The Brassicacea family includes many important crop species, such as broccoli, cabbage, and rapeseed. Therefore, pathogens and pests that interfere with these crops can have significant effects on food production, including the white blister rust, Albugo candida.
I worked in the lab of Dr Volkan Cevik at the University of Bath (where I study), where the focus is on the pathogen Albugo candida, and particularly compatible and incompatible interactions with the model plant Arabidopsis thaliana (a Brassicaceae). The lab has been working on the transcriptional activation pathway of the resistance gene WRR7 (White Rust Resistance 7) and immune signalling following pathogen infection. My work was involved in trying to understand one particular piece of this pathway involving Chromatin Remodeller 4 (CHR4) and how it affects WRR7 transcriptional dynamics.
Following Albugo infection, Parkes (2020) found that WRR7 expression is significantly up regulated in resistant Col-eds1.2 plants, but in the chr4 mutant plant EMS34, RT-qPCR experiments showed that following Albugo infection EMS34 mutants with the chr4.3 allele have less WRR7 expression compared to Col-eds1.2 at 3dpi, but similar expression levels at 5 dpi. This indicates that CHR4 is required for the responsiveness of WRR7 during early stages of the infection.
EMS34 mutant plants are susceptible, but Col-eds1.2 plants are resistant, leading to different levels of infection which can lead to biased expression data. Parkes (2020) also identified an additional mutant line, EMS138, with a non-synonymous SNP in the WRR7 gene (hereafter wrr7.2) causing an S8F mutation that impairs protein function but does not affect wrr7.2 up-regulation following pathogen infection. To investigate the variation of WRR7 expression due to chr4.3 mutation more precisely, EMS138 (wrr7.2) and EMS34 (chr4.3) lines were crossed and an F2 population was obtained. Initially, my work involved genotyping F2 plants using CAPS markers developed for specific genotypes and identifying F2 lines homozygous for wrr7.2 as well as chr4.3.
Once I indentified the desired mutant plants (hereafter Col-eds1.2 chr4.3 wrr7.2), we infected Col-eds1.2 chr4.3 wrr7.2 or Col-eds1.2 wrr7.2 with Albugo candida spores to trigger infection and transcriptional response, or with water as controls. We collected the samples and analysed their gene expression using RT-qPCR. For me, all of these practical techniques were new and provided valuable experience that I previously hadn’t gotten.
We found that there were basal transcriptional changes in the non-infected/control plants, with lower residual expression in the Col-eds1.2 chr4.3 wrr7.2 mutants compared to Col-eds1.2 wrr7.2 mutants. There were also post-activation transcriptional changes in the infected plants, with chr4.3 mutants showing lower transcription levels. On top of this, there were changes in WRR7 activation, with chr4.3 mutants showing lower initial activation. Overall, this all shows that chr4 mutation leads to both lower responsiveness to pathogen infection and lower basal magnitude of their response.
Throughout this process, the main problem encountered was the occassional failure in genotyping for double mutants, which could sometimes be pin-pointed to a particular error made in the process. The main solution was to re-genotype these plants which often would yield results.
The implications of this work could be important in food protection (as iterated before), but there is a more direct next step to be taken. This involves investigating the actual impact that mutations in CHR4 has on the histone modifications that it has been shown to edit, which in-turn impacts the DNA-histone dynamics and transcription of the WRR7 gene.
In the short-term, this project has massively boosted my confidence in the lab, and has given me great insight into what being in the lab everyday is like. It has given me an interest in plant pathology that wasn’t present before and has allowed me to think about doing a PhD in this area one day. I want to thank Dr Cevik for hosting me in his lab, Miss Zou for being a great supervisor, and the BSPP for their generous support in enabling this opportunity.
University of Bath