Notes on Work Programme and Methodology
Part 1
– We have an estimate of the number of copies of Lr34res-Venus in each transgenic plant. This was provided by NIAB using qPCR. We do not know if any of the T0 lines express the protein from these constructs. We need to know this before we can perform any experiment.
(PDF explaining this technique was attached to a previous email)
– We will compare the levels of expression of the T0 lines (40 in total) using:
1/ Epifluorescence microscopy. The “Venus” domain linked to Lr34 should fluoresce under the microscope if the construct is expressed. This is an excellent link to explain epifluorescence microscopy:
https://www.lifetechnologies.com/uk/en/home/life-science/cell-analysis/cell-analysis-learning-center/molecular-probes-school-of-fluorescence/fundamentals-of-fluorescence-microscopy/epifluorescence-microscope-basics.html
2/ qRT-PCR. Here is a good explanation of RT-PCR and qRT-PCR (the diagram is especially useful):
http://en.wikipedia.org/wiki/Reverse_transcription_polymerase_chain_reaction
– An epifluorescence microscope with the right filters is available in the laboratory. Samples of young T0 leaves will be mounted on slides and images taken of the fluorescence signal. The intensity of this signal will be compared to wild type.
– We will take samples of T0 leaves and extract RNA. We will make first-strand cDNA from this RNA. The cDNA will be used in a quantitative Reverse-Transcriptase Polymerase Chain Reaction (qRT-PCR) to compare levels of gene expression. Wild type plants should produce no product from the qRT-PCR. The strength of signal from the qRT-PCR should be proportional to the amount of Lr34res-Venus mRNA transcript produced.
Part 2
– Once we have established which lines produce Lr34res-Venus protein we will be able to test the hypothesis that Lr34res-Venus localises in response to pathogens.
– We will take leaves from T0 plants and infect these with conidia of Blumeria graminis f.sp. hordei (powdery mildew adapted to barley). This pathogen is chosen because it is available within the laboratory and generates a strong, localised immune response at sites of attempted infection.
– If the hypothesis is true Lr34res-Venus will accumulate close to the site of infection (similar to what has been observed with PEN3-GFP in Arabidopsis).
– To test our hypothesis we need high resolution fluorescence images where it is possible to compare the amount of fluorescence present at different areas of the same plasma membrane. We will compare the amount of Lr34res-Venus close to the interaction site with “control” areas far away. We will also compare uninfected cells.
– We will use a laser scanning confocal microscope (LSCM) for this purpose. The technical details can be found in the document I have attached (“confocal page.pdf”). This type of microscope can isolate the fluorescence signal from the plasma membrane. An epifluorescence microscope is not as good for this as it will see all of the fluorescence from other cell layers and not just the cell we are interested in.
Part 3
– We need to understand whether the Lr34res-Venus protein is functional. This is because the extra Venus domain may interfere with protein function. If Lr34res-Venus provides enhanced resistance to leaf rust then it means that the observations we make with the microscope are likely to also be true for the version of Lr34res that does not have the Venus domain. In other words, we have not altered the behaviour of Lr34 by adding the Venus domain.
– To do this test we need the help of a research group who work with leaf rust. Before we can ask for this help (a “collaboration”) we need to be sure that this effort is justified. The plants we have put Lr34res-Venus into may already be resistant. We need to confirm that the wild type Fielder cultivar carries a sensitive allele. We can do this be sequencing the Lr34 in the Fielder variety.
– We think that Fielder has the sensitive allele because a presentation at a meeting suggested this. The reference is in this picture (please copy it into text for your grant proposal and report):
– We do not have the sequence to confirm this and it is possible that Fielder was still segregating for the allele. It would be good practice to confirm that our starting strain had the expected allele.
– We can confirm this by:
1. Isolating genomic DNA from our control plants.
2. Designing PCR primers that will amplify the parts of Lr34 that are different between sensitive and resistant alleles.
3. Using these primers in a PCR to amplify these fragments.
4. Sending these fragments to sequence.
5. Comparing the sequences to known resistant and sensitive alleles.
– If our control Fielder plants are confirmed to carry a “sensitive” allele it is then possible to perform the disease resistance test. We could then approach a leaf rust laboratory (either at NIAB or elsewhere) for help in performing this test.
