.
Puccinia are pathogenic fungi known to cause infections in plants known as rusts which include over 120 genera and 6000 species (Sadys et.al, 2017) Their life stages include the production of sexual spores as well as up to four different asexual spores. Due to their large quantities and ultra-light nature, they are aerodynamically efficient and can travel long distances over short periods. Additionally, they possess thick walls and high pigmentation which protects them against UV radiation. This ensures that they remain viable for long distances. On advancement of infection, it manifests as a reddish/brown powder on the leaf surface due to formation of urediniospore masses. This can lead to water and nutrient loss through the restriction of photosynthetic areas, causing large yield losses in crops around the world.
In their obligately parasitic relationship, the fungi develop a specialised organ for the transfer of nutrients, from the plant host cells to the fungal thallus, called the haustorium. Through our initial literature review we found an haustorium-specific effector protein, Pst-12806, which affects chloroplast function in plants upon translocation. Effector proteins are expressed by plant pathogens and are involved in the infection of plant species (Mysore et.al, 2022) Expression of this protein plays a vital role in the pathogenesis of the fungus and in suppressing the basal immunity of the plant. It has been shown to reduce electron transport rate and photosynthesis in plants upon interaction with the wheat TaISP protein - a component of the cytochrome complex (Wang et.al, 2019)
Consequently, Puccinia spp. pose significant threat to wheat crop yields, with potential for disastrous economic and social consequences. Our solution proposes the design of a novel peptide-based inhibitor to prevent the growth of Puccinia spp. We began with a focus on determining appropriate targets to design our peptide-based inhibitor around. In this process, we screened existing literature and analysed protein databases using various bioinformatic tools such as GeneMania and NCBI’s BlastP (Modelling Page). This yielded an initial short list of proteins important to the pathogenesis and function of Puccinia spp. Additionally, we decided early on in our project to use a fluorescence based approach to analysing the interaction/binding affinity between our peptide and target protein. Consequently, we ran AlphaFold models to verify the feasibility of conjugating mVenus fluorophores to our proteins. This helped to narrow down our shortlist of protein targets to: PstSCR1, Pst_12806, PstHXT1 and Pgt-Iaam – all conjugated to a mVenus fluorophore.
The next stage of our project focussed on designing novel peptides to specifically bind and inhibit our shortlisted proteins. This involved modelling peptide-protein interactions on RossettaDock and PyMol. The feasibility of each peptide-fluorophore combination was not of significant importance – due to the minute differences in sequence/size between peptides. Additionally, we decided to include known protein binding partners for out short-listed proteins. These included TaISP and SERK3B. This resulted in a short list consisting of our novel peptides, TaISP and SERK3B – all conjugated to a mCerulean fluorophore.
Subsequently, we were able to recombinantly express our fluorescently tagged proteins in the laboratory. This involved using NEB TURBO and T7 Express E.coli strains transformed with plasmid pET-19b. Following this, the binding affinity between our protein targets and peptides, was tested using Fluorescence Resonance Energy Transfer (FRET). This allowed us to assess the feasibility of using our peptide as a potential Puccinia spp inhibitor.
.
.
15 µL of the assembly mixture aliquot consisting of: 320µL 5X Isothermal Master Mix, 0.64 µL 10 U/µL Phusion DNA Polymerase, 160 µL 40 U/µL Taq DNA Ligase and 699.36 µL water, was allowed to thaw in 4 microcentrifuge tubes and kept on ice. 5 µL DNA fragments of PstSCR1, PstHXTI, Pgt_IaaM and Pst_12806 were added to the assembly mixture. The mixture was incubated at 50ºC for 60 minutes.
