An internship abroad is an enrichment of the coursework of my bachelor course (Applied Biology), which I have executed at the John Innes Centre (JIC) in the Christopher Ridout lab group. I was supervised by Dr Henk-Jan Schoonbeek and worked also together with Hicret Asli Yalcin (PhD student) since my project was correlated to her PhD study. They work with Brassica napus and focus on genes involved in PAMP-triggered immunity (PTI) but also work with Arabidopsis thaliana.
The focus of my project at the JIC was characterising and identifying the role of candidate genes in PTI using A. thaliana plants as model plant. Candidate genes were based on Genomic Wide Association Studies (GWAS) analysis, where responses of 192 Brassica napus lines to the PAMP molecule BcNEP2 were screened. Significant hits in GWAS analysis gave insight of the location in the genome where a possible candidate gene should be located. Mutants in the Arabidopsis genes corresponding to the candidates genes were chosen based on the synteny between the two genomes. Part of the mutant lines were provided by Miss Yalcin, the other part was ordered from Nottingham Arabidopsis Stock Centre (NASC). We conducted three PTI and disease assays on 5 week old plants to investigate the role of each gene in response to two PAMPs, BcNEP2 and flg22. Since plants with mutations in normally working proteins will no longer show responses in the PTI assays, we aimed to use homozygous mutant plants. Identifying the genotype was done by extracting DNA and performing PCR reactions using specific selected primer pairs by Henk-Jan.
We found that most NASC-ordered plants turned out to be heterozygous and therefore not directly useable for phenotyping. After genotyping we conducted the three different assays on the limited number of plants available: oxidative burst (ROS), activity of plant peroxidases (POX) and the Botrytis cinerea disease assay. Gene 1 should be further investigated in its role in defence to B. cinerea as we found the biggest lesions on leaves of plants mutated in this gene. Unfortunately, one plant was available to use, making the result was not significant but still worthwhile to use for further research. As we used two B. cinerea strains, B05.10 as wildtype strain and ΔBcatrB4 as mutant, it would be recommended to use more mutant strains as resistance genes could be functional to specific B. cinerea pathogenicity factors. The role of gene 2 might be further investigated since we found significant high ROS productions to BcNEP2.
Multiple proteins might be active during certain PAMP recognition and that quantitative disease resistance could be achieved using BAK1, other co-receptor proteins. Asli and Henk-Jan had already obtained homozygous plants for this gene and used these for crossing experiments. Based on our results, the hypothesis that gene 3 has a role in PTI was confirmed. The effect of gene 3 was also clearly visible on plants infected with B. cinerea, significantly bigger lesions were found on gene 3 mutant plants. We also found F1 plants from crosses between mutants of gene 3 and mutant plant bak1-5 showed to significant higher ROS production, confirming that mutants in gene 3 and bak1-5 are not allelic.
During my project I was not able to use as many plants as hoped since less homozygous mutant plants were available for phenotype assays. Seeds of heterozygous and homozygous mutant plants will give more plants from also more lines so that similar research will give more representable results. I thoroughly enjoyed my time during my internship at the JIC. I have learnt so much about plant-pathogen interactions and plant disease resistance with proteins and genes involved, I did not expect to expand my knowledge about it so much. The project gave me more insight and hands-on experience in working with mutant plants and pathogens and performing analysis in the lab. It has made me considering doing my masters and PhD in a plant-pathogen related subject. I would like to thank Henk-Jan Schoonbeek and Asli Yalcin for their superior supervision and giving me the possibility to not only learn from my own project, but also from projects of other people in the JIC. I would also like Christopher Ridout for giving me the chance to work in the lab and setting up the project. And a big appreciation to the BSPP for funding this project and providing me with possibly one of the best experience in my life so far.
Teun van Lent
HAS University of Applied Sciences, The Netherlands