This summer I took part in a project in Professor Katherine Denby’s lab at the Centre for Novel Agricultural Products (CNAP), University of York, focused on improving plant defence responses to pathogen infection. Food insecurity is one of the most pressing issues of the current age, and biotic stresses account for at least 10% of global crop losses per year. With environmental and regulatory restrictions on pesticides, it is important that we uncover new ways to reduce crop loss from pathogen infection. One solution for improving plant resistance is to create transgenic lines that launch a faster and more effective defence response when infected. This is likely to have a less detrimental impact on overall plant fitness compared to approaches to increase defence gene expression all the time. Genetically engineering plants in this way has great potential as it allows plants to express beneficial characteristics without relying on the slower process of generating random variation and subsequent selection through breeding. However, it does rely on having a deep understanding of plant immunity and its regulation to be able to manipulate defence in planta.
Plant defence responses are known to be controlled by complex regulatory networks of transcription factors, allowing for fine tuning of the response to different pathogens and robustness of defence responses. Furthermore, pathogens are known to manipulate host plant physiology and we hypothesise that expression of positive regulators of defence during infection is reduced by the pathogen in order to increase the severity of infection. One such positive regulator is WRKY3 – a nuclear-localised transcription factor downregulated after infection with the fungal pathogen, Botrytis cinerea. In my project, I worked with transgenic Arabidopsis lines expressing WRKY3 under the control of ANAC055, SAP12 and ZAT11 promoters. These three genes are transcription factors which have been shown to be significantly upregulated in response to B. cinerea infection.
I confirmed the results of previous studies by analysing the expression of WRKY3 and the selected promoters in wild-type Arabidopsis plants by qPCR in both infected and uninfected leaves. The expression of WRKY3 demonstrated a significant decrease 40 hours post-infection with B. cinerea, while expression of ANAC055, SAP12 and ZAT11 increased significantly. As wild-type expression was behaving as expected, I moved on to study the transgenic lines.
Firstly, I checked for the presence of the correct transgenic constructs in each of the lines by PCR. All of the transgenic lines, excluding one proANAC055:WRKY3 line, had a copy of the transgenic WRKY3 gene in their genome. The transgenic copy was shorter than the endogenous copy due to it being constructed from cDNA and lacking the intron. As the proZAT11:WRKY3 lines were one generation behind the proANAC055:WRKY3 and the proSAP12:WRKY3 lines, lines containing single insertions of this transgene were selected for future analysis. The proANAC055:WRKY3 and proSAP12:WRKY3 lines used were homozygous for the re-wiring constructs.
Expression of WRKY3 in the leaves of these transgenic lines was assessed by qPCR. Four lines for each construct were tested, both uninfected and post-infection. For each, except for the single proANAC055:WRKY3 line with no detectable transgene, WRKY3 was seen to be upregulated after B. cinerea infection, but the degree of upregulation varied between the different lines. This variation may be useful in identifying a suitable threshold for re-wired WRKY3 expression.
I also carried out infection assays with B. cinerea to test for altered susceptibility in the transgenic lines, however most lines showed no obvious difference in susceptibility. Lesion area was significantly smaller than wild-type controls for one line of proSAP12:WRKY3, and this line also demonstrated the most significant upregulation of WRKY3 post-infection. This suggests that only high levels of WRKY3 upregulation will result in an altered disease resistance phenotype, and that additional untested transgenic lines should first be screened for high WRKY3 expression post-infection. In the future, there are transgenic lines in the lab containing re-wiring constructs of different transcription factors and promoters which should be explored.
During my time on this studentship project, I have gained invaluable laboratory skills that I will continue to use and build on in my future research career. I would like to thank everyone in Denby Lab for making this summer very special, in particular Dr Fabian Vaistij for all of his incredible support and guidance as my direct supervisor, and Professor Katherine Denby for granting me this amazing opportunity. I am looking forward to helping with their Green Impact project next year as I complete the 4th and final year of my Integrated Master’s degree!
Emma White
University of York
