Etablierung einer Methode zur markerfreien Genom-Editierung in Magnaporthe oryzae und Charakterisierung der Virulenzfaktoren MoPl1 und MoNudix
Wegner, Alex Leander; Schaffrath, Ulrich (Thesis advisor); Panstruga, Ralph (Thesis advisor)
Aachen : RWTH Aachen University (2022, 2023)
Dissertation / PhD Thesis
Dissertation, RWTH Aachen University, 2022
The fungus Magnaporthe oryzae is the causal agent of the rice blast disease infecting sweet grasses such as the crops rice, barley, millet and wheat whose yields account for a large proportion of the world's staple food supply. Up to now, crop losses could often be minimized by the use of fungicides. However, due to their intensive use, new resistances become established, making the control of the pathogen more difficult. Therefore, new approaches must be developed. To infect plants, the pathogen secretes virulence factors, which modulate the infection process and are used by the fungus to manipulate the plant immune system. Studying these virulence factors at the molecular level presents a novel approach to develop new defense strategies and to understand the fungus-plant interaction in detail. For the characterization of effectors and virulence factors, different methods, such as CRISPR/Cas9 mediated genome editing or the generation of mutants, constitutively expressing an effector-gene, are used and applied in reverse genetics. From the phenotypes of the different generated genotypes, initial conclusions can be drawn about the function of the protein encoded by the candidate gene. Since targeted gene deletion usually involves replacing a gene with a selection marker, the number of gene-deletions in the same M. oryzae mutant was severely limited by the number of well characterized selection markers. Therefore, the characterization of genes, e.g. occurring multiple times in the genome was restricted. In this work, the repertoire of selection markers for M. oryzae was successfully expanded and it was shown that the fungicidal compound fenhexamid in combination with the resistance-conferring gene FfERG27 can be used as a robust, cost-effective and efficient selection system for the selection of Magnaporthe-transformants. Furthermore, to avoid the problem of limiting selection markers, a method for marker-free selection of mutants of M. oryzae was established. This method is based on the idea of using telomeric vectors which encode a resistance marker gene for the selection of co-transformed M. oryzae protoplasts. The used telomeric vectors are known to be stably replicated only under selection pressure and get lost without it. The combination of the CRISPR/Cas9-technology together with the use of telomeric vectors as a transient selection marker paves the way for new tools to edit the phytopathogenic fungus, which will be discussed in this work. Using artificially generated gene deletion mutants and mutants constitutively expressing mRFP-tagged candidate genes, two genes were found to encode proteins that have an important function in the fungal infection process, the genes MoPL1 and MoNUDIX. The first gene is expressed during the necrotrophic stage of the infection and encodes a secreted pectate lyase. Both, the deletion and the constitutive expression of MoPL1 resulted in reduced virulence of the pathogen. It could be shown that the reduced virulence of the mutants correlated with a higher rate of infection sites, which were blocked by the formation of a papilla or a hypersensitive cell death response by the plant. In case of the gene deletion, the reduced virulence can be explained by the loss of the enzyme, since it is needed for the degradation of the cell wall. Expression of an enzymatically inactive version of the enzyme confirmed that the defense mechanism is not based on direct recognition of the enzyme as a PAMP (pathogen-associated molecular pattern). Thus, recognition of the degradation products is probable. The gene MoNUDIX encodes a protein with a NUDIX domain, whose possible role as a virulence factor has already been discussed in former studies. High amounts of transcripts from this gene were found almost exclusively at 48 hours after inoculation. Localization studies with an mRFP-tagged fusion protein suggested an accumulation of the protein in the nucleus and endoplasmic reticulum. According to this result, the protein might have a function in the fungus itself and is not secreted as it is known for many effectors. Using CRISPR/Cas9-technology and several resistance markers, deletion mutants of the gene present twice in the genome were generated for the first time. The deletion of both paralogues led to a strongly reduced virulence of M. oryzae in the interaction with barley, indicated by significantly smaller lesions on barley plants. Interestingly, the reduced virulence was not observed on rice. In this work, two additional proteins were identified that have an influence on the virulence of M. oryzae. The methodological improvements presented in this work by using fenhexamid for the selection of M. oryzae transformants and the use of telomere-vector driven selection for a selection without an integrative selection-marker will greatly facilitate the identification of further virulence factors in the future.
- Department of Biology 
- Department of Molecular Plant Physiology