Fusarium head blight (FHB) is a fungal disease of cereals which reduces crop yields and grain quality. It also poses a food safety risk due to contamination of grains with mycotoxins, but there is currently no effective control against this disease. Dr Neil Brown’s lab at the University of Bath aims to uncover the biological processes needed to establish FHB and potentially reveal new targets for fungicides.
The main agent of FHB on wheat is the ascomycete fungus Fusarium graminearum and during infection it must scavenge nutrients such as zinc from its host. Phylogenetic analyses suggest that F. graminearum encodes nine putative zinc importer proteins (ZIPs), some of which are upregulated during wheat infection, suggesting they are involved in FHB disease. My project aimed to evaluate the function of these proteins.
Firstly, we investigated whether complementation with F. graminearum ZIPs would recover the ability of mutant Saccharomyces cerevisiae strains lacking ZIPs to grow in low zinc conditions. Droplet assays were performed with wild-type and ZIP mutants to qualitatively compare their growth in zinc replete and zinc limited media. While the wild-type CM30 yeast grew well in zinc replete, unaltered and zinc limited conditions, the double (CM34) and triple (CM37) ZIP mutants grew very poorly in zinc limited conditions. We transformed CM34 and CM37 strains with individual F. graminearum ZIPs using the pESC-URA expression vector. We selected the transformed yeasts and carried out both droplet and liquid assays to evaluate the transformants’ recovery. The wild-type phenotype was not fully restored for any transformed mutant in the droplet assay, but growth in the zinc limited 25 µM CaEDTA condition was improved in CM37 when complemented with F. graminearum ZIP ZrfC. Slightly increased growth was also seen in the liquid assay for both the CM34 and CM37 strains complemented with ZrfC, however more replicates are needed to determine whether this difference is significant.
Secondly, we compared the growth of F. graminearum mutants lacking different ZIPs to growth of the wild-type PH-1 strain in different zinc conditions by measuring changes in the optical density. ΔZrfB (F. graminearum lacking ZrfB) had impaired growth relative to the wild-type strain in zinc limiting conditions. This suggests that this ZIP is involved in zinc acquisition. However, five other ZIP mutants grew no differently to the wild-type strain. This may be due to redundancy in ZIP function. F. graminearum strains lacking the putative ZIP-regulating transcription factor ZafA had severely impaired growth compared to the wild-type strain in zinc limited conditions. This suggests that ZafA is involved in zinc acquisition, and the extreme effect of its absence could indicate that it controls transcription of multiple functionally redundant ZIPs.
The results of the F. graminearum growth assays correlate with data from 15-day wheat infection experiments where ΔZrfB had the lowest mean extent of infection of all tested ZIP transporter mutants, while infection by the ΔZafA transcription factor mutant was nearly abolished. Together these results suggest that zinc acquisition is important for both growth and virulence of F. graminearum, and interrupting zinc acquisition could be a potential method for controlling FHB disease. ZafA does seem to be a transcription factor involved in zinc acquisition, while evidence suggests that ZrfB and ZrfC may function as ZIPs. Further research is needed to confirm whether the remainder of these genes do encode proteins that function as ZIPs, and to demonstrate their regulation by ZafA in response to zinc limitation.
I have enjoyed this period of research very much and am looking forward to pursuing further opportunities to be involved with plant pathology research in the future. I have had an invaluable insight into working in a research lab and gained experience in a range of procedures and techniques, including creating and characterising fungal mutants, performing droplet and liquid growth assays and carrying out wheat infection experiments. I would like to thank my supervisors Dr Neil Brown and Louise Johns for this exciting opportunity and the BSPP for funding this project.
University of Bath
Figure Above: Fusarium ZIP partially complements zinc transporter function in yeast. Droplet and liquid growth assays in zinc limiting media (CaEDTA zinc chelator) show increased growth of ScZIP mutants upon expression of ZrfC. Yeast strains: CM30 = wild-type, CM34 = ΔScZIP1/2, CM37 = ΔScZIP1/2/3, EV = empty vector control, A/B/C = complementation with ZrfA, ZrfB or ZrfC. Droplet assays on 25 µM CaEDTA after 72 hours at 30°C (A+C) and mean change in OD 600 in 50 µM CaEDTA over 48 hours (B+D).