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This year’s project has laid the foundations for another team to potentially continue our work with Puccinia spp. We have been able to establish optimised methods for culturing fungal proteins in bacteria and have curated a database of proteins we have yet to test. Future teams would be able to use our method of transforming fungal genomes into competent E. coli cells which has applications beyond our project and can be utilised by teams requiring expression of a variety of fungal proteins.
The list of proteins our team has worked on is as follows:
Pst_12806, PstSCR1 and HXT1 are proteins from Puccinia striiformis. TalSP is found in wheat plants.The cells used were T7 competent E. coli and TURBO competent E. coli cells.
TaISP is found as a subunit of cytochrome b6f complex, an enzyme in chloroplasts involved in the transfer of electrons during photosynthesis. TaISP is a target for proteins expressed by Pst 12806, a fungal genome, leading to impaired photosynthesis by disrupting the electron transport chain. Our work with TaISP and Pst 12806 involved inducing it in vitro demonstrating success with being able to replicate its effects in the lab to allow for further testing of this part in the future, such as purifying and crosslinking plant and fungal proteins to identify binding sites along with mass spectrometry analysis of the proteins to accurately determine specific binding sites.
PstSCR1 is a small cysteine-rich protein that is highly expressed during wheat infection acting as an apoplastic effector, helping the fungus evade the plant immune system within the apoplast. The exact method of action isn’t known, but this protein is interesting due to its increasing resistance in non-host plants. This translational research can help with exploring vaccination methods for wheat in the future and future teams can work towards replicating the method of resistance observed in non-host plants for wheat.
PsHXT1 is a hexose transporter gene highly expressed during infection, demonstrating that a method of action the rust takes upon infection is taking sugars from the host plant. Hexoses are seen to be the main form of sugar utilised by the fungus as PsHXT1 is the only highly induced sugar transporter gene to be seen during infection. This sugar transporter is seen to be highly conserved. When compared to other species, polymorphisms are low, with a less than 1% nucleotide difference seen. Future directions would involve coming up with mechanisms to silence PsHXT1 in conjunction with other sugar transporters to inhibit cereal rust metabolic pathways.
We have also devised methods for transforming fungal genomes in bacterial cells. We hoped to be able to have this bacterial vector as a stand-in due to culturing fungi - due to its comparative safety and ease of storing and culturing in the lab. Antibiotics can be used as a failsafe and also the implementation of a kill switch in the bacterial expression system is easy and able to be controlled externally. Future teams will be able to take advantage of our methodology when working with fungal genomes in the future.
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Through our exploratory modelling techniques. Our team shows the potential challenges and solutions to the problems future iGEM teams may make in the competition when using a protein protein interaction network to discern essential proteins for targeting. This model also highlights the necessary improvements for future teams to make in order to fully automate this pipeline we propose from proteome to essential protein targets.
Read more about our modelling approaches here.
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