Fusarium Head Blight (FHB), caused by Fusarium graminearum, is a pervasive floral disease of cereals from wheat to maize. As a top ten fungal pathogen, regular FHB epidemics are expensive and threaten food security. F. graminearum colonises wheat floral tissues, invading and destroying host cells resulting in unappetising, shrivelled grains. But appearances are the least concerning consequence, as the fungal pathogen secretes a pernicious cocktail of virulence factors notably the mycotoxin deoxynivalenol (DON) which is acutely toxic, causing nausea, diarrhoea and vomiting, threatening the health of livestock and humans alike. While good agronomic practice, fungicide spraying and post-harvest processing can keep mycotoxin levels low, the effects of this chronic exposure in our food system remains unclear. In tackling this pathogen, this summer I had the privilege of working in the Brown Lab at the University of Bath.
Previous research from the Brown lab on F. graminearum environment sensing identified a PTH11-like receptor that was upregulated during and essential for symptomless infection of wheat. Building on this finding and the crystal structure of the novel extracellular CFEM domain of a PTH11-like receptor (Prof. L-O. Essen, University of Marburg) we sought to investigate the impact of four different sets of mutations at residues thought to facilitate interaction between the receptor’s transmembrane and CFEM domains, which may play a role in receptor function, environment sensing and virulence. Edited-receptor genes and geneticin selectable markers were used to complement the PTH11-like gene deletion strain via split marker-mediated transformation. After several rounds of propagation on selective media, I extracted genomic DNA from the successful transformants and performed diagnostic PCRs to confirm homologous integration into the native gene locus. I was fortunate enough to obtain at least one correct transformant for each of the four edited-receptors from my first ever fungal transformation.
We proceeded with measuring the virulence phenotype of our receptor-edited strains alongside mock, parental (PH1), CFEM domain-deleted (ΔCFEM) and wild-type complemented (COM) strains. Virulence was measured by inoculating the wheat florets at anthesis. We measured the number of spikelets displaying infection through characteristic bleaching, as well as documenting any unexpected symptoms, such as brown lesions or atypical infection patterns by photography. Tallied spikelet counts were used to quantify virulence over the 12-day course of infection by employing an R script (apsnet.org) to calculate the average area under the disease progression curve, developing my programming and statistics skills with my own data.
Seeking to assess other measures of virulence and important consequences of the disease, at the end of the infection course wheat heads were cut, freeze-dried under negative pressure to remove moisture, before grinding into a powder in liquid nitrogen. The DON mycotoxin was then easily liberated by dissolving in water and the plant debris removed by centrifugation, before quantifying DON contamination in a competitive ELISA. The ELISA was an apt final test of my organisational, diluting and pipetting skills that I had honed over the summer. Between experiments, I set myself to analysing the model protein structure provided by Prof. Essen, comparing it with the primary structure in R for input into Missense 3D (missense3d.bc.ic.ac.uk), a publicly available tool for predicting the impact of missense mutations, greatly developing my bioinformatics skills.
The ΔCFEM strain was confirmed to have reduced virulence compared to the wild-type strain. But ultimately, the four receptor-edited F. graminearum strains did not show statistically significant differences in the spread of infection, or DON contamination, in comparison to a strain complemented with the wild-type PTH11-like receptor, which rescued virulence though not to the same level as the parental strain. Missense3D also predicted little structural damage. Therefore, further studies of the structural impact of these receptor mutations and additional replication of wheat infection studies are now required to confirm the importance of these residues to CFEM domain interactions and receptor function.
This productive summer has given me an arsenal of essential hands-on molecular genetics, plant pathology and dry-lab skills that I know will prove vital going forward, as I look towards establishing a career in plant-health research. I would like to thank my supervisor Neil Brown for his tireless support and patience during this project, Louise and Pamela for their help, and the BSPP for making this invaluable opportunity possible.
University of Bath
MAIN IMAGE above: Model structure of F. graminearum G protein-coupled receptor FGRRES_16221, courtesy of Prof. Essen, University of Marburg. CFEM-domain (Red), transmembrane domain (cream), visualised in ChimeraX.
A) Photos of wheat heads at 12 days post infection (DPI). Receptor-edited strains (denoted by edited-receptor No._colony No.) are presented alongside mock infected, parental (PH1), CFEM-deleted (ΔCFEM) and wild-type complemented (COM) strain. B) Concentrations of DON (ppm/g) for each strain. Error bars represent standard deviation.