to assess their contribution to virulence.