To prepare competent cells, 5 mL of LB media was inoculated in a 50 mL falcon tube with a single colony from each NEB TURBO and T7 Express E. coli strains. The inoculated media was incubated in a shaking incubator for
18 hours, after which they were centrifuged at 3000rcf for 5 minutes. The supernatant was removed and the pellets were resuspended in 5 mL of PBS. 0.5 mL of this culture was used to inoculate a 50 mL flask of LB media
and incubated in a shaking incubator until an OD600 between 0.4 - 0.6 was achieved. Following this, the cells were poured into a sterile 50 mL falcon tube and placed in ice for 10 minutes. They were then centrifuged at
3000rcf for 5 minues and the supernatant was discarded. The resulting pellet was resuspended in 20 mL of 0.1 M CaCl2 and left on ice for 1 hour. The resuspension was centrifuged at 3000rcf for 5 minutes to obtain a pellet
and the supernatant was discarded. The cells were resuspended in 10 mL of 0.1 M CaCl2, 15% glycerol.
2 µl of ligation mixture was chilled in a 1.5 mL microcentrifuge tube. 50 µL of competent cells, prepared earlier, were added to the DNA by gentle mixing. The mixture was placed on ice for 30 minutes and then heat
shocked at 42ºC for 30 seconds. 950 µL of room temperature media was added to the tube which was placed at 37ºC for 60 minutes. The mixture was shaken at 250 rpm and 50 µL was spread onto selection plates. Control
plates were set up according to Table 1. The plates were incubated overnight at 37ºC and inspected for growth the next day. Single colonies were streaked from each of the three transformed cells.
| LB + Ampicillin | LB only (No Ampicillin) |
|---|---|
| NEB TURBO cells without plasmid | NEB TURBO cells without plasmid |
| T7 cells without plasmid | T7 cells without plasmid |
| T7 cells with SCR1 plasmid | Dots of the 3 cell cultures with plasmid (SCR1, HXT1, Pst_12806) |
| TURBO cells with HXT1 plasmid | |
| TURBO cells with Pst_12806 plasmid |
3 plates were prepared with LB and ampicillin for PstHXT1, PstSCR1, and Pst_12806. A grid was drawn on each plate with 8 cells. 15 µL of cell lysis buffer was added to each of 24 microcentrifuge tubes. A pipette tip
was used to pick a single colony from the relevant cell culture plate and dotted onto cell on the grid plate. The tip was then placed in the cell lysis solution and the resulting samples were boiled at 100 ºC for 15
minutes. Following this, the samples were centrifuged at 13, 000g. PCR was conducted on the supernatant and visualised on agarose gels.
Two protein purification techniques were employed; gravity column purification and the AKTA start chromatography system. SCR1 protein was purified using gravity column purification and then AKTA start chromatography.
For gravity column purification, a chromatography column was loaded with 1 mL of Slurry, composed of equal parts (by volume)of resin and 20% ethanol. 20% ethanol was then allowed to flow through the column. Following
this, the column was equilibriated by adding 5 CV (column volume) of binding buffer. This was done twice. Previously prepared clarified lysate was then loaded into the column, using a syringe. It was prepared via
sonication, then centrifugation and filtration of cell culture. Subsequent, flowthrough was collected for later analysis. Next, 5 CV of binding buffer was loaded and after it flowed through, 5 CV of wash buffer was
added. 5 CV of elution buffer was finally added, with the flow through being collected to catch any protein that was eluted out.
For the AKTA start chromatography system, we first set up the system by running proprietary unicorn software to allow for remote control of the AKTA start from a computer. The tubing was inserted into the peristaltic
pump, the sample inlet was placed into 30 mL of equilibration buffer, Buffer A inlet was placed into equilibration buffer and Buffer B inlet was placed into elution buffer. To connect the column to the system,
a manual run of of 0.5 mL per minute was started. The flowpath of the wash valve was changed from ‘waste’ to ‘column’ and the column was connected, starting with the top first after checking to see if buffer was
dripping from the connector. The bottom was then connected and the column was checked for leaks. To begin purification of the protein, the sample inlet was moved from the equilbriation buffer and placed into the sample
tube. Below the fraction detector, enough tubes were loaded on the a rack. Using the computer, we ran the recommended method of ⅕ mL column 20 CV gradient manual flow through collection. This process is automatic and
our intervention was only needed at the end of the sample loading to avoid drying the column and during the wash phase to hold the wash longer, allowing UV to reach baseline detection.
