Tailored coumarin biosynthesis for plant protection

Beesley, Alexander; Conrath, Uwe (Thesis advisor); Schirawski, Jan (Thesis advisor)

Aachen : RWTH Aachen University (2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2022


The adaptation of crop pathogens to currently used control measures increases the demand for novel strategies to ensure plant protection. In previous work in the Conrath laboratory, the coumarin scopoletin emerged as a promising natural plant protectant. Scopoletin is effective against the devastating soybean pathogen Phakopsora pachyrhizi. Furthermore, soybean plants hyper-accumulating scopoletin are less susceptible to the Sudden death syndrome (SDS) disease caused by Fusarium virguliforme. In the first part of this PhD thesis, I investigated possible modes of action of scopoletin in plant protection. F. virguliforme infection severity in soybean plants was evaluated using a qRT-PCR-based method and revealed no differences in infection severity between wild-type and scopoletin-hyperaccumulating, transgenic plants. Instead, the mode of action of scopoletin in the increased SDS tolerance seemed to be based on the enhanced capacity of transgenic plants to scavenge mycotoxin-induced reactive oxygen species (ROS) production thereby reducing cell death. In the second part of the thesis, I tried to find ways to boost the scopoletin levels in plants for enhancing plant protection. By utilising the Arabidopsis transcription factor MYB72, the expression of genes in the phenylpropanoid pathway was boosted in all transgenic species of plant analysed, that is Arabidopsis, tobacco, and soybean. This increase in phenylpropanoid gene expression translated to an increase in the level of scopoletin and its glucoside scopolin. Transgenic tobacco plants co-overexpressing AtMYB72 and AtF6’H1, the latter encoding Arabidopsis feruloyl-CoA 6’-hydroxylase, did not only lead to the hyper-accumulation of scopoletin but also of salicylic acid. Consistently, these transgenic tobacco plants were more resistant to TMV. However, strong hyperaccumulation also coincided with adverse effects like dwarfing in transgenic plants. To overcome these detrimental effects, the production of scopoletin derivatives was explored to potentially reduce the amount of the strong antioxidant scopoletin. Therefore, sideretin was tested for potential use as a plant protectant. In transgenic soybean plants overexpressing sideretin-biosynthesis genes, susceptibility to P. pachyrhizi was strongly reduced. With increasing complexity of genetic engineering of coumarin derivative biosynthesis, cloning multigene DNA-constructs became more challenging. To overcome these challenges, ways to improve cloning of multigene DNA-constructs were explored utilising first steps in metabolon-building. Utilising ribosomal skipping by using the 2A peptide enabled two peptides to be formed from one DNA coding sequence, thereby providing different advantages e.g., reducing the usage of promoters and tightly regulating co-expression. In sum, my work provides novel insight into the successful utilisation of increased and tailored coumarin biosynthesis for plant protection.