Due to time constraints, the team decided to use cell lysates in preparation for FRET analysis. NEB T7 E.coli cultures for Pst12806 and TaiSP were sonicated according to the following protocol:
Our SCR1 pellet was suspended in lysis buffer and put on ice. For 1 g of our pellet, we used 10 mL of lysis buffer. Using a serological pipette attached to a pipette gun, the pellet was resuspended.
The resuspended pellet was transferred into a 100 mL glass beaker. The sonicator was then set up by screwing on the sonicator tip on to the bottom of the sonicator probe. We adjusted the table to ensure
the tip was submerged in sample but not contacting the beaker. The beaker on ice was then placed on the table inside the sonicator with parafilm on top. A hole was made to allow tip entry. The sonicator
was then set to pulses of 2 seconds on and 2 seconds off. This is done until the total time a pulse is being applies (‘on’ time) is 8 minutes. Next, we transferred the sample into centrifuge tube and
centrifuged it at 10 000 rcf for 10 minutes.
Samples of cell lysates containing: Pst12806-mCerulean and TaISP-mVenus were prepared, in duplicate, on a 96 well plate. They were loaded according to an 8 sample serial dilution, at a dilution factor of 4:5, and loaded across two rows of the 96 well plate. Additionally a control containing purified mVenus and mCerulean protein was identically serially diluted in a third row.
.
.
cPCR
Overall, we were able to demonstrate successful transformation of our Pst12806/TaISP ligated pET19b plasmids into NEB T7 E.coli cells.
Figure 1 represents the results of our cPCR screening for cells undergoing transformation with Pst12806 recombinant plasmid. It confirms the successful transformation of Colony 3.
This is indicated by the presence of a DNA band at 1300bp, which corresponds to the predicted length of our Pst12806 DNA (1239bp).
Similarly, Figure 2 represents the results of our cPCR screening for cells undergoing transformation with TaISP recombinant plasmid. It confirms the successful transformation of colony 2 and 6.
This is indicated by the presence of a DNA band at approximately 1595bp which corresponds to the predicted length of our TaISP DNA (1467bp)
Protein Expression
Quantification of Pst12806 and TaISP expression levels were not successfully measured.
FRET analysis
Analysis of the FRET results reveals no usable or statistically significant findings.
.
.
Through our experimental analysis we showed successful recombinant expression and purification of the two fluorescently tagged proteins: Pst12806-mVenus and TaISP-mCerulean. This is supported by colony PCR results, as seen in Figures 1 and 2. In Figure 1, the protein of interest was detected within one colony- indicated by a strong band at1300bp in lane 4 of the gel. Similarly, in Figure 2, bands corresponding to the protein of interest were detected for two colonies.
However, due to constraints within the project, the specific expression levels for Pst12806 and TaISP were not measured. Conventional purification and visualisation steps, were foregone in place of sonication. This allowed us to prepare whole cell lysates for FRET analysis but prevented validation of specific Pst12806 and TaISP expression. This represents a flaw in experimental design. Future iterations of the experiment should implement protein purification followed by quality control via SDS-PAGE. This would allow for verified and enriched expression of our proteins.
Following this, FRET analysis was conducted. Although we had success transforming our colonies, FRET analysis revealed Pst12806 concentrations were not adequately high enough to produce meaningful and statistically significant results. Additionally flaws setting up adequate control samples, voided our FRET results.
.
.
To improve this experiment, we would need to see a significant increase in Pst12806 expression levels. Future rounds of this experiment could focus on the optimisation of Pst12806 expression and implementation
of affinity chromatography. Additionally, optimisation of the Pst12806 fusion protein and pET19b plasmid could play a significant role in enhancing protein expression.
.
.
.
.